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WO2025131063A1 - Antibody drug conjugates targeting b7-h3 and trop2 and the use thereof - Google Patents

Antibody drug conjugates targeting b7-h3 and trop2 and the use thereof Download PDF

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Publication number
WO2025131063A1
WO2025131063A1 PCT/CN2024/140994 CN2024140994W WO2025131063A1 WO 2025131063 A1 WO2025131063 A1 WO 2025131063A1 CN 2024140994 W CN2024140994 W CN 2024140994W WO 2025131063 A1 WO2025131063 A1 WO 2025131063A1
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Prior art keywords
antibody
amino acid
region
acid sequence
seq
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French (fr)
Inventor
Bo Wang
Bin Li
Li Li
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Fortvita Biologics Inc
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Fortvita Biologics Inc
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    • A—HUMAN NECESSITIES
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    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
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    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
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    • 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
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    • 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
    • A61K47/6875—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 being a hybrid immunoglobulin
    • A61K47/6877—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 being a hybrid immunoglobulin the antibody being an immunoglobulin containing regions, domains or residues from different species
    • A—HUMAN NECESSITIES
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    • 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
    • A61K47/6875—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 being a hybrid immunoglobulin
    • A61K47/6879—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 being a hybrid immunoglobulin the immunoglobulin having two or more different antigen-binding sites, e.g. bispecific or multispecific immunoglobulin
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    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C—CHEMISTRY; METALLURGY
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    • 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
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    • 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
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
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Definitions

  • the invention relates to antibodies targeting B7-H3 and TROP2, and antibody-drug conjugates (ADCs) constructed based thereon, and to compositions containing the antibody-drug conjugates.
  • ADCs antibody-drug conjugates
  • the invention also relates to therapeutic and diagnostic uses of these antibodies or ADCs.
  • ADC antibody-drug conjugate
  • B7-H3 is a type I transmembrane protein of the B7 family. It is not constitutively expressed in many immune cells (e.g., natural killer (NK) cells, T cells, and antigen-presenting cells (APCs)) ; however, its expression can be induced. Moreover, the expression of B7-H3 is not limited to immune cells. B7-H3 transcripts are expressed in various human tissues (including colon, heart, liver, placenta, prostate, small intestine, testis, and uterus) as well as in osteoblasts, fibroblasts, epithelial cells, and other non-lymphoid lineage cells that may indicate immune and non-immune functions (Nygren et al. Front Biosci.
  • B7-H3 is also expressed in various human cancers, including prostate cancer, clear cell renal cell carcinoma, glioma, melanoma, lung cancer, breast cancer, and so on.
  • ADCs antibody-drug conjugates
  • ADCs including DS-7300a
  • DS-7300a still have some drawbacks, such as the need for improved efficacy, low internalization efficiency, side effects that need to be improved, uneven product DAR value distribution affecting drug efficacy, low DAR values, and/or short half-life, and the like.
  • TROP2 a trophoblast cell surface antigen, also known as tumor-associated calcium signal transduction protein (TACSTD2) , is overexpressed in various human epithelial cancers, including breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, and ovarian cancer (Yezhe Cheng et al., Frontiers in Oncology, 2022 Dec 23; 12: 951589) .
  • TACSTD2 tumor-associated calcium signal transduction protein
  • ADCs anti-TROP2 antibody-drug conjugates
  • Trodelvy an anti-human TROP2 antibody-SN-38 conjugate, or DS-1062a
  • ORR objective response rate
  • TNBC drug-resistant triple-negative breast cancer
  • Trodelvy may have a relatively high off-target effect.
  • DS-1062a Dato-DXd, AstraZeneca and Daiichi Sankyo
  • DS-1062a has shown significant efficacy in treating patients with lung cancer without gene mutation and triple-negative breast cancer, it still has issues such as uneven DAR distribution affecting drug efficacy and target toxicity in normal tissues.
  • ADCs of monoclonal antibodies also face the issue of drug tolerance due to the downregulation of target expression.
  • the invention provides an immunoconjugate comprising an antibody targeting B7-H3 and TROP2 (e.g., a bispecific antibody of the invention that specifically binds B7-H3 and TROP2) and an additional payload.
  • B7-H3 and TROP2 e.g., a bispecific antibody of the invention that specifically binds B7-H3 and TROP2
  • the immunoconjugate is an antibody-drug conjugate.
  • the present invention provides bispecific antibody-drug conjugates (ADCs) targeting B7-H3 and TROP2.
  • ADCs antibody-drug conjugates
  • the ADC has one or more of the following advantages:
  • the present invention provides an antibody-drug conjugate of formula (I) :
  • Ab is an antibody or fragment thereof that specifically binds B7-H3 and TROP2 (e.g., human B7-H3 and human TROP2) ;
  • L is a linker
  • D is a drug, preferably an anti-tumor compound
  • p is an integer selected from 1 to 16, such as an integer selected from 4 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,
  • Ab in formula (I) is a bispecific antibody that specifically binds to B7-H3 and TROP2 comprising a first antigen-binding region that specifically binds to TROP2 and a second antigen-binding region that specifically binds to B7-H3, wherein the second antigen-binding region that specifically binds to B7-H3 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein
  • the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1
  • the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2
  • the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3
  • the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 7
  • the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 8
  • the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 9.
  • the second antigen-binding region that specifically binds B7-H3 of said bispecific antibodies comprises a VH and a VL, wherein said VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence set forth in SEQ ID NO: 4, and/or said VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity the amino acid sequence set forth in SEQ ID NO: 10.
  • the second antigen-binding region that specifically binds B7-H3 of said bispecific antibodies comprises a VH and a VL, wherein the VH and VL comprise or consist of, respectively, the amino acid sequences shown below: SEQ ID NO: 4 and SEQ ID NO: 10, respectively.
  • the first antigen-binding region that specifically binds TROP2 of said bispecific antibodies comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID No: 13; HCDR2 comprises or consists of the amino acid sequence of SEQ ID No: 14; HCDR3 comprises or consists of the amino acid sequence of SEQ ID No: 15; LCDR1 comprises or consists of the amino acid sequence of SEQ ID No: 19; LCDR2 comprises or consists of the amino acid sequence of SEQ ID No: 20; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID No: 21.
  • the first antigen-binding region that specifically binds TROP2 of said bispecific antibodies comprises a VH and a VL, wherein said VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity thereto, and/or said VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity thereto.
  • the bispecific antibody comprises a first Fc region and a second Fc region, wherein the first Fc region and the second Fc region are the same or different.
  • the first Fc region and second Fc regions are, respectively, human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc, e.g., comprise or consist of the amino acid sequence of SEQ ID NO: 30 or 31 or an amino acid sequence having at least 90%, e.g., 95%, 96%, 97%, 98%or 99%or more identity thereto.
  • the first and second Fc regions of the bispecific antibody have introduced therein mutations that promote heterodimerization of the first and second Fc regions.
  • the mutation of the Fc region is introduced based on the Knob-into-Hole technique, wherein the corresponding Knob mutation and Hole mutation are introduced in the first Fc region and the second Fc region, respectively.
  • one Fc region comprises the amino acid substitutions S354C and T366W and the other Fc region comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to the EU index) , e.g., wherein the S354C and Y349C form a non-natural disulfide bond.
  • the mutation of the Fc region is introduced based on the Innobody technology.
  • the Fc region further comprises a mutation that reduces binding to the Fc ⁇ receptor, such as the L234A/L235A mutation.
  • one Fc region polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 27 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to the amino acid sequence set forth in SEQ ID No: 27, and the other Fc region polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 28 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to the amino acid sequence set forth in SEQ ID No: 28.
  • the first antigen-binding region of the bispecific antibody is a Fab fragment, and/or the second antigen-binding region is a Fab.
  • the Fab which is the first antigen binding region or the second antigen binding region, comprises CH1, wherein the CH1 is CH1 from IgG1, IgG2, IgG3, or IgG4, preferably CH1 from IgG1.
  • the CH1 comprises
  • (i) comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 32, or
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 32.
  • the Fab which is the first antigen binding region or the second antigen binding region, comprises a light chain constant region, wherein the light chain constant region is a Kappa light chain constant region or a Lambda light chain constant region.
  • the Kappa light chain constant region comprises
  • (i) comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 11, or
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 11.
  • the first antigen binding region that specifically binds TROP2 comprises a first Fab linked at its C-terminus of CH1 to the N-terminus of the first Fc region (with or without a connector, e.g., a hinge region) and the second antigen binding region that specifically binds B7-H3 comprises a second Fab linked at its C-terminus of CH1 to the N-terminus of the second Fc region (with or without a connector, e.g., a hinge region) .
  • the bispecific antibody is an IgG-like antibody having the configuration shown in Figure 1.
  • the bispecific antibody comprises a first Fab as a first antigen-binding region that specifically binds TROP2 and a second Fab as a second antigen-binding region that specifically binds to B7-H3, wherein the bispecific antibody comprises or consists of
  • Heavy chain 1 from N-terminus to C-terminus comprises or consists of: heavy chain variable region of the Fab that specifically binds TROP2 -heavy chain constant region CH1-First Fc region, wherein heavy chain constant region CH1 is linked at its C-terminus to the N-terminus of the first Fc region with or without a connector (e.g., hinge region) ;
  • Light chain 1 from N-terminus to C-terminus comprises or consists of: a light chain variable region of the Fab that specifically binds TROP2 -light chain constant region;
  • Heavy chain 2 from N-terminus to C-terminus comprises or consists of: heavy chain variable region of the Fab that specifically binds B7-H3 -heavy chain constant region CH1-second Fc region, wherein heavy chain constant region CH1 is linked at its C-terminus to the N-terminus of the second Fc region with or without a connector (e.g., hinge region) ;
  • Light chain 2 from N-terminus to C-terminus comprises or consists of: a light chain variable region of the Fab that specifically binds B7-H3 -light chain constant region,
  • each domain is linked directly;
  • said first Fc region comprises mutations Y349C, T366S, L368A and Y407V, and said second Fc region comprises mutations S354C and T366W, and vice versa; optionally, the first and second Fc regions further comprise L234A/L235A mutations, respectively.
  • said Heavy chain 1 comprising or consisting of the amino acid sequence shown as SEQ ID NO: 18, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity thereto;
  • Light chain 1 comprising or consisting of the amino acid sequence shown as SEQ ID NO: 23, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity thereto;
  • Heavy chain 2 comprising or consisting of the amino acid sequence shown as SEQ ID NO: 6, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity thereto; ; and
  • Light chain 2 comprising or consisting of the amino acid sequence shown as SEQ ID NO: 12, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity thereto.
  • the anti-tumor compound is a cytotoxic agent, such as camptothecins, auristatins, maytansinoids, taxanes, anthracyclines, vinca alkaloids, MEK inhibitors, or KSP inhibitors.
  • cytotoxic agent such as camptothecins, auristatins, maytansinoids, taxanes, anthracyclines, vinca alkaloids, MEK inhibitors, or KSP inhibitors.
  • D is represented by formula (D-1) :
  • R 1 is selected from H, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 1 -C 6 haloalkyl, C 2 -C 6 haloalkenyl and C 2 -C 6 haloalkynyl;
  • R 2 is selected from H, halo, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, -OR 4 and -SR 4 ;
  • R 3 is selected from H, halo, CN, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl and -OR 4 ; or R 2 and R 3 together form -O (CH 2 ) n O-or -O (CF 2 ) n O-, wherein n is 1 or 2;
  • R 4 is selected from H or C 1 -C 4 alkyl.
  • a wavy line in a structure indicate the connection position of the structure, for example, the wavy line in formula (D-1) indicates the connection position of D-1 linked to L.
  • R 1 is H
  • R 2 is C 1 -C 6 alkyl
  • R 3 is -F.
  • D is represented by formula (D-2) :
  • R 1 , R 2 and R 3 are as defined above.
  • D is represented by formula (D-3) or formula (D-4) :
  • -L- is represented by the following structure:
  • Z is selected from wherein m is an integer selected from 1 to 10; the carbonyl at the right end of Z is covalently linked to E;
  • E is a peptide residue comprising 2-10 amino acids, wherein the peptide residue is optionally substituted with one or more polyol groups, wherein the N-terminus of the peptide residue is covalently linked to Z;
  • Q is -O-or -S-;
  • L 1 is absent or - (C 1 -C 10 alkylene) -;
  • L 2 is absent, -N (R 5 ) C (O) - (C 1 -C 10 alkylene) -*or -C (O) N (R 5 ) - (C 1 -C 10 alkylene) -*; wherein *indicates that the terminus is covalently linked to Q; and
  • R 5 is H or C 1 -C 6 alkyl.
  • E is a peptide residue consisting of 2, 3, or 4 amino acids, the amino acids are selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine and leucine, and wherein the glutamine or glutamic acid is optionally substituted with one polyol group, and -L 2 -L 1 -is - (C 1 -C 6 alkylene) -, - (C 1 -C 6 alkylene) -N (R 5 ) C (O) - (C 1 -C 6 alkylene) -*or - (C 1 -C 6 alkylene) -C (O) N (R 5 ) - (C 1 -C 10 alkylene) -*, wherein *indicates that said terminus is covalently linked to Q ;
  • R 5 is H or C 1 -C 6 alkyl.
  • E is -Gln-Val-Ala-, -Gly-Val-Ala-, -Gln-Phe-Ala-, -Gly-Phe-Ala-or wherein R 6 is H or C 1 -C 6 alkyl, wherein these E groups are covalently linked to Z through the left N-terminus, and
  • -Z-E-NH-CH 2 -Q-L 2 -L 1 - is represented by the following structure
  • the antibody-drug conjugate is represented by the following formula
  • the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof has an average DAR of from 5 to 11, for example, from 7.5-8.5.
  • the antibody-drug conjugate is represented by the following formula
  • Ab is the bispecific antibody of the present invention
  • q is as defined for p in formula (I) above,
  • the antibody-drug conjugate has an average DAR of from 5 to 11, for example, from 7.5-8.5.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof as described above, and optionally one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immune modulators and optionally pharmaceutically acceptable excipients.
  • the present invention provides a pharmaceutical combination comprising the antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof as described above, and one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immune modulators.
  • therapeutic agents such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immune modulators.
  • the present invention provides a method of preventing or treating a tumor in a subject, comprising administering to the subject an effective amount of the antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof as described above, or the pharmaceutical composition as described above, or the pharmaceutical combination as described above.
  • the tumor is a cancer
  • the cancer has B7-H3 and/or TROP2 at elevated level (such as at nucleic acid or protein level) compared with the corresponding tissue of a healthy subject or a healthy tissue adjacent to the cancer tissue of the patient.
  • the cancer is selected from lung cancer (including non-small cell lung cancer and small cell lung cancer) , colon cancer, oral squamous cell carcinoma, breast cancer, melanoma, head and neck tumors, prostate cancer, esophageal cancer, cervical cancer, renal cancer, bladder cancer, ovarian cancer or pancreatic cancer.
  • lung cancer including non-small cell lung cancer and small cell lung cancer
  • colon cancer oral squamous cell carcinoma
  • breast cancer melanoma
  • head and neck tumors prostate cancer
  • esophageal cancer cervical cancer
  • renal cancer renal cancer
  • bladder cancer ovarian cancer or pancreatic cancer.
  • the method further comprises administering one or more therapies, such as a therapeutic approach and/or other therapeutic agents, to a patient, preferably the therapeutic approach includes radiation therapy or surgery, or the therapeutic agents include chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immune modulators.
  • therapies such as a therapeutic approach and/or other therapeutic agents
  • the therapeutic approach includes radiation therapy or surgery
  • the therapeutic agents include chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immune modulators.
  • the antibody-drug conjugate of the present invention has the following advantages, the antibody-drug conjugate:
  • Fig. 1 shows a schematic structural diagram of bispecific antibody HZ5C2.9/hRS7.13.
  • Fig. 2 shows the results of the binding experiments of bispecific antibody HZ5C2.9/hRS7.13 and the parental antibody to NCI-H358 cells.
  • Fig. 3 shows the results of the endocytosis experiments of bispecific antibody HZ5C2.9/hRS7.13 and parental antibody.
  • Fig. 4 shows the results of the binding experiments of the bispecific antibody ADC molecule and the parent antibody ADC molecule to cells.
  • Fig. 5 shows the in vitro killing effect of bispecific antibody ADC molecules and parent antibody ADC molecules on several tumor cell lines.
  • Fig. 6A shows the tumor inhibiton effect of bispecific antibody ADC molecules and parent antibody ADC molecules in NCI-H358 mouse graft tumor model.
  • Fig. 6B shows the body weight change of mice in a tumor inhibition experiment using the NCI-H358 mouse graft tumor model.
  • Fig. 7A shows the tumor inhibition effect of bispecific antibody ADC molecules and parent antibody ADC molecules in HCC1806 mouse graft tumor model.
  • Fig. 7B shows the body weight change of mice in a tumor inhibition experiment using HCC1806 mouse graft tumor model.
  • Fig. 8 shows a schematic of the bispecific antibody ADC molecule.
  • B7-H3 is a type I transmembrane glycoprotein belonging to a member of the B7/CD28 superfamily, and is similar in sequence to the extracellular domain of PD-L1.
  • B7-H3 has 316 amino acids and contains one putative signal peptide consisting of 28 amino acids, one extracellular region consisting of 217 amino acids, one transmembrane region and one cytoplasmic domain consisting of 45 amino acids with a molecular weight of about 45-66 kDa.
  • B7-H3 In humans, the extracellular structure of B7-H3 may be an IgV-IgC-like domain (2Ig-B7-H3) or an IgV-IgC-IgV-IgC-like domain (4Ig-B7-H3) due to exon replication.
  • the sequence of cynomolgus monkey B7-H3 has about 90%homology with its human counterpart.
  • B7-H3 is human B7-H3.
  • B7-H3 is a protein under UniProt database accession number Q5ZPR3.
  • anti-B7-H3 antibody refers to an antibody that is capable of binding B7-H3 protein with sufficient affinity.
  • the antibodies may be used as diagnostic and/or therapeutic agents in targeting B7-H3, or to construct immunoconjugates, such as antibody drug conjugates.
  • anti-TROP2 antibody refers to an antibody that is capable of binding to TROP2 protein with sufficient affinity.
  • the antibodies may be used as diagnostic and/or therapeutic agents in targeting TROP2, or for the construction of immunoconjugates, such as antibody drug conjugates.
  • TROP2 is human TROP2. In some embodiments, TROP2 is the protein under Accession No. UniProt data Accession No. P09758.
  • amino acid positions of all variable regions of the heavy and light chains are numbered according to the Kabat numbering system described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, MD (1991) and referred to herein as "Kabat numbering" .
  • EU numbering when used in reference to amino acid positions in domains (e.g., constant regions, e.g., Fc regions) of antibodies other than the variable regions, numbering is according to the EU numbering system described in Kabat, E. A. et al, Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991) and referred to herein as "EU numbering" .
  • position numbering and/or amino acid residues are assigned to a particular antibody isotype, it is intended to apply to the corresponding position and/or amino acid residue of any other antibody isotype, as is known to those skilled in the art.
  • antibody is used herein in the broadest sense to refer to proteins comprising an antigen-binding site, encompassing natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) , single chain antibodies, intact antibodies, and antibody fragments.
  • the terms “whole antibody” , “full-length antibody” , “complete antibody” and “intact antibody” are used interchangeably herein to refer to a naturally occurring glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region consists of 3 domains CH1, CH2 and CH3 (and optionally CH4) .
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain CL.
  • the VH and VL regions may be further subdivided into hypervariable regions (being Complementarity Determining Regions (CDRs) ) interspersed with relatively conserved regions (being Framework Regions (FRs) .
  • CDRs Complementarity Determining Regions
  • FRs Framework Regions
  • Each VH and VL consists of three CDRs and 4 FRs, arranged from N-terminal to C-terminal in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the constant regions are not directly involved in binding of the antibody to the antigen, but exhibit multiple effector functions.
  • Half-antibody or half-polymer refers to a monovalent antigen-binding polypeptide.
  • the half-antibody or half-polymer comprises a VH/VL unit and optionally at least a portion of an immunoglobulin constant domain.
  • a half-antibody or a half-polymer comprises one immunoglobulin heavy chain, or antigen-binding fragment thereof, associated with one immunoglobulin light chain.
  • the half-antibody or half-polymer is monospecific, i.e., binds a single antigen or epitope.
  • the half-antibody binds to TROP2 and does not bind to B7-H3.
  • the half-antibody binds to B7-H3 and does not bind to TROP2.
  • a half-antibody may have an antigen-binding domain consisting of a single variable domain, e.g., derived from camelidae.
  • antibody constant regions or antibody constant domains may be selected according to the intended function of the antibody molecule.
  • the constant region may be an IgA, IgD, IgE, IgG or IgM region, especially an immunoglobulin constant domain of human IgG, e.g. a constant domain of human IgG1, IgG2, IgG3 or IgG4, preferably a constant domain of human IgG1.
  • a Fab fragment of an antibody may comprise CH1 from IgG1 and CL constant regions.
  • the Fc region of an antibody may comprise the CH2 and CH3 domains from IgG1.
  • the immunoglobulin constant region may have a native sequence or a variant sequence.
  • Fc domain or "Fc region” is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • a native immunoglobulin "Fc domain” comprises two or three constant domains, namely the CH2 domain, the CH3 domain, and the optional CH4 domain.
  • the immunoglobulin Fc domain comprises the second and third constant domains (CH2 and CH3 domains) originated from the two heavy chains of IgG, IgA, and IgD class antibodies; or comprises the second, third and fourth constant domains (CH2 domain, CH3 domain and CH4 domain) originated from the two heavy chains of the IgM and IgE classes antibodies.
  • the numbering of amino acid residues in the Fc region or heavy chain constant region is according to the EU numbering system (also known as the EU index) as described in Kabat et al, Sequences of Proteins of Immunological Interes, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present.
  • Two Fc regions can be dimerized to form a dimeric Fc, and two different Fc heterodimerizations forms a heterodimeric Fc.
  • the terms "Fc region” , “Fc portion” and “dimeric Fc (e.g., heterodimeric Fc) do not include the heavy chain variable region VH and the light chain variable region VL and the heavy chain constant region CH1 and the light chain constant region CL of an immunoglobulin, but may in some cases include a hinge region of the heavy chain constant region at the N-terminal.
  • the human IgG heavy chain Fc region extends from Asp221 or from Cys226 or from Asp231 to the carboxy-terminus of the heavy chain.
  • a human IgG1 Fc region polypeptide (comprising a portion of hinge region) comprises or consists of the following amino acid sequence:
  • a human IgG1 Fc region polypeptide (comprising a portion of hinge region) comprises or consists of the following amino acid sequence, wherein E is at position 356 and M is at position 358:
  • the Fc region is originated from an Fc region of human origin. In one embodiment, the Fc region comprises all or part of a human constant region. The antibody Fc region is directly involved in complement activation, C1q binding, C3 activation, and Fc receptor binding. In one embodiment, the Fc region is a human Fc region. In one embodiment, the Fc region belongs to the subclass human IgG 4. In one embodiment, the Fc region belongs to the subclass human IgG1.
  • a “heterodimeric Fc scaffold” refers to a scaffold comprising or formed from two different Fc regions by dimerization, which can be linked at its N-terminus or C-terminus to a domain (e.g., heavy and/or light chain variable regions of an antibody or antigen-binding fragment of an antibody that can bind to a target molecule, or soluble portions of a ligand or receptor that can bind to a target molecule) binding to an antigen for use in constructing multispecific antibodies, e.g., bispecific antibodies.
  • a domain e.g., heavy and/or light chain variable regions of an antibody or antigen-binding fragment of an antibody that can bind to a target molecule, or soluble portions of a ligand or receptor that can bind to a target molecule
  • CH1 region refers to the portion of an antibody heavy chain polypeptide that extends from EU position 118 to EU position 220 (EU numbering system) .
  • the CH1 domain comprises or consists of the amino acid sequence of
  • antibody fragment includes a portion of an intact antibody.
  • the antibody fragment is an antigen-binding fragment.
  • antigen-binding fragment of an antibody is a molecule distinct from a full-length antibody that comprises a portion of the full-length antibody, but is capable of binding to an antigen of the full-length antibody or competes for binding to an antigen with the full-length antibody (i.e., with the full-length antibody from which the antigen-binding fragment is derived) .
  • Antigen-binding fragments may be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen binding fragments include, but are not limited to, Fab, Fab', F (ab') 2, Fv, single chain Fv, diabodies, single domain antibodies (sdAb) , or nanobodies.
  • Fab fragment or “Fab” are used interchangeably herein to refer to an immunoglobulin fragment consisting of two polypeptide chains, comprising an immunoglobulin heavy chain variable region VH, a heavy chain constant domain CH1, a light chain variable region VL, and a light chain constant domain CL, wherein one polypeptide chain comprises, from N-terminus to C-terminus, VH and one constant region selected from CH1 and CL, and the other polypeptide chain comprises, from N-terminus to C-terminus, VL and another constant region selected from CL and CH1, wherein the VH and VL domains pair to form an antigen-binding site or an antigen-binding region.
  • the Fab polypeptide chain comprising the heavy chain constant region CH1 is also referred to as the "Fab heavy chain” ; accordingly, the Fab polypeptide chain comprising the light chain constant region CL is also referred to as "Fab light chain” .
  • CDR region is a region in an antibody variable domain that is highly variable in sequence and forms a structurally defined loop ( “hypervariable loop” ) and/or comprises antigen contact residues ( “antigen contact point” ) .
  • CDRs are primarily responsible for binding to epitopes.
  • the CDRs of the heavy and light chains are generally referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from N-terminus.
  • the CDRs located in the variable domain of the antibody heavy chains are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located in the variable domain of the antibody light chains are referred to as LCDR1, LCDR2, and LCDR3.
  • each CDR can be determined using any one or a combination of many well-known antibody CDR assignment systems including, e.g., Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al.
  • CDR or “CDR sequence” encompasses CDR sequences determined in any of the schemes described above or the combination thereof. CDRs may also be determined based on the same Kabat numbering position as a reference CDR sequence (e.g., any one of the exemplary CDRs of the invention) .
  • the CDRs of the antibodies of the invention are determined by Kabat schemes for boundaries, or by AbM schemes, or by a combination thereof.
  • the HCDR1 of the VH is determined by the AbM scheme
  • HCDR2 and HCDR3 are determined by the Kabat scheme
  • the LCDRs of the VL are determined by the Kabat scheme respectively.
  • the HCDR1 of the VH is determined by the AbM scheme
  • HCDR2 and HCDR3 are determined by the Kabat scheme
  • the LCDRs of the VL are determined by the Kabat scheme respectively.
  • hinge region refers to the portion of an antibody heavy chain polypeptide that connects the CH1 and CH2 regions in the wild-type antibody heavy chain, e.g., the IgG1 hinge region, e.g., according to EU numbering, the sequence of D221 to P230.
  • Other hinge region of IgG subclasses can be determined by alignment with cysteine residues in the hinge region of IgG1 subclass sequence.
  • CH1 may comprise a portion of the hinge region.
  • Fc region may comprise a portion of the hinge region.
  • Amino acid mutations are denoted by (original amino acid, amino acid position, mutated amino acid) .
  • T366W means that T located at EU numbering position 366 is substituted with W.
  • the mutations in a combination are linked by an "and” or “/” .
  • L234A/L235A indicates that both mutations L234A and L235A are included. It should be noted that when describing mutations, a particular position encompasses its corresponding amino acid position on other polypeptide chains as well.
  • “conservative alterations” include substitutions, deletions, or additions to the polypeptide sequence that does not substantially alter the desired functional activity of the polypeptide sequence. For example, conservative substitutions often result in the substitution of an amino acid for a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • alanine (A) alanine (A) , glycine (G) ; 2) aspartic acid (D) , glutamic acid (E) ; 3) asparagine (N) , glutamine (Q) ; 4) arginine (R) , lysine (K) ; 5) isoleucine (I) , leucine (L) , methionine (M) , valine (V) ; 6) phenylalanine (F) , tyrosine (Y) , tryptophan (W) ; 7) serine (S) , threonine (T) ; and 8) cysteine (C) , methionine (M) .
  • the term "conservative sequence changes" is used to refer to amino acid modifications that do not significantly affect or alter the antigen-binding characteristics of interest of the antibody molecules of the present invention containing the amino acid sequence.
  • conservatively modified variants will retain at least 80%, 85%, 90%, 95%, 98%, 99%or more, e.g., 100-110%or more, binding affinity for the antigen of interest relative to the parent antibody.
  • target refers to the substance for binding against which the binding molecule is directed.
  • the target may be an antigen, or may be a ligand or receptor.
  • antigen refers to a molecule that elicits an immune response. Such an immune response may involve antibody production or activation of specific immune cells, or both.
  • the antigen may be derived from recombinant or genomic DNA.
  • epipe refers to the portion of an antigen that specifically interacts with an antibody molecule.
  • antigen-binding region refers to any portion of an antibody or antigen-binding fragment thereof, e.g., a multispecific antibody or bispecific antibody, that binds a particular target or antigen.
  • the antigen-binding region may be, for example, an antibody or immunoglobulin per se or an antibody fragment.
  • antigen-binding regions may or may not have tertiary structure independent of the remainder of the multispecific or bispecific antibody, and may or may not bind to their antigen/epitope as separate entities.
  • multispecific antibody refers to an antibody having at least two antigen-binding regions, each of which binds to a different epitope of the same antigen or to a different epitope of different antigens.
  • Multispecific antibodies are antibodies that have binding specificities for at least two different antigens or epitopes.
  • bispecific antibodies having binding specificity for a first antigen and a second antigen.
  • the term "bispecific antibody” comprises antigen binding domains that specifically bind to two antigens or two epitopes. Unless otherwise indicated, the order of antigen binding by the bispecific antibody in the listed bispecific antibody names is arbitrary. That is, in some embodiments, the terms “anti-TROP2/B7-H3 bispecific antibody” and “anti-B7-H3/TROP2 bispecific antibody” are used interchangeably.
  • the bispecific antibody comprises two half-antibodies, wherein each half antibody comprises a single heavy chain variable region and optionally at least a portion of a heavy chain constant region and a single light chain variable region and optionally at least a portion of a light chain constant region.
  • the bispecific antibody comprises two half-antibodies, wherein each half-antibody comprises a single heavy chain variable region and a single light chain variable region and does not comprise more than one single heavy chain variable region and does not comprise more than one single light chain variable region. In some embodiments, the bispecific antibody comprises two half-antibodies, wherein each half-antibody comprises a single heavy chain variable region and a single light chain variable region, and wherein the first half-antibody binds to a first antigen/epitope and does not bind to a second antigen and the second half-antibody binds to a second antigen/epitope and does not bind to the first antigen.
  • first antigen-binding region in a multispecific antibody or bispecific antibody, it is meant a binding region that binds to a first antigen, and is not intended to limit the number of such antigen-binding regions contained in the antibody, e.g., one or more than one first antigen-binding region may be included in a multispecific antibody or bispecific antibody.
  • a bispecific antibody comprises a first antigen-binding region and a second antigen-binding region, but may comprise one or more than one first antigen-binding region and one or more than one second antigen-binding region.
  • the binding domain constituting the target/antigen-binding region is or is derived from a binding domain of the antibody that specifically binds antigen, e.g. a fragment of the antigen-binding region that specifically binds antigen, e.g. Fab, is or is derived from a corresponding fragment of the antibody, e.g. Fab, or the heavy chain variable region and/or the light chain variable region of the antigen-binding region is or is derived from the heavy chain variable region and/or the light chain variable region of the antibody, or 1, 2, 3, 4, 5 or 6 CDRs of the antigen-binding region are CDRs of the antibody.
  • the term "derived from” means that the fragment in the antigen-binding region is substantially identical to the fragment of the antibody from which it is derived, but has a mutation, such as a substitution, deletion or addition, at one or more sites. In a specific embodiment, the mutation is not in a CDR of the antibody.
  • the multispecific or bispecific antibodies of the present invention may comprise a connector.
  • the term "connector” as used herein refers to any molecule that enables direct attachment of different moieties of a multispecific antibody. Examples of connectors to establish covalent linkages between different moieties of the multispecific antibody include peptide connectors and non-proteinaceous polymers, including but not limited to polyethylene glycol (PEG) , polypropylene glycol, polyalkylene oxide, or copolymers of polyethylene glycol or polypropylene glycol.
  • PEG polyethylene glycol
  • peptide connector refers to a sequence of amino acids, wherein said sequence links together the amino acid sequences of the various moieties of the multispecific antibody.
  • the peptide connector has a length sufficient to link the two entities in such a way that they maintain their conformation relative to each other so as not to interfere with the desired activity.
  • the peptide connector may or may not comprise predominantly the following amino acid residues: Gly, Ser, Ala or Thr.
  • effector functions refers to those biological activities attributed to the Fc region of an immunoglobulin that vary with the isotype of the immunoglobulin.
  • immunoglobulin effector functions include: C1q binding and Complement Dependent Cytotoxicity (CDC) , Fc receptor binding, antibody dependent cell mediated cytotoxicity (ADCC) , Antibody Dependent Cellular Phagocytosis (ADCP) , cytokine secretion, immune complex mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g., B cell receptors) , and B cell activation.
  • ...valent antibody refers to the number of antigen binding sites present in an antibody molecule.
  • bivalent, trivalent, and tetravalent antibodies refer to the presence of 2, 3, and 4 antigen binding sites, respectively, in an antibody molecule.
  • a “knob-into-hole (KIH) " mutation or “KIH” mutation is used herein to refer to the introduction of mutations in the first Fc-polypeptide and the second Fc-polypeptide, respectively, using the “KIH” technique to form a bulge ( “knob” ) and a complementary cavity ( “hole” ) at the interface of the first Fc-polypeptide and at the interface of the second Fc-polypeptide. It is known in the art that "knob-into-hole” techniques can engineer the interface between different chains of an antibody molecule to facilitate proper conjugation of the individual chains of the antibody molecule.
  • this technique involves introducing a "bulge/knob" at the interface of one strand and a corresponding "cavity/hole” at the interface of the other strand to be paired with, so that the bulge can be placed in the cavity.
  • One preferred interface comprises the CH3 domain of the heavy chain constant domain of one chain and the CH3 domain of the heavy chain constant domain of the other chain to be paired with.
  • the bulge may be constructed by replacing the small amino acid side chain from the interface of the CH3 domain of the heavy chain constant domain of one chain with a larger side chain (e.g., tyrosine or tryptophan) .
  • Compensatory cavities of the same or similar size to the buldge are constructed at the interface of the CH3 domains of the heavy chain constant domains of the other chain to be paired by replacing large amino acid side chains with smaller side chains (e.g., alanine or threonine) .
  • Another alternative interface is the CL domain of the Fab fragment comprising the light chain and the CH1 domain of the heavy chain described above, which promotes the correct heterodimerization between the two chains of the Fab fragment by constructing a bulge-cavity interaction.
  • KIH mutations may further comprises cysteine mutations in the two CH3 regions, so as to form non-natural disulfide bond.
  • binding means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions.
  • the ability of an antigen binding site to bind to a particular antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art such as by Radioimmunoassay (RIA) or Bio-Layer Interferometry (BLI) or MSD assay or Surface Plasmon Resonance (SPR) .
  • ELISA enzyme-linked immunosorbent assay
  • RIA Radioimmunoassay
  • BLI Bio-Layer Interferometry
  • MSD assay Surface Plasmon Resonance
  • affinity or "binding affinity” refers to the inherent binding affinity that reflects the interaction between members of a binding pair.
  • the affinity of a molecule X for its partner Y can be generally represented by the dissociation constant (K D ) , which is the ratio of the dissociation and association rate constants (K dis and K on , respectively) .
  • K D dissociation constant
  • K dis and K on dissociation and association rate constants
  • sequence identity between sequences is performed as follows. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., for optimal alignment, gaps can be introduced in the first and second amino acid sequences or in one or both of nucleic acid sequences, or non-homologous sequences can be discarded for comparison purposes) .
  • the length of the aligned reference sequence is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100%of the length of the reference sequence. Amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at this position.
  • immunoconjugate is meant that the payload is attached to the antibody or antigen-binding fragment thereof by a linker so that the antibody or antigen-binding fragment thereof can act as a carrier to transport the payload to a target site in a targeted manner.
  • payload refers to an active moiety conjugated to an antibody or antibody fragment of the invention, and may include any moiety used to attach an antibody or antibody fragment.
  • the payload can be a drug, such as a small molecule drug, a radionuclide, DNA, RNA, an enzyme, or a polypeptide, among others.
  • the immunoconjugate encompasses an Antibody Drug Conjugate (ADC) , an antibody immunostimulatory conjugate drug (ISAC) , an Antibody Oligonucleotide Conjugate (AOC) , an antibody polypeptide conjugate drug (APC) , an antibody nuclide conjugate drug (RDC) , or an antibody degrading conjugate drug (ADeC) , among others.
  • ADC Antibody Drug Conjugate
  • ADC antibody immunostimulatory conjugate drug
  • AOC Antibody Oligonucleotide Conjugate
  • APC antibody polypeptide conjugate drug
  • RDC antibody nuclide conjugate drug
  • ADeC antibody degrading conjugate drug
  • Suitable payloads or active moieties for conjugation to the antibody include, for example, cytotoxic agents, chemotherapeutic agents, innate immune agonists (e.g., Toll-like receptor agonists (TLR) class ISAC drugs SBT6050, SBT6290, BDC-1001; STING agonist ISAC drugs XMT-2056, Treg cell regulatory ISAC drugs ADCT-301, etc. ) , immune modulators, therapeutic oligonucleotides (siRNA, PMO, etc. ) , or radionuclides, etc.
  • TLR Toll-like receptor agonists
  • the immunoconjugate of the invention is an antibody drug conjugate, i.e., ADC.
  • antibody-drug conjugate refers to a compound/molecule obtained by linking an antibody to a drug (e.g. asmall molecule drug) via a linker.
  • linker refers to a structural fragment that links a drug (e.g., a small molecule drug) to an antibody moiety. It is understood that the linker, prior to attachment to the antibody or antigen-binding fragment thereof, has a functional group that can form a bond or linkage with a functional group of the antibody or antigen-binding fragment thereof.
  • linker-payload refers to a compound formed by linking a payload, such as a drug (e.g., a small molecule drug) , to a linker.
  • a payload such as a drug (e.g., a small molecule drug)
  • site-specific conjugation refers to conjugating a drug specifically to a specific site of an antibody via a linker.
  • alkyl refers to a fully saturated branched or unbranched hydrocarbon group.
  • the alkyl group preferably contains 1 to 16 carbon atoms, for example 1 to 12 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms or 1 to 4 carbon atoms.
  • alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2-dimethylpentyl, 2, 3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like.
  • alkylene refers to an alkyl group as defined above, but which is divalent, i.e., has two single bonds linked to two other groups.
  • alkylene groups include -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 -, -CH (-CH 2 CH 3 ) -or -CH 2 CH (-CH 3 ) -.
  • alkenyl refers to a straight or branched chain hydrocarbon group containing 2 to 16 carbon atoms and containing at least one double bond and no triple bond.
  • the alkenyl group preferably contains 2 to 12 carbon atoms, 2 to 10 carbon atoms, 2 to 8 carbon atoms, 2 to 6 carbon atoms, or 2 to 4 carbon atoms.
  • Representative examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, and the like.
  • alkynyl refers to a straight or branched hydrocarbon group containing 2 to 16 carbon atoms and comprising at least one triple bond. Alkynyl groups preferably contain 2-12 carbon atoms, 2-10 carbon atoms, 2-8 carbon atoms, 2-6 carbon atoms, or 2-4 carbon atoms. Representative examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like.
  • halogen refers to fluoro (-F) , chloro (-Cl) , bromo (-Br) , and iodo (-I) .
  • haloalkyl refers to an alkyl group, as defined herein, substituted with one or more halo groups, as defined herein.
  • the haloalkyl group may preferably be a monohaloalkyl group, a dihaloalkyl group or a polyhaloalkyl group (including a perhaloalkyl group) .
  • the monohaloalkyl group may contain one iodo, bromo, chloro or fluoro in the alkyl group.
  • the dihaloalkyl and polyhaloalkyl groups may contain two or more of the same halogen atoms in the alkyl group or a combination of different halo groups.
  • the polyhaloalkyl contains up to 12, 10 or 8 or 6 or 4 or 3 or 2 halo groups.
  • haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, and dichloropropyl.
  • Perhaloalkyl refers to alkyl groups in which all hydrogen atoms are replaced by halogen atoms.
  • haloalkenyl refers to an alkenyl group, as defined herein, substituted with one or more halo groups as defined herein.
  • haloalkynyl refers to an alkynyl group, as defined herein, which is substituted with one or more halo groups as defined herein.
  • halo as defined for “haloalkyl” is applicable to “haloalkenyl” and “haloalkynyl” .
  • polyol group refers to an alkyl group as defined above containing multiple (e.g., 2 to 10, e.g., 3, 4, 5, 6, 7 or 8) hydroxyl groups, optionally containing one or more (e.g., 2, 3 or 4) other groups (e.g., amino, carbonyl) .
  • Non-limiting examples of "polyol groups” include, for example,
  • amino acid refers to both naturally occurring and synthetic amino acids.
  • the amino acids may be either L or D isomers.
  • the conventional amino acids referred to herein are written following a conventional approach. See, for example, Immunology-ASynthesis (2nd Edition, E. S. Golub and D. R. Gren, eds., Sinauer Associates, Sunderland, Mass. (1991) ) which is incorporated herein by reference.
  • amino acids are generally represented by the single and three letter abbreviations commonly known in the art.
  • glycine may be represented by Gly, alanine by Ala, valine by Val, glutamine by Gln, glutamic acid by Glu, phenylalanine by Phe and leucine by Leu.
  • pharmaceutically acceptable salt refers to a salt that retains the biological effects and properties of the ADC conjugates of the invention, and which is not biologically or otherwise undesirable.
  • the ADC conjugates of the invention may exist in the form of their pharmaceutically acceptable salt, including acid addition salts and base addition salts.
  • pharmaceutically acceptable non-toxic acid addition salts refer to salts formed by the ADC conjugates of the present invention with organic or inorganic acids including, but not limited to, hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, malic acid, and the like.
  • organic or inorganic acids including, but not limited to, hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzen
  • Non-toxic base addition salts mean salts formed by the ADC conjugates of the invention with organic or inorganic bases, including but not limited to alkali metal salts, such as lithium, sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; organic base salts, for example ammonium salts, formed with organic bases containing N groups.
  • alkali metal salts such as lithium, sodium or potassium salts
  • alkaline earth metal salts such as calcium or magnesium salts
  • organic base salts for example ammonium salts, formed with organic bases containing N groups.
  • solvate refers to an associated complex of one or more solvent molecules with an ADC antibody-drug conjugate of the invention.
  • Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N-dimethylformamide, dimethylsulfoxide, and the like.
  • DAR drug to antibody ratio
  • DAR refers to the ratio of drug moiety (D) conjugated to the Ab moiety as described herein to the Ab moiety in an ADC molecule.
  • the DAR may be determined by p in formula I.
  • DAR may be from 1 to 16, e.g. 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
  • DAR may also be calculated as the average DAR of a population of molecules in a product, i.e., the overall ratio of drug moiety (D) conjugated to the Ab moiety described herein to the Ab moiety as measured by detection methods (e.g., by conventional methods such as mass spectrometry, ELISA assays, electrophoresis, and/or HPLC) in a product, such DAR being referred to herein as average DAR.
  • detection methods e.g., by conventional methods such as mass spectrometry, ELISA assays, electrophoresis, and/or HPLC
  • the average DAR value of a conjugate of the invention is 1 to 16, e.g., 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, e.g., 1.0-8.0, 2.0-6.0, e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.
  • therapeutic agent comprises any substance effective in preventing or treating tumors (such as cancer) , including a chemotherapeutic agent, a cytokine, angiogenesis inhibitor, a cytotoxic agent, other antibodies, a small molecule drug or an immunomodulatory agent (such as an immunosuppressant) .
  • cytotoxic agent used in the invention refers to a substance that inhibits or prevents the cell function and/or causes cell death or destruction.
  • “Chemotherapeutic agents” include chemical compounds useful in treatment of cancer or immune system disease.
  • drug refers to an organic compound capable of modulating a biological process, in particular altering or preventing a pathological process.
  • prodrug refers to a chemically modified active or inactive compound that, upon administration to a subject, undergoes a physiological effect in vivo (e.g., hydrolysis, anabolism, etc. ) to become the active drug.
  • a physiological effect in vivo e.g., hydrolysis, anabolism, etc.
  • Techniques for making and using prodrugs are well known to those skilled in the art.
  • Angiogenesis inhibitor refers to a compound that blocks or interferes to some extent with vascular development.
  • the angiogenesis inhibitor may be, for example, a small molecule or an antibody that binds to a growth factor or a growth factor receptor involved in promoting angiogenesis.
  • small molecule drugs refers to organic compounds with low molecular weight that can regulate biological processes, especially change or prevent pathological processes.
  • Small molecule is defined as a molecule with molecular weight less than 10kD, generally less than 2kD and preferably less than 1kD, more preferably less than 500D.
  • Small molecules include but are not limited to inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimics and antibody mimics. As a therapeutic agent, small molecules can penetrate cells more easily than large molecules, and are less susceptible to degradation and less prone to trigger immune response.
  • immunomodulators refer to natural or synthetic active agents or drugs that inhibit or regulate (e.g., activate) immune response.
  • the immune response can be humoral or cellular.
  • Immunomodulators include immunosuppressants or immunoagonist.
  • immunemodulators of the invention include immune checkpoint inhibitors or immune checkpoint agonists.
  • Anti-tumor compounds are pharmaceutically active compounds that have an effect on tumors, including but not limited to cytotoxic or chemotherapeutic agents, such as those disclosed in WO 2021/173773, e.g., camptothecins, e.g., exatecan (topoisomerase I inhibitor, Exatecan) , Dxd (a novel topoisomerase I inhibitor, Exatecan derivative) , auristatins, e.g., monomethyl auristatin E (MMAE) , maytansinoids, e.g., small molecule microtubule inhibitor, DM1, taxanes, e.g., paclitaxel or docetaxel, anthracyclines, epothilones, mitomycins, combretastatin, vinca alkaloids, calicheamicins, duocarmycin, Tubulysins, amatoxins, bleomycin, MEK inhibitors, KSP inhibitors, and the like
  • the anti-tumor compounds may be substituted with isotopes including, but not limited to, deuterium, tritium, and the like.
  • isotopes including, but not limited to, deuterium, tritium, and the like.
  • the carbon-deuterium bonds replace carbon-hydrogen bonds, since the former are more stable than the latter, the replacement can directly affect the certain properties of drugs such as absorption, distribution, metabolism, excretion, etc. thereby improving the efficacy, safety, and tolerability of the drugs.
  • the "anti-tumor compound" of the present application may encompass compounds substituted with deuterium.
  • Substituted with deuterium is meant that a hydrogen in the molecule is replaced with deuterium, for example one or more hydrogens, for example 1 to 10 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) hydrogens are replaced with deuterium.
  • an effective amount refers to the amount or dose of the antibody, ADC molecule or composition or combination of the invention, which will produce the expected effect in patients needing such treatment or prevention after being administered to patients in a single or multiple doses.
  • therapeutically effective amount and “preventively effective amount” may be included.
  • “Therapeutically effective amount” refers to an amount that can effectively achieve the desired results at the required dose and for the required period of time.
  • the therapeutically effective amount is also such an amount, where any toxic or harmful effect of the antibody, ADC or composition or combination is less than the therapeutic beneficial effect.
  • “Therapeutically effective amount” preferably inhibits measurable parameters (such as tumor volume) by at least about 20%, more preferably by at least about 30%, or even more preferably by at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%or even 100%compared with untreated objects.
  • Preventively effective amount refers to an amount that can effectively achieve the desired prevention results at the required dose and for the required period of time. Generally, since the preventive dose is used before or at an earlier stage of the disease in the objects, the preventively effective amount will be less than the therapeutically effective amount.
  • host cell refers to the cells in which foreign nucleic acids are introduced, including the descendants of such cells.
  • Host cells include “transformants” and “transformed cells” , which include primary transformed cells and offspring derived from them, regardless of the number of passages.
  • the nucleic acid content of the descendants may not be exactly the same as that of the parent cell, but may contain mutations.
  • the mutant descendants with the same function or biological activity screened or selected from the initially transformed cells are included herein.
  • label refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and facilitates the detection of the conjugated or fused reagent.
  • the label itself can be detectable (for example, radioisotope label or fluorescent label) or can catalyze the chemical changes of detectable substrate compounds or compositions in the case of enzymatic labeling.
  • the term is intended to cover the direct labeling of probes or antibodies by coupling (i.e., physically connecting) detectable substances to probes or antibodies and the indirect labeling of probes or antibodies by reacting with another directly labeled reagent.
  • “Individuals” or “subjects” include mammals. Mammals include, but are not limited to, domestic animals (such as cattle, sheep, cats, dogs and horses) , primates (such as human and non-human primates, such as monkeys) , rabbits, and rodents (such as mice and rats) . In some embodiments, the individuals or subjects are human.
  • Isolated antibodies or other molecules are antibodies or molecules that have been separated from components of their natural environment or the environment in which they are expressed.
  • the antibody is purified to more than 95%or 99%purity, such as as determined by electrophoresis (for example, SDS-PAGE, isoelectric focusing (IEF) , capillary electrophoresis) or chromatography (for example, ion exchange or reverse phase HPLC) .
  • electrophoresis for example, SDS-PAGE, isoelectric focusing (IEF) , capillary electrophoresis
  • chromatography for example, ion exchange or reverse phase HPLC
  • An "isolated" nucleic acid is a nucleic acid molecule that has been separated from components of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.
  • anti-tumor effect refers to a biological effect that can be exhibited by a variety of means, including, but not limited to, for example, a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • the cancer may be in an early, intermediate or advanced stage or metastatic cancer.
  • Cancers suitable for treatment by the molecules of the invention include, but are not limited to, such as lung cancer (including non-small cell lung cancer and small cell lung cancer) , colon cancer, oral squamous cell carcinoma, breast cancer, melanoma, head and neck tumors, prostate cancer, esophageal cancer, cervical cancer, renal cancer, bladder cancer, ovarian cancer or pancreatic cancer, including metastatic forms of these cancers.
  • tumor refers to the growth and proliferation of all neoplastic cells, whether malignant or benign, as well as all pre-cancerous and cancerous cells and tissues. “Tumor” encompasses both solid and hematological tumors and metastatic lesions. The terms “cancer” , “cancerous” and “tumor” are not mutually exclusive when referred to herein.
  • Tumor immune escape refers to the process by which tumors escape immune recognition and clearance. As such, as a therapeutic concept, tumor immunity is “treated” when such escape diminishes, and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage and tumor clearance.
  • pharmaceutically acceptable excipient refers to diluents, adjuvants (e.g., freund's adjuvant (complete and incomplete) ) , vehicles, carriers, stabilizers or the like with which the active substance is administered.
  • composition refers to a composition that is present in a form that allows for the biological activity of the active ingredients contained therein to be effective, and that does not contain additional ingredients that have unacceptable toxicity to the subject to which the composition is administered.
  • pharmaceutical combination or combination product refers to non-fixed combination products or fixed combination products, including but not limited to (pharmaceutical) kits and pharmaceutical compositions.
  • unfixed combination means that the active ingredients (for example, (i) the antibody molecule or ADC of the present invention, and (ii) other therapeutic agents) are administered to patients simultaneously, without specific time limits or at the same or different time intervals, in sequence, in separate entities, where these two or more active agents are administered to provide effective levels of prevention or treatment in patients.
  • the antibody molecule or ADC and other therapeutic agents of the invention used in the pharmaceutical combination are administered at a level not exceeding the level when they are used alone.
  • fixed combination means that two or more active agents are administered simultaneously to patients in the form of a single entity. It is preferred to select the dose and/or time interval of two or more active agents, so that the combined use of each component can produce greater effect than the single use of any one component in the treatment of disease or disorder. Each component can take its own form of formualtion, which can be the same or different.
  • combination therapy refers to the administration of two or more therapeutic agents or therapeutic modes (such as radiotherapy or surgery) to treat the diseases described herein.
  • administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed proportion of active ingredients.
  • administration includes the joint administration of each active ingredient in multiple or separate containers (such as tablets, capsules, powders and liquids) .
  • the powder and/or liquid can be reconstituted or diluted to the required dose before administration.
  • this administration also includes the use of each type of therapeutic agent at approximately the same time or at different times in a sequential manner. In either case, the treatment strategy will provide the beneficial effect of pharmaceutical combination in treating the disease or condition described herein.
  • treatment refers to slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.
  • prevention includes the inhibition of the onset or progression of a disease or disorder or a symptom of a particular disease or disorder.
  • subjects with family history of cancer are candidates for preventive regimens.
  • prevention refers to the administration of a drug prior to the onset of signs or symptoms of a cancer, particularly in subjects at risk of cancer.
  • vector refers to a nucleic acid molecule capable of proliferating another nucleic acid to which it is linked.
  • the term includes vectors that serve as self-replicating nucleic acid structures as well as vectors binding to the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are called "expression vectors" herein.
  • Subject/patient/individual sample refers to a collection of cells or fluids obtained from a patient or subject.
  • the source of the tissue or cell samples can be solid tissues, e.g., from fresh, frozen and/or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood component; body fluids such as cerebrospinal fluids, amniotic fluids, peritoneal fluids (ascites) , or interstitial fluids; cells from a subject at any time during pregnancy or development.
  • Tissue samples may comprise compounds which are naturally not mixed with tissues, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.
  • tumor samples herein include, but are not limited to, tumor biopsies, fine needle aspirates, bronchial lavage, pleural fluid, sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor samples or frozen tumor samples.
  • the present invention provides a multispecific antibody that specifically binds to TROP2 and B7-H3.
  • the multispecific antibodies of the present invention comprise a first binding specificity to TROP2 and a second binding specificity to B7-H3, and optionally other binding specificities.
  • the multispecific antibody is a bispecific antibody.
  • the present invention relates to a bispecific antibody that specifically binds to TROP2 and B7-H3.
  • one aspect of the present invention relates to a multispecific antibody comprising
  • first antigen-binding region specifically binds to TROP2
  • second antigen-binding region specifically binds to B7-H3, and optionally other antigen-binding region (s) .
  • one aspect of the present invention relates to a bispecific antibody comprising
  • first antigen-binding region specifically binds to TROP2 and/or the second antigen-binding region specifically binds to B7-H3.
  • Bispecific antibodies of the invention can be prepared using bispecific antibody formats or techniques known in the art. Specific exemplary bispecific formats that may be used in the context of the present invention are described, for example, in Labrijn, et al., Bispecific antibodies: a mechanical review of the pipeline. Nature Reviews Drug Discovery, 2019, 18 (8) : 1-24.
  • the bispecific antibody format includes an IgG-like antibody (Fan et al (2015) Journal of Hematology &Oncology. 8: 130) .
  • the most common type of IgG-like antibodies comprises two Fab regions and two Fc regions, the heavy and light chains of each Fab may be derived from a separate monoclonal antibody.
  • the bispecific antibody of the invention is an IgG-like bispecific antibody comprising as one antigen-binding region a Fab fragment that specifically binds to TROP2 and as the other antigen-binding region a Fab fragment that specifically binds to B7-H3.
  • the following provides a detailed description of the components of the multispecific antibodies, such as bispecific antibodies, of the present invention. Those skilled in the art will understand that, unless the context clearly indicates otherwise, any combination of any technical features of these components is within the scope of the present invention. Moreover, those skilled in the art will understand that, unless the context clearly indicates otherwise, the antibodies of the present invention (including antibodies in any form) can comprise any such combination.
  • the first antigen-binding region applicable for the anti-TROP2/B7-H3 bispecific antibodies of the present invention may comprise or consist of an anti-TROP2 antibody or antigen binding fragment thereof (e.g. an TROP2 antibody disclosed in US9849176B2 (which is incorporated herein as a whole) , e.g. Sacituzumab hRS7) , as long as it is capable of specifically binding to TROP2, including, but not limited to, e.g. a full length antibody, a half antibody, a Fab, a Fab', a Fab'-SH, a Fv, a single chain antibody (e.g. scFv) , a (Fab') 2, a single domain antibody such as VHH, dAb (domain antibody) , a heavy chain antibody, or a linear antibody that specifically binds to TROP2, and the like.
  • an anti-TROP2 antibody or antigen binding fragment thereof e.g. an TROP2 antibody disclosed in US9849176
  • the antigen-binding region that specifically binds to TROP2 is originated from an antibody that specifically binds to TROP2, e.g., an TROP2 antibody disclosed in US9849176B2, e.g., Sacituzumab hRS7.
  • the antigen-binding region that specifically binds to TROP2 comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds to TROP2, e.g., an TROP2 antibody disclosed in US9849176B2, e.g., Sacituzumab hRS7.
  • the antigen-binding region that specifically binds to TROP2 comprises 1, 2, or 3 heavy chain variable region CDRs, i.e., HCDR1, HCDR2, and HCDR3, of a known antibody that specifically binds to TROP2, e.g., an TROP2 antibody disclosed in US9849176B2, e.g., Sacituzumab hRS7.
  • the antigen-binding region that specifically binds to TROP2 comprises 1, 2, or 3 light chain variable region CDRs, i.e., LCDR1, LCDR2, and LCDR3, of a known antibody that specifically binds to TROP2, e.g., an TROP2 antibody disclosed in US9849176B2, e.g., Sacituzumab hRS7.
  • the antigen-binding region that specifically binds to TROP2 comprises 3 heavy chain variable region CDRs and 3 light chain variable region CDRs of a known antibody that specifically binds to TROP2, e.g., an TROP2 antibody disclosed in US9849176B2, e.g., Sacituzumab hRS7.
  • the antigen-binding region that specifically binds to TROP2 comprises the heavy chain variable region and/or the light chain variable region of a known antibody that specifically binds to TROP2, such as an TROP2 antibody disclosed in US9849176B2, e.g., Sacituzumab hRS7.
  • the antigen-binding region that specifically binds to TROP2 comprises a Fab of a known antibody that specifically binds to TROP2 such as an TROP2 antibody disclosed in US9849176B2, e.g., Sacituzumab hRS7.
  • the antigen-binding region that specifically binds to TROP2 comprises 3 Complementarity Determining Regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the antigen-binding region that specifically binds to TROP2 comprises 3 Complementarity Determining Regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3. In some embodiments, the antigen-binding region that specifically binds to TROP2 comprises 3 Complementarity Determining Regions (HCDRs) from the heavy chain variable region and 3 Complementarity Determining Regions (LCDRs) from the light chain variable region.
  • HCDRs Complementarity Determining Regions
  • LCDRs Complementarity Determining Regions
  • the antigen-binding region that specifically binds to TROP2 comprises a heavy chain variable region (VH) . In some aspects, the antigen-binding region that specifically binds to TROP2 comprises a light chain variable region (VL) . In some aspects, the antigen-binding region that specifically binds to TROP2 comprises a heavy chain variable region (VH) and a light chain variable region (VL) . In some embodiments, the heavy chain variable region comprises 3 Complementarity Determining Regions (CDRs) from a heavy chain variable region, HCDR1, HCDR2 and HCDR3. In some embodiments, the light chain variable region comprises 3 Complementarity Determining Regions (CDRs) from a light chain variable region, LCDR1, LCDR2 and LCDR3.
  • CDRs Complementarity Determining Regions
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, the VH, and/or the VL, comprised by the antigen-binding region that specifically binds to TROP2 of the present invention are respectively defined herein.
  • (i) comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 16, or
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 16;
  • (iii) comprises or consists of an amino acid sequence having one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 16, preferably said amino acid changes do not occur in the CDR regions.
  • (i) comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 22, or
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 22;
  • (iii) comprises or consists of an amino acid sequence having one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 22, preferably said amino acid changes do not occur in the CDR regions.
  • the 3 complementarity determining regions (HCDRs) from the heavy chain variable region of the antigen-binding region that specifically binds to TROP2 described in the present invention, HCDR1, HCDR2 and HCDR3 are selected from
  • HCDRs can be determined according to any scheme for determining CDRs, such as determined respectively by the Kabat, AbM, Chothia, Contact, or IMGT schemes or combination thereof;
  • the HCDR1 is determined by AbM scheme
  • the HCDR2 and HCDR3 are determined by the Kabat scheme respectively.
  • the 3 complementarity determining regions (LCDRs) from the light chain variable region of the antigen-binding region that specifically binds to TROP2 described in the present invention, LCDR1, LCDR2, and LCDR3 are selected from
  • LCDRs can be determined according to any scheme for determining CDRs, such as determined respectively by the Kabat, AbM, Chothia, Contact, or IMGT schemes or combination thereof;
  • the LCDRs1-3 are determined by the Kabat scheme respectively.
  • the antigen-binding region that specifically binds to TROP2 of the present invention comprises 3 complementarity determining regions (HCDRs) comprised by the heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 16 and 3 complementarity determining regions (LCDRs) comprised by the light chain variable region consisting of the amino acid sequence of SEQ ID NO: 22;
  • HCDRs complementarity determining regions
  • LCDRs complementarity determining regions
  • HCDRs and LCDRs can be determined according to any scheme for determining CDRs, such as determined respectively by the Kabat, AbM, Chothia, Contact, or IMGT schemes or combination thereof;
  • the HCDR1 is determined by AbM scheme
  • the HCDR2, HCDR3 and LCDRs1-3 are determined by the Kabat scheme respectively.
  • the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 13 or the HCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 13.
  • the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 14 or the HCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 14.
  • the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 15 or the HCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 15.
  • the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 19 or the LCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 19.
  • the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 20 or the LCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 20.
  • the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 21 or the LCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 21.
  • the antigen-binding region that specifically binds to TROP2 described in the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and/or LCDR3 as described above.
  • the antigen-binding region that specifically binds to TROP2 described in the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as described above.
  • the antigen-binding region that specifically binds to TROP2 described in the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 13; the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 14; the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 15; the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 19; the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 20; and the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 21.
  • the antigen-binding region that specifically binds to TROP2 as described in the present invention comprises HCDR1 set forth in SEQ ID NO: 13, HCDR2 set forth in SEQ ID NO: 14, HCDR3 set forth in SEQ ID NO: 15; LCDR1 set forth in SEQ ID NO: 19, LCDR2 set forth in SEQ ID NO: 20 and LCDR3 set forth in SEQ ID NO: 21.
  • the antigen-binding region that specifically binds to TROP2 as described in the present invention comprises a VH and a VL, wherein
  • the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity thereto
  • the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity thereto.
  • the antigen-binding region that specifically binds to TROP2 as described in the present invention comprises a VH and a VL, wherein the VH and VL comprise or consist of, respectively, the amino acid sequences shown below: SEQ ID NO: 16 and 22, respectively.
  • the antigen-binding region that specifically binds to TROP2 as described in the present invention is an anti-TROP2 Fab.
  • the second antigen-binding region applicable for the anti-TROP2/B7-H3 bispecific antibodies of the present invention may comprise or consist of an anti-B7-H3 antibody or antigen binding fragment thereof, as long as it is capable of specifically binding to B7-H3, including, but not limited to, e.g. a full length antibody, a half antibody, a Fab, a Fab', a Fab'-SH, a Fv, a single chain antibody (e.g. scFv) , a (Fab') 2, a single domain antibody such as VHH, dAb (domain antibody) , a heavy chain antibody, or a linear antibody that specifically binds to B7-H3, and the like.
  • an anti-B7-H3 antibody or antigen binding fragment thereof as long as it is capable of specifically binding to B7-H3, including, but not limited to, e.g. a full length antibody, a half antibody, a Fab, a Fab', a Fab'-SH
  • the antigen-binding region that specifically binds to B7-H3 comprises 3 Complementarity Determining Regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the antigen-binding region that specifically binds to B7-H3 comprises 3 Complementarity Determining Regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3. In some embodiments, the antigen-binding region that specifically binds to B7-H3 comprises 3 Complementarity Determining Regions (HCDRs) from the heavy chain variable region and 3 Complementarity Determining Regions (LCDRs) from the light chain variable region.
  • HCDRs Complementarity Determining Regions
  • LCDRs Complementarity Determining Regions
  • the antigen-binding region that specifically binds to B7-H3 comprises a heavy chain variable region (VH) . In some aspects, the antigen-binding region that specifically binds to B7-H3 comprises a light chain variable region (VL) . In some aspects, the antigen-binding region that specifically binds to B7-H3 comprises a heavy chain variable region (VH) and a light chain variable region (VL) . In some embodiments, the heavy chain variable region comprises 3 Complementarity Determining Regions (CDRs) from a heavy chain variable region, HCDR1, HCDR2 and HCDR3. In some embodiments, the light chain variable region comprises 3 Complementarity Determining Regions (CDRs) from a light chain variable region, LCDR1, LCDR2 and LCDR3.
  • CDRs Complementarity Determining Regions
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, the VH, and/or the VL, comprised by the antigen-binding region that specifically binds to B7-H3 of the present invention are respectively defined herein.
  • (i) comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 4, or
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 4;
  • (iii) comprises or consists of an amino acid sequence having one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 4, preferably said amino acid changes do not occur in the CDR regions.
  • (i) comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 10, or
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 10;
  • (iii) comprises or consists of an amino acid sequence having one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 10, preferably said amino acid changes do not occur in the CDR regions.
  • the 3 complementarity determining regions (HCDRs) from the heavy chain variable region of the antigen-binding region that specifically binds to B7-H3 described in the present invention, HCDR1, HCDR2 and HCDR3 are selected from
  • HCDRs can be determined according to any scheme for determining CDRs, such as determined respectively by the Kabat, AbM, Chothia, Contact, or IMGT schemes or combination thereof;
  • the HCDR1 is determined by AbM scheme
  • the HCDR2 and HCDR3 are determined by the Kabat scheme respectively.
  • the 3 complementarity determining regions (LCDRs) from the light chain variable region of the antigen-binding region that specifically binds to B7-H3 described in the present invention, LCDR1, LCDR2, and LCDR3 are selected from
  • LCDRs can be determined according to any scheme for determining CDRs, such as determined respectively by the Kabat, AbM, Chothia, Contact, or IMGT schemes or combination thereof;
  • the LCDRs1-3 are determined by the Kabat scheme respectively.
  • the antigen-binding region that specifically binds to B7-H3 of the present invention comprises 3 complementarity determining regions (HCDRs) comprised by the heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 4 and 3 complementarity determining regions (LCDRs) comprised by the light chain variable region consisting of the amino acid sequence of SEQ ID NO: 10;
  • HCDRs complementarity determining regions
  • LCDRs complementarity determining regions
  • HCDRs and LCDRs can be determined according to any scheme for determining CDRs, such as determined respectively by the Kabat, AbM, Chothia, Contact, or IMGT schemes or combination thereof;
  • the HCDR1 is determined by AbM scheme
  • the HCDR2, HCDR3 and LCDRs1-3 are determined by the Kabat scheme respectively.
  • the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1 or the HCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 1.
  • the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2 or the HCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 2.
  • the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3 or the HCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 3.
  • the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 7 or the LCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 7.
  • the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 8 or the LCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 8.
  • the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 9 or the LCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 9.
  • the antigen-binding region that specifically binds to B7-H3 described in the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and/or LCDR3 as described above.
  • the antigen-binding region that specifically binds to B7-H3 described in the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1; the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2; the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3; the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 7; the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 8; and the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 9.
  • the antigen-binding region that specifically binds to B7-H3 as described in the present invention comprises HCDR1 set forth in SEQ ID NO: 1, HCDR2 set forth in SEQ ID NO: 2, HCDR3 set forth in SEQ ID NO: 3; LCDR1 set forth in SEQ ID NO: 7, LCDR2 set forth in SEQ ID NO: 8 and LCDR3 set forth in SEQ ID NO: 9.
  • the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 4, and the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 10.
  • the antigen-binding region that specifically binds to B7-H3 as described in the present invention comprises a VH and a VL, wherein the VH and VL comprise or consist of, respectively, the amino acid sequences shown below: SEQ ID NO: 4 and 10, respectively.
  • the antigen-binding region that specifically binds to B7-H3 as described in the present invention is an anti-B7-H3 Fab.
  • the first antigen-binding region and/or the second antigen-binding region of the invention is a Fab fragment.
  • a Fab fragment suitable for use as an antigen-binding region of the multispecific antibody such as bispecific antibody as described herein consists of two polypeptide chains comprising the antibody VH, CH1, VL and CL domains, wherein the VH is paired with VL and the CH1 is paired with CL to form the antigen-binding region.
  • one chain comprises or consists of VH and CH1 (i.e., VH-CH1) from N-terminus to C-terminus
  • the other chain comprises or consists of VL and CL (i.e., VL-CL) from N-terminus to C-terminus.
  • the Fab may be linked to the N-terminus of the Fc domain of the antibody via the C-terminus of the chain comprising the VH.
  • the Fab comprises a VH-CH1 chain and a VL-CL chain and may be linked to an antibody Fc domain via the C-terminus of CH1 of the VH-CH1 chain.
  • the linkage is a direct linkage, or through a connector.
  • Fab chain comprising VH-CH1 is also referred to as a Fab heavy chain
  • Fab chain comprising VL-CL is also referred to as a Fab light chain.
  • the CH1 is a CH1 from IgG1, IgG2, IgG3, or IgG4, preferably CH1 from IgG1. In some embodiments, the CH1
  • (i) comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 32, or
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 32;
  • (iii) comprises or consists of an amino acid sequence having one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 32.
  • the CL is a Kappa light chain constant region or a Lambda light chain constant region. In some embodiments, CL is a Kappa light chain constant region. In some embodiments, the CL
  • (i) comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 11;
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 11;
  • (iii) comprises or consists of an amino acid sequence having one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 11.
  • the first antigen-binding region is a Fab that specifically binds to TROP2, wherein the Fab fragment is from an anti-TROP2 antibody, and comprises the heavy chain variable region VH and the light chain variable region VL of the anti-TROP2 antibody.
  • the Fab that specifically binds to TROP2 as the first antigen-binding region comprises the 6 CDRs of the antigen-binding regions that specifically bind to TROP2 described herein.
  • the Fab that specifically binds to TROP2 as the first antigen-binding region comprises a VH or a VL of the antigen-binding region that specifically binds to TROP2 as described herein, or comprises a VH and a VL of the antigen-binding region that specifically binds to TROP2 as described herein.
  • the second antigen-binding region is a Fab that specifically binds to B7-H3, wherein the Fab fragment is from an anti-B7-H3 antibody, and comprises the heavy chain variable region VH and the light chain variable region VL of the anti-B7-H3 antibody.
  • the Fab that specifically binds to B7-H3 as the second antigen-binding region comprises the 6 CDRs of the antigen-binding regions that specifically bind B7-H3 as described herein.
  • the Fab that specifically binds B7-H3 as the second antigen-binding region comprises a VH or a VL of the antigen-binding region that specifically binds to B7-H3 as described herein, or comprises a VH and a VL of the antigen-binding region that specifically binds to B7-H3 as described herein.
  • the bispecific antibody of the present invention further comprises an Fc region, wherein the Fc regions comprised may be the same or different.
  • the antibody molecular of the present invention comprises the first Fc region and the second Fc region, wherein the first Fc region and the second Fc region are the same or different.
  • the first Fc region and the second Fc region are different and are capable of dimerizing to form a heterodimeric Fc scaffold.
  • the Fc region refers to the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region, and may include native sequence Fc regions and variant Fc regions.
  • the native sequence Fc region encompasses the naturally occurring Fc sequences of various immunoglobulins, such as the Fc regions of various Ig subclass and allotypes thereof (Gestur Vidarsson et al, IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi: 10.3389/fimmu. 2014.00520) .
  • the Fc region of the present invention comprises the antibody CH2 and CH3.
  • the antibody Fc region may also have an IgG hinge region or a partial IgG hinge region at the N-terminus, e.g., an IgG1 hinge region or a partial IgG1 hinge region, e.g., according to EU numbering, the sequence of D221 to P230.
  • a mutation may be comprised in the hinge region.
  • the numbering of amino acid residues in the Fc region is according to the EU numbering system, also known as the EU index, as described in Kabat, E. A. et al, Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991) , NIH Publication 91-3242.
  • the Fc region is a human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, human IgG3 Fc or human IgG4 Fc.
  • the Fc region comprises or consists of the amino acid sequence SEQ ID NO: 30 or SEQ ID NO: 31, or an amino acid sequence having at least 90%identity, e.g., 95%, 96%, 97%, 98%, 99%or more identity to said amino acid sequence.
  • the Fc region comprised by the multispecific antibodies of the present invention may comprise mutations that facilitate heterodimerization of a first Fc region with a second Fc region.
  • mutations are introduced in the CH3 regions of both Fc regions.
  • the CH3 region of the first Fc region and the CH3 region of the second Fc region are engineered in a complementary manner such that each CH3 region (or the heavy chain comprising it) can no longer homodimerize with itself but is forced to heterodimerize with other CH3 regions that are complementarily engineered (such that the CH3 regions of the first and the second Fc regions heterodimerize and no homodimers are formed between the two first CH3 regions or the two second CH3 regions) .
  • the respective Knob mutations and Hole mutations are introduced in the first Fc region and the second Fc region. See, e.g., US 5, 731, 168; US 7, 695, 936; Ridgway et al, Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001) for the technique.
  • the Knob mutations and Hole mutations may further comprise mutations to cysteine residue and thus to introduce disulfide bond. Disulfide bonds can be used to stabilize the final antibody product or the ADC of the antibody.
  • the threonine residue at position 366 is replaced with a tryptophan residue (T366W) (knob mutation) ; in the CH3 region of the other Fc region, the tyrosine residue at position 407 was replaced with a valine residue (Y407V) (hole mutation) , optionally the threonine residue at position 366 was replaced with a serine residue (T366S) and the tyrosine residue at position 407 was replaced with a valine residue (Y407V) (numbering according to the EU index) .
  • T366W tryptophan residue
  • Y407V valine residue
  • T366S serine residue
  • tyrosine residue at position 407 was replaced with a valine residue (Y407V) (numbering according to the EU index) .
  • the Fc regions may further comprise cysteine residue substitution, so that to obtain non-natural disulfide bond linkage.
  • the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (in particular, the serine residue at position 354 is replaced with a cysteine residue)
  • the tyrosine residue at position 349 is replaced with a cysteine residue (Y349C) (numbering according to the EU index) .
  • the threonine residue at position 366 is replaced with a tryptophan residue (T366W) and the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (in particular, the serine residue at position 354 is replaced with a cysteine residue) ;
  • the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (hole mutation)
  • the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the EU index)
  • optionally the tyrosine residue at position 349 is replaced with a cysteine residue (Y349
  • one Fc region comprises the amino acid substitutions T366W and the other Fc region comprises the amino acid substitutions T366S, L368A and Y407V (numbering according to the EU index) .
  • one Fc region comprises the amino acid substitutions S354C and T366W and the other Fc region comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to the EU index) .
  • Mutations may also be introduced in the first Fc region and the second Fc region based on the Innobody technique. See, e.g., PCT/CN2021/143141 for this technique, which is incorporated herein as its entirty.
  • the first CH3 region comprises the S364R/K mutation (preferably S364R) , and optionally one or more additional mutations.
  • the second CH3 region comprises a K370S/T/A/V mutation (preferably K370S) , and optionally one or more additional mutations.
  • the first CH3 region includes the S364R/K mutation, and the second CH3 region includes the K370S/T/A/V mutation.
  • the first CH3 region comprises the S364R mutation, and the second CH3 region comprises the K370S mutation.
  • the first CH3 region comprises S364R/K (preferably S364R) and D399K/R (preferably D399K) mutations.
  • the second CH3 region comprises a K370S/T/A/V (preferably K370S) mutation and a K409D/E (preferably K409D) mutation.
  • the first CH3 region comprises S364R/K + D399K/R and the second CH3 region comprises K370S/T/A/V + Y349T/S/A/V.
  • the first CH3 region comprises S364R + D399K and the second CH3 region comprises K370S + Y349T.
  • the first CH3 region further comprises E375N/Q (preferably E375N) and/or T350V/A (preferably T350V) .
  • the second CH3 region further comprises K409D/E (preferably K409D) , Q347D/E (preferably Q347D) and/or T350V/A (preferably T350V) .
  • the first CH3 region comprises S364R + D399K and the second CH3 region comprises K370S + Y349T + K409D.
  • the first CH3 region further comprises E357N.
  • the second CH3 region further comprises Q347D.
  • the first CH3 region also includes E357N, and the second CH3 region also includes Q347D.
  • the first CH3 region and the second CH3 region further comprise T350V, respectively, or both comprises T350V.
  • the first CH3 region comprises S364R + D399K and the second CH3 region comprises K370S + Y349T + K409D + Q347D.
  • the first CH3 region comprises S364R + D399K + E357N and the second CH3 region comprises K370S + Y349T + K409D + Q347D.
  • the first CH3 region comprises S364R + D399K +E357N + T350V
  • the second CH3 region comprises K370S + Y349T + K409D + Q347D +T350V.
  • the first CH3 region comprises K409E/D (preferably K409E) .
  • the second CH3 region comprises D399K/R (preferably D399K) or K370T/S/A/V (preferably K370T) .
  • the first CH3 region comprises K409E/D (preferably K409E) and the second CH3 region comprises D399K/R (preferably D399K) .
  • the first CH3 region further comprises T411R/K (preferably T411R) .
  • the second CH3 region further comprises K370T/S/A/V (preferably K370T) .
  • the CH3 region comprises K409E/D + T411R/K and the second CH3 region comprises D399K/R + K370T/S/A/V. In some embodiments, the CH3 region comprises K409E +T411R and the second CH3 region comprises D399K + K370T.
  • the first and the second CH3 regions have the following combination of mutations:
  • the Fc region is modified in characteristics of an effector function of the Fc region (e.g., complement activation function of the Fc region) .
  • the effector function has been reduced or eliminated relative to a wild-type isotype Fc region.
  • effector function is reduced or eliminated by a method selected from the group consisting of: use of a Fc isoform which naturally has reduced or eliminated effector function or Fc region modification.
  • the Fc region has reduced effector function mediated by the Fc region, such as reduced or eliminated ADCC or ADCP or CDC effector function, e.g. comprising mutations to achieve the above.
  • the antibody molecules of the invention may also comprise modifications in the Fc domain that alter binding affinity to one or more Fc receptors, depending on the intended use of the the antibody molecules of the invention.
  • the Fc receptor is an Fc gamma receptor, particularly a human Fc gamma receptor.
  • the Fc region comprises a mutation that reduces binding to an Fc gamma receptor.
  • the Fc region used in the invention has mutations that reduce binding to Fc gamma receptors, such as L234A/L235A.
  • the Fc fragment may have a mutation that results in increased serum half-life, such as a mutation that improves binding of the Fc fragment to FcRn.
  • the Fc fragmetns comprises an amino acid sequence as shown in SEQ ID NO: 29, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to the amino acid sequence set forth in SEQ ID NO: 29 and comprising L234A/L235A mutations.
  • the multipecific antibodies such as bispecific antibodies of the present invention comprise two Fc regions that heterodimerize, wherein one Fc-region polypeptide comprises the mutations S354C and T366W, while the other Fc-region polypeptide comprises the mutations Y349C, T366S, L368A and Y407V.
  • the multipecific antibodies such as bispecific antibodies of the present invention comprise two Fc regions that heterodimerize, wherein one Fc region polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 27, while the other Fc region polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 28.
  • the multipecific antibodies such as bispecific antibodies of the present invention comprise two Fc regions that heterodimerize, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to the amino acid sequence set forth in SEQ ID NO: 27, while the other Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to the amino acid sequence set forth in SEQ ID NO: 28.
  • the multipecific antibodies such as bispecific antibodies of the present invention comprise two Fc regions that heterodimerize, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to the amino acid sequence set forth in SEQ ID NO: 27 and comprising the mutations S354C and T366W and optionally L234A/L235A mutations, while the other Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to the amino acid sequence set forth in SEQ ID NO: 28 and comprising the mutations Y349C, T366S, L368A and Y407V and optionally L234A/L235A mutations.
  • the Fc region further comprises additional mutations that facilitate purification of the heterodimer.
  • the anti-B7-H3/TROP2 bispecific antibody of the invention has one or more of the following properties:
  • the bispecific antibodies of the invention can specifically bind to cells expressing TROP2 and/or B7-H3, such as tumor cells;
  • the bispecific antibody of the invention can be endocytosed by cells such as tumor cells, for example, the endocytotic activity is stronger than that of the parent monoclonal antibody.
  • the heavy chain variable region of the antigen binding region that specifically binds TROP2 as described herein is linked to heavy chain constant region CH, e.g., wherein the C-terminus of the heavy chain variable region is linked to the N-terminus of the heavy chain constant region CH.
  • the light chain variable region of the antigen binding region that specifically binds TROP2 as described herein is linked to a light chain constant region CL, e.g., wherein the C-terminus of the light chain variable region is linked to the N-terminus of the light chain constant region CL.
  • the heavy chain variable region is linked to the heavy chain constant region CH and the light chain variable region is linked to the light chain constant region CL in the antigen binding region that specifically binds TROP2 as described herein.
  • the heavy chain variable region of the antigen binding region that specifically binds B7-H3 as described herein is linked to heavy chain constant region CH, e.g., wherein the C-terminus of the heavy chain variable region is linked to the N-terminus of the heavy chain constant region CH.
  • the light chain variable region of the antigen binding region that specifically binds B7-H3 as described herein is linked to a light chain constant region CL, e.g., wherein the C-terminus of the light chain variable region is linked to the N-terminus of the light chain constant region CL.
  • the heavy chain variable region is linked to the heavy chain constant region CH and the light chain variable region is linked to the light chain constant region CL in the antigen binding region that specifically binds B7-H3 as described herein.
  • the heavy chain constant region comprises the CH1 and the Fc region as described herein, connected via or not via a hinge region. In some embodiments, the heavy chain constant region consists of the CH1 and the Fc region.
  • the present invention provides a bispecific antibody molecule comprising one Fab fragment that specifically binds to TROP2 as described herein, one Fab fragment that specifically binds to B7-H3 as described herein, and an Fc dimer as described herein, wherein the Fab fragment that specifically binds to TROP2 and one Fc as described herein forms a half-antibody that specifically binds to TROP2 and the Fab fragment that specifically binds to B7-H3 and one Fc as described herein forms a half-antibody that specifically binds to B7-H3.
  • the bispecific antibody is an IgG-like antibody having the configuration shown in Figure 1.
  • the first and second Fc regions comprise a Knob-into-Hole mutation, and optionally a mutation that reduces affinity for the Fc ⁇ receptor, e.g., L234A/L235A mutation.
  • the bispecific antibody comprises or consists of a heavy chain 1 and a light chain 1, and a heavy chain 2 and a light chain 2, wherein heavy chain 1 and light chain 1 constitutes the first half-antibody, and heavy chain 2 and light chain 2 constitutes the second half-antibody; wherein heavy chain 1 comprises a heavy chain variable region of the first antigen binding region and a first heavy chain constant region; light chain 1 comprises a light chain variable region of the first antigen binding region and a first light chain constant region; and heavy chain 2 comprises a heavy chain variable region of the second antigen-binding region and a second heavy chain constant region; light chain 2 comprises a light chain variable region of the second antigen-binding region and a second light chain constant region.
  • the bispecific antibody of the invention specifically binds TROP2 and B7-H3 and comprises or consists of:
  • Heavy chain 2 comprising or consisting of: from N-terminus to C-terminus, heavy chain variable region of the Fab that specifically binds B7-H3 -heavy chain constant region CH1-second Fc region, wherein heavy chain constant region CH1 is linked at its C-terminus to the N-terminus of the second Fc region with or without a connector (e.g., hinge region) ;
  • Light chain 2 comprising or consisting of: from N-terminus to C-terminus, a light chain variable region of the Fab that specifically binds B7-H3 -light chain constant region,
  • said first Fc region comprises S354C and T366W
  • said second Fc region comprises mutations Y349C, T366S, L368A and Y407V, and vice versa; optionally, the first and second Fc regions further comprise mutations to reduce affinity for the Fc ⁇ receptor, e.g., L234A/L235A mutations, respectively.
  • the domains of the bispecific antibody of the present invention are described herein, e.g., the Fab, the Fc, the VH and VL as described herein.
  • the bispecific antibody of the invention that specifically bind to TROP2 and B7-H3 comprises or consists of:
  • Heavy chain 1 comprising or consisting of the amino acid sequence shown as SEQ ID NO: 18, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity thereto;
  • Light chain 1 comprising or consisting of the amino acid sequence shown as SEQ ID NO: 23, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity thereto;
  • Heavy chain 2 comprising or consisting of the amino acid sequence shown as SEQ ID NO: 6, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity thereto;
  • Light chain 2 comprising or consisting of the amino acid sequence shown as SEQ ID NO: 12, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity thereto.
  • the present invention provides a method for preparing the antibody molecules of the present invention, which includes culturing the host cells under conditions suitable for expressing nucleic acids encoding the antibody molecules of the present invention, and optionally isolating the antibodies. In a certain embodiment, the method also includes recovering the antibody molecules of the present invention from the host cells.
  • the method for preparing the antibody comprises culturing a host cell comprising a nucleic acid encoding the antibodies (e.g., any one and/or more polypeptide chains) or an expression vector comprising the nucleic acid, under conditions suitable for expression of the antibody or its chain, and optionally recovering the antibodies from the host cell (or host cell culture medium) .
  • a host cell comprising a nucleic acid encoding the antibodies (e.g., any one and/or more polypeptide chains) or an expression vector comprising the nucleic acid, under conditions suitable for expression of the antibody or its chain, and optionally recovering the antibodies from the host cell (or host cell culture medium) .
  • a nucleic acid of the invention encodes any one chain of an antibody molecule of the invention.
  • the present invention relates to nucleic acids encoding any of the above bispecific antibodies.
  • the polypeptide encoded by the nucleic acid is capable of exhibiting human TROP and/or B7-H3 antigen binding capacity when expressed from a suitable expression vector.
  • the present invention relates to a vector comprising the nucleic acid of the present invention.
  • the vector is an expression vector, such as a eukaryotic expression vector.
  • Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or Yeast Artificial Chromosomes (YACs) .
  • the vector is, for example, a pcDNA vector, such as pcDNA3.1.
  • the expression vector may be transfected or introduced into a suitable host cell.
  • a variety of techniques may be used to achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection or other conventional techniques.
  • protoplast fusion cells are cultured in a medium and screened for suitable activity.
  • the methods and conditions for culturing the resulting transfected cells and for recovering the produced antibody molecules are known to those skilled in the art and can be varied or optimized based on the specific expression vector and mammalian host cell used, in accordance with this disclosure and known methods in the existing technology.
  • cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow for the selection of transfected host cells.
  • the host cell comprises a nucleic acid of the invention or comprises an expression vector of the invention.
  • the host cell is eukaryotic.
  • the host cell is selected from a yeast cell, a mammalian cell such as a CHO cell (e.g., CHO-S, such as ExpiCHO-S) or 293 cell (e.g., 293F or HEK293 cell or Expi293 cell) ) or other cell suitable for the production of an antibody or fragment thereof.
  • the host cell is prokaryotic, e.g., a bacterium, such as E. coli.
  • the antibody molecules prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc.
  • the actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, and these will be apparent to those skilled in the art.
  • the purity of the antibody molecules of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.
  • the present invention provides an antibody-drug conjugate of formula (I) :
  • Ab is an antibody or fragment thereof (e.g., antigen-binding fragment) that specifically binds TROP2 and B7-H3 (e.g., human TROP2 and human B7-H3) ;
  • L is a linker
  • D is a drug, preferably an anti-tumor compound
  • p is an integer selected from 1 to 16, such as an integer selected from 1-10, 1-9, 2-8, 4-10, 6-8,3-7, 4-6, 2-6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12.
  • p refers to the number of -L-P attached to the Ab in theantibody-drug conjugate molecule of formula (I).
  • the Ab in formula (I) of the present invention is an antibody of thepresent invention, e.g., a bispecific antibody of the present invention.
  • D in formula (I) of the present invention may be any anti-tumorcompound, and is not particularly limited, as long as it has an anti-tumor effect and has a substituent,or a structural moiety, which can be linked to a linker structure.
  • the anti-tumorcompound may be a pharmaceutically active compound that acts on a tumor.
  • it is preferable that a part or whole of the linker may be cleaved in a tumorcell to release the antitumor compound, thereby exhibiting an antitumor effect.
  • the linker iscleaved at the linker to the drug, the antitumor compound is released in an unmodified or modifiedstructure and exhibits its antitumor effect.
  • the anti-tumor compound can be, for example, a cytotoxic orchemotherapeutic agent, e.g., camptothecins, e.g., exatecan (topoisomerase I inhibitor, Exatecan),Dxd (a novel topoisomerase I inhibitor, Exatecan derivative), auristatins, e.g., monomethylauristatin E (MMAE), maytansinoids, e.g., small molecule microtubule inhibitor, DM1, taxanes,e.g., paclitaxel or docetaxel, anthracyclines, epothilones, mitomycins, combretastatin, vincaalkaloids, calicheamicins, duocarmycin, Tubulysins, amatoxins, bleomycin, MEK inhibitors, KSPinhibitors, and the like.
  • a cytotoxic orchemotherapeutic agent e.g., camptothecins, e.g
  • D is represented by formula (D-1):
  • R 1 is selected from H, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 1 -C 6 haloalkyl,C 2 -C 6 haloalkenyl and C 2 -C 6 haloalkynyl;
  • R 2 is selected from H, halo, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, -OR 4 ⁇ -SR 4 ;
  • R 3 is selected from H,halo, CN, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl and -OR 4 ; or R 2 and R 3 together form -O (CH 2 ) n O- or-O (CF 2 ) n O-, wherein n is 1 or 2;
  • R 4 is selected from H or C 1 -C 4 alkyl.
  • R 1 is H
  • R 2 is C 1 -C 6 alkyl
  • R 3 is -F.
  • D is represented by formula (D-2):
  • R 1 , R 2 and R 3 are as defined above.
  • D is represented by formula (D-3) :
  • D is represented by formula (D-4) :
  • -L- is represented by the following structure: -Z-E-NH-CH 2 -Q-L 2 -L 1 -wherein Z is linked to Ab, L 1 is linked to D;
  • Z is selected from wherein m is an integer selected from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7 or 8, preferably is 5; the carbonyl at the right end of Z is covalently linked to E;
  • E is a peptide residue comprising 2-10 amino acids, wherein the peptide residue is optionally substituted with one or more polyol groups, wherein the N-terminus of the peptide is covalently linked to Z;
  • Q is -O-or -S-;
  • L 1 is absent or - (C 1 -C 10 alkylene) -;
  • L 2 is absent, -N (R 5 ) C (O) - (C 1 -C 10 alkylene) -*or -C (O) N (R 5 ) - (C 1 -C 10 alkylene) -*; wherein *indicates that said terminus is covalently linked to Q; and
  • R 5 is H or C 1 -C 6 alkyl.
  • E is a peptide residue consisting of 2, 3, or 4 amino acids.
  • the amino acids are selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine and leucine, and wherein the glutamine or glutamic acid is optionally substituted with one polyol group.
  • E is -Gln-Val-Ala-, -Gly-Val-Ala-, -Gln-Phe-Ala-, -Gly-Phe-Ala-or wherein R 6 is H or C 1 -C 6 alkyl, wherein these E groups are covalently linked to Z through the left N-terminus.
  • -L 2 -L 1 - is - (C 1 -C 6 alkylene) -, - (C 1 -C 6 alkylene) -N (R 5 ) C (O) - (C 1 -C 6 alkylene) -*or - (C 1 -C 6 alkylene) -C (O) N (R 5 ) - (C 1 -C 10 alkylene) -*, wherein *indicates that the terminus is covalently linked to Q;
  • R 5 is H or C 1 -C 6 alkyl.
  • -L 2 -L 1 - is - (C 1 -C 6 alkylene) -.
  • -Z-E-NH-CH 2 -Q-L 2 -L 1 - is represented by the following structure
  • the payload (e.g., drug) of the immunoconjugate of the invention is attached to the modified antibody or antibody fragment through the thiol group of the free cysteine of the antibody or antigen binding fragment thereof (optionally via a linker) .
  • the payload (e.g., drug) of the immunoconjugate of the invention is linked to the sulfhydryl group of the cysteine through a cleavable or non-cleavable linker.
  • Ab is linked to L through a sulfur atom on its sulfhydryl group
  • the antibody-drug conjugate of formula (I) may be represented by the following formula:
  • the antibody-drug conjugate has an average DAR of from 5 to 11 or from 7.5 to 8.5.
  • the antibody-drug conjugate is selected from
  • Ab is the bispecific antibody of the present invention, preferably HZ5C2.9/hRS7.13; q is as defined for p in formula (I) above,
  • the antibody-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof has an average DAR of, for example, from 5 to 11, from 6 to 10, from 7 to 9 or from 7.5 to 8.5.
  • the S atom connected to Ab in the above ADC comes from the antibody Ab.
  • Ab opens the disulfide bond under the action of a reducing agent such as TCEP to generate a thiol (-SH) , which is then connected to the maleimide moiety of the linker.
  • the invention provides a method of using the antibodies of the present invention to prepare ADCs.
  • the "ADC" in the present invention is defined as an antibody coupled to an active substance (D) having biological and/or pharmaceutical activity via a linker (L) .
  • the method comprises coupling an antibody (Ab) of the present invention to one or more active substances D via one or more linkers (L) as defined herein.
  • the method comprises preparing an Ab for an ADC comprising culturing a host cell comprising a nucleic acid encoding the Ab (e.g., any one and/or more polypeptide chains) or an expression vector comprising the nucleic acid, under conditions suitable for expression of the Ab or its chain, and optionally recovering the Ab from the host cell (or host cell culture medium) .
  • a host cell comprising a nucleic acid encoding the Ab (e.g., any one and/or more polypeptide chains) or an expression vector comprising the nucleic acid
  • the method comprises the steps of:
  • step (b) adding a linker-payload to the reaction solution of step (a) for coupling to obtain a crude product
  • linker-payload is reacted with the Ab to provide the-L-D moiety in the compound of formula I, and when -L-D is clearly defined, the structure of the linker-payload can be determined or selected.
  • the buffer solution of step a) is a PBS buffer, preferably at a pH of 5.0-9.0, e.g. 6.0-8.0.
  • the reducing agent of step a) is TCEP.
  • the linker-payload has the structure: Z’ -E-NH-CH 2 -Q-L 2 -L 1 -D, wherein E, Q, L 2 , L 1 , D are as defined above, Z'is and m is as defined above.
  • the steps are performed under the specific reaction conditions disclosed in the examples.
  • the ADC may be prepared according to the schematic shown in Fig. 8.
  • the resulting ADC is also contemplated by the present invention.
  • the present invention provides a composition comprising any of the bispecific antibody molecules and/or the ADC molecules described herein, or a pharmaceutically acceptable salt thereof, preferably the composition is a pharmaceutical composition or pharmaceutical formulation.
  • the composition further comprises a pharmaceutically acceptable excipient.
  • a composition e.g., a pharmaceutical composition, comprises a combination of a bispecific antibody molecules and/or an ADC molecule of the invention, and one or more other therapeutic agents.
  • the present invention also includes compositions (including pharmaceutical compositions) comprising a salt of a bispecific antibody molecules or fragment thereof of the present invention.
  • the present invention also includes compositions (including pharmaceutical compositions) comprising the ADC molecules of the invention, or pharmaceutically acceptable salts thereof.
  • These compositions may also contain suitable pharmaceutically acceptable excipients such as pharmaceutically acceptable carriers, pharmaceutically acceptable vehicles, including buffers, as are known in the art.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion medium, isotonic agents and absorption delaying agents, and the like that are physiologically compatible.
  • compositions of the present invention may be in a variety of forms.
  • forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions) , powders or suspensions, liposomal formulations, and suppositories.
  • liquid solutions e.g., injectable and infusible solutions
  • powders or suspensions e.g., powders or suspensions
  • liposomal formulations e.g., liposomal formulations
  • suppositories e.g., suppositories.
  • the preferred form depends on the intended mode of administration and therapeutic use.
  • Medicaments comprising the bispecific antibody molecules or ADCs described herein may be prepared by mixing the bispecific antibody molecules or ADC molecules of the present invention having the desired purity with one or more optional pharmaceutically acceptable excipients, preferably in the form of lyophilized formulations or aqueous solutions.
  • compositions or formulations of the present invention may also contain more than one active ingredient as required for the particular indication being treated, preferably those active ingredients having complementary activities that do not adversely affect each other.
  • active ingredients may be desirable to further provide other therapeutic agents, including chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immune modulators (e.g., immune checkpoint inhibitors or agonists) , and the like.
  • the active ingredients are suitably present in combination in an amount effective for the intended use.
  • sustained release formulations can be prepared.
  • Appropriate examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody or ADC molecule, which matrices are in the form of shaped articles, e.g., films, or microcapsules.
  • the invention further provides a pharmaceutical combination or a pharmaceutical combination product, which comprises the bispecific antibody molecules and/or ADC of the present invention, and one or more other therapeutic agents (such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immune modulators (e.g., immune checkpoint inhibitors or agonists) etc. ) .
  • therapeutic agents such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immune modulators (e.g., immune checkpoint inhibitors or agonists) etc.
  • Another object of the present invention is to provide a kit comprising the pharmaceutical combination of the invention, preferably in the form of drug dose unit. Therefore, the dose unit can be provided according to the regimen or interval of the administration.
  • the kit of the present invention comprises:
  • a first container comprising a pharmaceutical composition containing the bispecific antibody molecules or ADC molecules of the present invention
  • a second container comprising a pharmaceutical composition containing other therapeutic agent (s) .
  • the present inventin provides a method of preventing or treating a disease in a subject, comprising administering to the subject an effective amount of the bispecific antibody molecule, the ADC molecule, pharmaceutical composition, pharmaceutical combination or kit of the present invention.
  • the disease is B7-H3 and/or TROP2 related disease. In some embodiments, the disease is related to aberrant expression or aberrant activity of B7-H3 and/or TROP2.
  • the disease is a tumor, e.g., cancer.
  • the tumor e.g., cancer
  • the tumor is B7-H3 positive, e.g., it comprises tumor cells that express B7-H3.
  • the patient's tumor comprises tumor cells that express B7-H3.
  • the patient's tumor cells express B7-H3, e.g., moderately express B7-H3, preferably highly express B7-H3.
  • the tumor (e.g., cancer) patient has B7-H3 in the tumor tissue (e.g., at an elevated level, e.g., at a nucleic acid or protein level or activity) , e.g., as compared with the B7-H3 level in the same tissue of a healthy subject or in a healthy tissue adjacent to the tumor tissue of the patient.
  • the patient has B7-H3 in the tumor cells (e.g., at an elevated level, e.g., a nucleic acid or protein level or activity) , e.g., as compared with the B7-H3 level in the same cells of a healthy subject or in healthy cells adjacent to the patient's tumor cells.
  • the tumor e.g., cancer
  • the tumor is TROP2 positive, e.g., it comprises tumor cells that express TROP2.
  • the patient's tumor comprises tumor cells that express TROP2.
  • the patient's tumor cells express TROP2, e.g., moderately express TROP2, preferably highly express TROP2.
  • the tumor (e.g., cancer) patient has TROP2 in the tumor tissue (e.g., at an elevated level, e.g., at a nucleic acid or protein level or activity) , e.g., as compared with the TROP2 level in the same tissue of a healthy subject or in a healthy tissue adjacent to the tumor tissue of the patient.
  • the patient has TROP2 in the tumor cells (e.g., at an elevated level, e.g., a nucleic acid or protein level or activity) , e.g., as compared with the TROP2 level in the same cells of a healthy subject or in healthy cells adjacent to the patient's tumor cells.
  • an elevated level e.g., a nucleic acid or protein level or activity
  • the tumor e.g., cancer
  • the tumor is B7-H3 positive and TROP2 positive, e.g., it comprises tumor cells that express B7-H3 and TROP2.
  • the patient's tumor comprises tumor cells that express B7-H3 and TROP2.
  • the patient's tumor cells express B7-H3 and TROP2, e.g., moderately or highly express B7-H3 and TROP2.
  • the tumor (e.g., cancer) patient has B7-H3 and TROP2 in the tumor tissue (e.g., at an elevated level, e.g., at a nucleic acid or protein level or activity) , e.g., as compared with the B7-H3 and TROP2 level in the same tissue of a healthy subject or in a healthy tissue adjacent to the tumor tissue of the patient.
  • B7-H3 and TROP2 in the tumor tissue e.g., at an elevated level, e.g., at a nucleic acid or protein level or activity
  • the patient has B7-H3 and TROP2 in the tumor cells (e.g., at an elevated level, e.g., a nucleic acid or protein level or activity) , e.g., as compared with the B7-H3 and TROP2 level in the same cells of a healthy subject or in healthy cells adjacent to the patient's tumor cell ⁇ s.
  • an elevated level e.g., a nucleic acid or protein level or activity
  • the tumors such as cancers
  • the tumors include solid tumors and hematological tumors as well as metastatic lesions.
  • examples of solid tumors include malignant tumors.
  • the cancer may be in an early, intermediate or advanced stage or metastatic cancer.
  • the ADC molecules of the present invenntion are capable of killing tumor cells, and/or inhibiting tumor cell proliferation
  • the tumor cell are tumor cells expressing B7-H3 and/or TROP2, e.g., cells of the following cancers: lung cancer including non-small cell lung cancer and small cell lung cancer, colon cancer, oral squamous cell carcinoma, breast cancer, melanoma, head and neck tumors, prostate cancer, esophageal cancer, cervical cancer, renal cancer, bladder cancer, ovarian cancer or pancreatic cancer.
  • the tumor is tumor immune escape.
  • the tumor is a cancer, such as lung cancer including non-small cell lung cancer and small cell lung cancer, colon cancer, oral squamous cell carcinoma, breast cancer, melanoma, head and neck tumors, prostate cancer, esophageal cancer, cervical cancer, renal cancer, bladder cancer, ovarian cancer or pancreatic cancer.
  • lung cancer including non-small cell lung cancer and small cell lung cancer, colon cancer, oral squamous cell carcinoma, breast cancer, melanoma, head and neck tumors, prostate cancer, esophageal cancer, cervical cancer, renal cancer, bladder cancer, ovarian cancer or pancreatic cancer.
  • the subject can be a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., a subject having or at risk of having a disease as described herein) .
  • the subject has or is at risk of having a disease described herein (e.g., cancer) .
  • the subject receives or has received other treatment, such as chemotherapy treatment and/or radiation therapy.
  • the subject has previously received or is receiving immunotherapy.
  • the present invention provides the bispecific antibody molecule, ADC molecules or pharmaceutical compositions or pharmaceutical combinations or kits as described above for use in therapy, e.g. for use as described herein, e.g. for the prevention or treatment of a related disease or disorder mentioned herein.
  • combination therapies of the present invention encompass both combined administration (e.g., two or more therapeutic agents contained in the same formulation or separate formulations) , and separate administration, in which administration of the ADC molecules of the present invention can occur prior to, simultaneously with, and/or after administration of the other therapeutic agents and/or active agents.
  • the route of administration of the pharmaceutical composition is according to known methods, e.g., orally, by intravenous injection, intraperitoneally, intracerebrally (intraparenchymal) , intracerebroventricularly, intramuscularly, intraocularly, intraarterially, intraportally or intralesionally; by a sustained release system or by an implanted device.
  • the composition may be administered by bolus injection or by continuous infusion or by an implanted device.
  • composition may also be administered topically via an implant membrane, sponge, or another suitable material onto which the desired molecule is absorbed or encapsulated.
  • an implant device when an implant device is used, the device may be implanted into any suitable tissue or organ and the desired molecule may be delivered via diffusion, timed-release bolus, or continuous administration.
  • one or more DNA molecules encoding the following amino acid sequences can be used: a first heavy chain with the amino acid sequence SEQ ID NO: 6, a first light chain with SEQ ID NO: 12, a second heavy chain with SEQ ID NO: 18, and a second light chain with SEQ ID NO: 23, and are transiently or stably transfect with an expression system for secretion of antibodies to prepare the bispecific antibody HZ5C2.9/hRS7.13 of the present invention.
  • Figure 1 shows a schematic diagram of the structure of the aforementioned antibody.
  • the culture medium can be conveniently applied to a MabSelect column (GE Healthcare) or KappaSelect column (GE Healthcare) that has been equilibrated with a compatible buffer such as phosphate-buffered saline (pH 7.4) .
  • a compatible buffer such as phosphate-buffered saline (pH 7.4) .
  • the column is washed to remove non-specifically bound components.
  • the bound antibody can be eluted, for example, by a pH gradient (e.g., from 20 mM Tris buffer pH 7.0 to 10 mM sodium citrate buffer pH 3.0, or from phosphate-buffered saline pH 7.4 to 100 mM glycine buffer pH 3.0) .
  • a pH gradient e.g., from 20 mM Tris buffer pH 7.0 to 10 mM sodium citrate buffer pH 3.0, or from phosphate-buffered saline pH 7.4 to 100 mM glycine buffer pH 3.
  • the antibody fractions are detected, for example, by UV absorption or SDS-PAGE, and then the antibody fractions are pooled. Further purification can be optionally carried out according to the intended use.
  • the purified antibody can be concentrated and/or sterile filtered using conventional techniques. Soluble aggregates and polymers can be effectively removed using conventional techniques, including size exclusion, hydrophobic interaction, ion exchange, multi-mode or hydroxyapatite chromatography, etc.
  • the purified antibody can be immediately frozen at below -30°C or lyophilized.
  • the heavy chain sequence of the TROP2-Arm and the light chain sequence of the TROP2-Arm, as well as the heavy chain sequence of the B7-H3-Arm and the light chain sequence of the B7-H3-Arm, were inserted into the vector pcDNA3.1 (Invitrogen, V790-20) , respectively obtaining the heavy chain plasmid and light chain plasmid of the anti-TROP2 end, and the heavy chain plasmid and light chain plasmid of the anti-B7-H3 end.
  • the heavy chain plasmid and light chain plasmid of the anti-TROP2 end, and the heavy chain plasmid and light chain plasmid of the anti-B7-H3 end were transiently transfected into ExpiCHO (Invitrogen, A29133) cells, respectively. After 7 days, the cell fermentation broth was harvested, filtered and clarified, and captured using a Hitrap Mabselect Sure chromatography column (GE Healthcare, 11-0034-95) to obtain the TROP2 antibody parent and B7-H3 antibody parent.
  • the antibody parents were mixed in a molar ratio of 1: 1, and an appropriate amount of reducing agent GSH was added to react overnight at room temperature. The reducing agent was removed by ultrafiltration to terminate the reaction. MonoS cation exchange chromatography column (GE Healthcare, 17-5168-01) was used for fine purification, with the anti-B7-H3 parent liquid being 20 mM sodium phosphate buffer (pH 6.6) and the anti-TROP2 parent liquid being 20 mM sodium phosphate buffer containing 1M sodium chloride (pH 6.6) , with an elution gradient of 0-50% (30 column volumes) . The eluted protein solution was ultrafiltered and exchanged into PBS (Gibco, 70011-044) , and the purity was detected by SEC-HPLC.
  • PBS Gibco, 70011-044
  • the expression vector was constructed by placing the heavy chain variable region and light chain variable region of the anti-B7-H3 humanized antibody HZ5C2.9, anti-TROP2 antibody hRS7.13, and control antibody (negative control antibody IgG) (see sequence list information) at the N-terminus of the human IgG1 heavy chain constant region (SEQ ID NO: 24) and light chain kappa constant region (SEQ ID NO: 11) respectively. Then, it was constructed into the pcDNA3.1 expression vector with an N-terminal signal peptide to obtain the heavy and light chain expression vectors.
  • HEK293 cells Invitrogen
  • the cell density was about 3 ⁇ 10 6 cells/ml.
  • a final volume of 1/10 (v/v) of Opti-MEM medium (Gibco catalog number: 31985-070) was taken as the transfection buffer, added with the constructed expression vector, mixed well, filtered with a 0.22 ⁇ m filter head, and set aside.
  • An appropriate amount of polyethyleneimine (PEI) (Polysciences, 23966) was added to the above plasmid (the mass ratio of plasmid to PEI was 1: 3) , mixed and incubated at room temperature for 10 minutes to obtain a DNA/PEI mixture.
  • the DNA/PEI mixture was gently poured into HEK293 cells and mixed, cultured at 37°C, 8%CO 2 for 24 hours, then supplemented with VPA (Sigma, catalog number: P4543-100G) at a final concentration of 2mM, and 2% (v/v) of Feed solution (1g/L Phytone Peptone + 1g/L Difco Select Phytone) , and continue to culture for 6 days.
  • VPA Sigma, catalog number: P4543-100G
  • Feed solution (1g/L Phytone Peptone + 1g/L Difco Select Phytone)
  • the cell culture broth was centrifuged at 13000rpm for 20 minutes, the supernatant was collected, and the supernatant was purified according to the manufacturer's instructions using a pre-packed column Hitrap Mabselect Sure (GE, 11-0034-95) , and the concentration was determined.
  • 100 ⁇ g of purified protein was takend, adjusted to the concentration to 1mg/mL
  • the equilibrium dissociation constant of the binding of the antibodies of the invention with human B7-H3 and human Trop2 were determined using biomolecular thin-film interferometry (ForteBio) .
  • the ForteBio affinity assay was performed according to established methods (Estep, P et al., High throughput solution Based measurement of antibody antigen affinity and epitope binning. MAbs, 2013.5 (2) : pp. 270-8) .
  • the senor was equilibrated in the analysis buffer for 30 minutes offline, then a baseline was established by online detection for 60 seconds, and the purified antibodies obtained as described above were loaded online onto the AHQ sensor (ForteBio) for ForteBio affinity measurement.
  • the sensor loaded with antibodies was then exposed to human B7-H3 protein (Sinobiological, 29849-H08H) or human Trop2 protein (Sinobiological, 90893-C08H) , after which the sensor was transferred to the analysis buffer for dissociation rate measurement.
  • the K D values were analyzed using ForteBio analysis software.
  • the binding of the Trop2/B7-H3 bispecific antibody HZ5C2.9/hRS7.13 and the parental mAbs (HZ5C2.9 and hRS7.13) to the human lung cancer cell line NCI-H358 (Cobioer, CBP60136) was determined by flow cytometry (FACS) . Specifically, the cells to be tested were counted and diluted to a concentration of 2 ⁇ 10 6 cells/ml, and 50 ⁇ l/well was added to a U-bottom 96-well plate. After centrifugation at 500 g for 5 minutes, the cell culture medium was removed.
  • the Trop2/B7-H3 bispecific antibody HZ5C2.9/hRS7.13 and the parental mAb were added to the U-bottom plate and the cells were resuspended, with 50 ⁇ l of antibody starting at a concentration of 140 nM and then diluted threefold in a series, for a total of 10 concentration points for each well.
  • the plate was incubated on ice for 30 minutes. After centrifugation at 500 g for 5 minutes to remove the supernatant, the cells were washed once with PBS. Then, 100 ⁇ l of goat anti-human IgG PE-labeled secondary antibody (SouthernBiotech, 51380) was added to each well. The plate was incubated in the dark on ice for 30 minutes.
  • NCI-H358 cells were trypsinized, and the cell density was adjusted to 4 ⁇ 10 4 /ml; 50 ⁇ l/well was added to an opaque white 96-well plate and incubated overnight in a 37°C CO 2 incubator; the test antibodies were diluted to 4 nM with complete culture medium, and this was used as the initial concentration with a gradient dilution of 1/3 ratio; Fab-ZAP (Advanced Targeting Systems, IT-51-250) was diluted to 40 nM with complete culture medium, and this was used as the initial concentration with a gradient dilution of 1/3 ratio; an equal volume of antibody and Fab-ZAP was mixed at a ratio of 1: 1 and incubated at room temperature for 20 to 30 minutes; after incubation, 50 ⁇ l/well was added to the cells, mixed well, and then continued for incubation at 37°C; on Day 4, the plate was removed, and 100 ⁇ l of CellTiter-Glo detection reagent was added to each well, shaken at 150 rpm
  • ADC coupling with small molecular compounds was further designed and synthesized.
  • Ab is antibody HZ5C2.9/hRS7.13; q is for example an integer selected from 4 to 10, for example 8, and HZ5C2.9/hRS7.13-NT3 obtained had an average DAR of 7.98 as determined in the experiments described below.
  • step (b) An excess of linker-toxin (NT3, dissolved in DMSO) was added, reacted with the reduced antibody in step (a) , and the reaction mixture was placed at room temperature for 1-2 hours, wherein
  • the purity of the ADC was obtained by SEC-HPLC based on the ratio of the peak areas of monomer peak, aggregate, oligomer at UV 280nm. After calculation, the purity was 98.96%for HZ5C2.9/hRS7.13-NT3.
  • the preparation process was similar to that of HZ5C2.9/hRS7.13-NT3, except that the bispecific antibody was replaced by the parent B7-H3 mAb HZ5C2.9.
  • the average DAR was determined to be 7.85 and the purity was 97.27%.
  • the preparation process was similar to that of HZ5C2.9/hRS7.13-NT3, except that the bispecific antibody was replaced by the parent Trop2 mAb hRS7.13.
  • the average DAR was determined to be 7.85 and the purity was 97.14%.
  • the preparation process was similar to that of HZ5C2.9/hRS7.13-NT3, except that the bispecific antibody was changed to the control antibody IgG.
  • the average DAR was determined to be 7.94 and the purity was 99.35%.
  • Example 6 Cell binding assay for Trop2/B7-H3 bispecific antibody ADC molecules
  • the binding activity of HZ5C2.9/hRS7.13-NT3 to the targets was determined by flow cytometry using a human lung cancer cell line NCI-H322 (Cobioer, CBP60134) and a Trop2&B7-H3 negative cell KM12 (Meisen, CTCC-001-0684) .
  • the process was the same as Example 3.
  • ADC molecules were tested using the Cell Titer-Glo (Promega, G9242) assay kit in multiple tumor Cell lines co-expressing Trop2&B7-H3: NCI-H358, NCI-H322, SUM149PT (Meisen, CTCC-001-0949) , SCC25 (Cobioer, CBP61033) and normal cell MCF10A (Cobioer, CBP60419) .
  • the killing effects of the Trop2/B7-H3 bispecific antibody ADC molecule on a plurality of tumor cell lines were stronger than that of the parent mAb ADC.
  • Trop2 mAb ADC hRS7.13-NT3 has stronger cytotoxicity, while the cytotoxicity of bispecific ADC HZ5C2.9/hRS7.13-NT3 was significantly weaker than that of hRS7.13-NT3.
  • the bispecific ADC had a tumor inhibition effect better than that of the parent mAb ADC, and can siginificantly weaken the on-target toxicity of the Trop2 ADC to normal tissues.
  • Example 8 Antitumor effect of Trop2/B7-H3 bispecific antibody ADC molecules in NCI-H358 mouse graft tumor model
  • CB17-SCID mice were inoculated with NCI-H358 cells to determine the anti-tumor efficacy of the molecules of the present invention.
  • Female mice of SPF grade 14-17 g, from Vital River Laboratory Animal Technology Co., Ltd., Beijing) were used for the experiments.
  • NCI-H358 cells were routinely subcultured for subsequent in vivo experiments. Cells were harvested by centrifugation, and NCI-H358 cells were dispersed with PBS (1X) to prepare a cell suspension having a cell concentration of 2.5x10 7 cells/ml.
  • a model of NCI-H358 tumor-bearing mice was established by inoculating 0.2 ml of the cell suspension subcutaneously on day 0 into the right abdominal region of CB17-SCID mice. All mice were randomized into 5 groups (5 mice per group) on Day 7 after tumor cell inoculation. Administration doses and mode were shown in table 4. Administration was performed on day 7 after inoculation, and tumor volume and body weight were monitored twice per week, as shown in Fig. 6A, 6B. The monitoring continued until Day 50.
  • TGI tumor growth inhibition rate
  • TGI% 100%* (control group tumor volume -treatment group tumor volume) / (control group tumor volume -control group tumor volume prior to administration) .
  • the tumor growth inhibition rate results were shown in table 5 and Fig. 6A: on Day 50 after inoculation, the single-dose of HZ5C2.9/hRS7.13-NT3 reached 93%tumor growth inhibition rate, which was better than 29%and 16%of the tumor growth inhibition rates of HZ5C2.9-NT3 and hRS7.13-NT3. Meanwhile, the body weights of the mice were detected, and the results were shown in Fig 6B, the body weights of the mice had no significant difference.
  • Example 9 Antitumor effect of Trop2/B7-H3 bispecific antibody ADC molecules in HCC1806 mouse graft tumor model
  • HCC1806 HCC 1806 human breast cancer cells, from ATCC, Cat. No. CBP60373, Nanjing Cobioer Biotech Co., Ltd.
  • Female mice of SPF grade 14-17 g, from Vital River Laboratory Animal Technology Co., Ltd., Beijing) were used for the experiments.
  • TGI% Relative tumor growth inhibition rate
  • Tumor volume determination the maximum major axis (L) and maximum broad axis (W) of the tumor was measured using a vernier caliper, tumor volume was calculated as follows:

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Abstract

The present invention relates to bispecific antibodies targeting B7-H3 and TROP2, as well as antibody-drug conjugates based on them and compositions containing the antibody-drug conjugate. The present invention also relates to the therapeutic and diagnostic use of these antibody drug conjugates.

Description

Antibody Drug Conjugates targeting B7-H3 and TROP2 and the use thereof
Cross-reference to related applications
This application is based on the Chinese patent application with the Application No. 202311785547.2, filed on December 22, 2023, and claims priority to the aforementioned application, the entire contents of which are hereby incorporated by reference into this application.
Technical Field
The invention relates to antibodies targeting B7-H3 and TROP2, and antibody-drug conjugates (ADCs) constructed based thereon, and to compositions containing the antibody-drug conjugates. The invention also relates to therapeutic and diagnostic uses of these antibodies or ADCs.
Background
Therapies using cytotoxic agents are important therapies for treating cancer, but low selectivity cytotoxic agents often kill normal cells, causing severe toxic side effects. Biomacromolecule drugs, such as antibodies or antibody fragments, although highly targeting, have limited therapeutic effects on solid tumors, thereby limiting their applications. The antibody-drug conjugate (ADC) is a conjugate of an antibody and a small molecule drug, combines the targeting effect of the antibody with the high activity of the small molecule drug (such as a cytotoxic agent) , and has the advantages of high curative effect, high safety, and the like.
B7-H3 is a type I transmembrane protein of the B7 family. It is not constitutively expressed in many immune cells (e.g., natural killer (NK) cells, T cells, and antigen-presenting cells (APCs)) ; however, its expression can be induced. Moreover, the expression of B7-H3 is not limited to immune cells. B7-H3 transcripts are expressed in various human tissues (including colon, heart, liver, placenta, prostate, small intestine, testis, and uterus) as well as in osteoblasts, fibroblasts, epithelial cells, and other non-lymphoid lineage cells that may indicate immune and non-immune functions (Nygren et al. Front Biosci. 3: 989-93 (2011) ) . However, protein expression in normal tissues is usually maintained at low levels and may be subject to post-transcriptional regulation. B7-H3 is also expressed in various human cancers, including prostate cancer, clear cell renal cell carcinoma, glioma, melanoma, lung cancer, breast cancer, and so on.
Research on antibody-drug conjugates (ADCs) targeting B7-H3 has been conducted (see, for example, US20200338209A1; Scribner et al., Mol Cancer Ther (2020) 19 (11) : 2235–2244; Yamato et al., Mol Cancer Ther. 2022 Apr 1; 21 (4) : 635–646) . One of the most typical blockbuster new drugs in this field is DS-7300a developed by Daiichi Sankyo Co., Ltd, which has high efficacy against various cancers (Yamato et al., Mol Cancer Ther. 2022 Apr 1; 21 (4) : 635–646) . However, existing ADCs, including DS-7300a, still have some drawbacks, such as the need for improved efficacy, low internalization efficiency, side effects that need to be improved, uneven product DAR value distribution affecting drug efficacy, low DAR values, and/or short half-life, and the like.
TROP2, a trophoblast cell surface antigen, also known as tumor-associated calcium signal transduction protein (TACSTD2) , is overexpressed in various human epithelial cancers, including breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, and ovarian cancer (Yezhe Cheng et al., Frontiers in Oncology, 2022 Dec 23; 12: 951589) .
Preclinical and clinical studies have demonstrated the use of anti-TROP2 antibody-drug conjugates (ADCs) (e.g., Trodelvy, an anti-human TROP2 antibody-SN-38 conjugate, or DS-1062a) for cancer treatment. Clinical results have shown promising therapeutic effects in the treatment of refractory solid tumors with Trodelvy. The objective response rate (ORR) of Trodelvy in patients with drug-resistant triple-negative breast cancer (TNBC) reached 33%. One of the important mechanisms of action of Trodelvy is that SN-38 (payload) is connected via a pH-sensitive linker, which can break in the acidic tumor microenvironment and specifically release SN-38. However, because the linker of Trodelvy is not stable enough, the maleimide-mediated linker breaks through thiol exchange under physiological conditions, making the serum half-life of Trodelvy relatively short (about 1 day) . Therefore, Trodelvy may have a relatively high off-target effect. While DS-1062a (Dato-DXd, AstraZeneca and Daiichi Sankyo) has shown significant efficacy in treating patients with lung cancer without gene mutation and triple-negative breast cancer, it still has issues such as uneven DAR distribution affecting drug efficacy and target toxicity in normal tissues.
In addition, ADCs of monoclonal antibodies also face the issue of drug tolerance due to the downregulation of target expression.
Therefore, there is a need in this field to develop new bispecific antibodies based on B7-H3 and TROP2, as well as ADC molecules based on them, preferably with advantages such as high efficacy, high safety (including low side effects) , and/or high product uniformity.
Summary of The Invention
The invention provides an immunoconjugate comprising an antibody targeting B7-H3 and TROP2 (e.g., a bispecific antibody of the invention that specifically binds B7-H3 and TROP2) and an additional payload.
In some aspects, the immunoconjugate is an antibody-drug conjugate.
Accordingly, the present invention provides bispecific antibody-drug conjugates (ADCs) targeting B7-H3 and TROP2.
In some embodiments, the ADC has one or more of the following advantages:
(1) can target two antigens simultaneously due to the contained bispecific antibody,
(2) has higher efficiency of endocytosis,
(3) has higher affinity, etc.
In an aspect, the present invention provides an antibody-drug conjugate of formula (I) :
Ab- (L-D) p (I)
or a pharmaceutically acceptable salt or solvate thereof,
wherein:
Ab is an antibody or fragment thereof that specifically binds B7-H3 and TROP2 (e.g., human B7-H3 and human TROP2) ;
L is a linker;
D is a drug, preferably an anti-tumor compound; and
p is an integer selected from 1 to 16, such as an integer selected from 4 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,
wherein Ab in formula (I) is a bispecific antibody that specifically binds to B7-H3 and TROP2 comprising a first antigen-binding region that specifically binds to TROP2 and a second antigen-binding region that specifically binds to B7-H3, wherein the second antigen-binding region that specifically binds to B7-H3 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein
the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2, the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3, the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 7, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 8, and the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 9.
In some embodiments, the second antigen-binding region that specifically binds B7-H3 of said bispecific antibodies comprises a VH and a VL, wherein said VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence set forth in SEQ ID NO: 4, and/or said VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity the amino acid sequence set forth in SEQ ID NO: 10.
In some embodiments, the second antigen-binding region that specifically binds B7-H3 of said bispecific antibodies comprises a VH and a VL, wherein the VH and VL comprise or consist of, respectively, the amino acid sequences shown below: SEQ ID NO: 4 and SEQ ID NO: 10, respectively.
In some embodiments, the first antigen-binding region that specifically binds TROP2 of said bispecific antibodies comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID No: 13; HCDR2 comprises or consists of the amino acid sequence of SEQ ID No: 14; HCDR3 comprises or consists of the amino acid sequence of SEQ ID No: 15; LCDR1 comprises or consists of the amino acid sequence of SEQ ID No: 19; LCDR2 comprises or consists of the amino acid sequence of SEQ ID No: 20; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID No: 21.
In some embodiments, the first antigen-binding region that specifically binds TROP2 of said bispecific antibodies comprises a VH and a VL, wherein said VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity thereto, and/or said VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity thereto.
In some embodiments, the first antigen-binding region that specifically binds TROP2 of said bispecific antibodies comprises a VH and a VL, wherein the VH and VL comprise or consist of, respectively, the amino acid sequences shown below: SEQ ID NO: 16 and SEQ ID NO: 22, respectively.
In some embodiments, the bispecific antibody comprises a first Fc region and a second Fc region, wherein the first Fc region and the second Fc region are the same or different. In some embodiments, the first Fc region and second Fc regions are, respectively, human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc, e.g., comprise or consist of the amino acid sequence of SEQ ID NO: 30 or 31 or an amino acid sequence having at least 90%, e.g., 95%, 96%, 97%, 98%or 99%or more identity thereto. In some embodiments, the first and second Fc regions of the bispecific antibody have introduced therein mutations that promote heterodimerization of the first and second Fc regions. In some embodiments, the mutation of the Fc region is introduced based on the Knob-into-Hole technique, wherein the corresponding Knob mutation and Hole mutation are introduced in the first Fc region and the second Fc region, respectively. In some embodiments, in the bispecfic antibodies, one Fc region comprises the amino acid substitutions S354C and T366W and the other Fc region comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to the EU index) , e.g., wherein the S354C and Y349C form a non-natural disulfide bond.
In some embodiments, in the bispecfic antibodies, the mutation of the Fc region is introduced based on the Innobody technology.
In some embodiments, in the bispecfic antibodies, the Fc region further comprises a mutation that reduces binding to the Fcγ receptor, such as the L234A/L235A mutation.
In some embodiments, in the bispecfic antibodies, one Fc region polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 27 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to the amino acid sequence set forth in SEQ ID No: 27, and the other Fc region polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 28 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to the amino acid sequence set forth in SEQ ID No: 28.
In some embodiments, in the bispecfic antibodies, the first antigen-binding region of the bispecific antibody is a Fab fragment, and/or the second antigen-binding region is a Fab. In some embodiments, in the bispecfic antibodies, the Fab, which is the first antigen binding region or the second antigen binding region, comprises CH1, wherein the CH1 is CH1 from IgG1, IgG2, IgG3, or IgG4, preferably CH1 from IgG1. In some embodiments, the CH1 comprises
(i) comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 32, or
(ii) comprises or consists of the amino acid sequence of SEQ ID NO: 32.
In some embodiments, in the bispecfic antibodies, the Fab, which is the first antigen binding region or the second antigen binding region, comprises a light chain constant region, wherein the light chain constant region is a Kappa light chain constant region or a Lambda light chain constant region. In some embodiments, the Kappa light chain constant region comprises
(i) comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 11, or
(ii) comprises or consists of the amino acid sequence of SEQ ID NO: 11.
In some embodiments, in the bispecfic antibodies, the first antigen binding region that specifically binds TROP2 comprises a first Fab linked at its C-terminus of CH1 to the N-terminus of the first Fc region (with or without a connector, e.g., a hinge region) and the second antigen binding region that specifically binds B7-H3 comprises a second Fab linked at its C-terminus of CH1 to the N-terminus of the second Fc region (with or without a connector, e.g., a hinge region) .
Ins some embodiments, the bispecific antibody is an IgG-like antibody having the configuration shown in Figure 1.
In some embodiments, the bispecific antibody comprises a first Fab as a first antigen-binding region that specifically binds TROP2 and a second Fab as a second antigen-binding region that specifically binds to B7-H3, wherein the bispecific antibody comprises or consists of
Heavy chain 1: from N-terminus to C-terminus comprises or consists of: heavy chain variable region of the Fab that specifically binds TROP2 -heavy chain constant region CH1-First Fc region, wherein heavy chain constant region CH1 is linked at its C-terminus to the N-terminus of the first Fc region with or without a connector (e.g., hinge region) ;
Light chain 1: from N-terminus to C-terminus comprises or consists of: a light chain variable region of the Fab that specifically binds TROP2 -light chain constant region;
Heavy chain 2: from N-terminus to C-terminus comprises or consists of: heavy chain variable region of the Fab that specifically binds B7-H3 -heavy chain constant region CH1-second Fc region, wherein heavy chain constant region CH1 is linked at its C-terminus to the N-terminus of the second Fc region with or without a connector (e.g., hinge region) ;
Light chain 2: from N-terminus to C-terminus comprises or consists of: a light chain variable region of the Fab that specifically binds B7-H3 -light chain constant region,
preferably, each domain is linked directly;
optionally, said first Fc region comprises mutations Y349C, T366S, L368A and Y407V, and said second Fc region comprises mutations S354C and T366W, and vice versa; optionally, the first and second Fc regions further comprise L234A/L235A mutations, respectively.
In some embodiments, said Heavy chain 1 comprising or consisting of the amino acid sequence shown as SEQ ID NO: 18, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity thereto; Light chain 1 comprising or consisting of the amino acid sequence shown as SEQ ID NO: 23, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity thereto; Heavy chain 2 comprising or consisting of the amino acid sequence shown as SEQ ID NO: 6, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity thereto; ; and Light chain 2 comprising or consisting of the amino acid sequence shown as SEQ ID NO: 12, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity thereto.
In some embodiments, the anti-tumor compound is a cytotoxic agent, such as camptothecins, auristatins, maytansinoids, taxanes, anthracyclines, vinca alkaloids, MEK inhibitors, or KSP inhibitors.
In some embodiments, D is represented by formula (D-1) :
wherein R1 is selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C2-C6 haloalkenyl and C2-C6 haloalkynyl;
R2 is selected from H, halo, C1-C6 alkyl, C1-C6 haloalkyl, -OR4 and -SR4; R3 is selected from H, halo, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR4; or R2 and R3 together form -O (CH2) nO-or -O (CF2) nO-, wherein n is 1 or 2;
R4 is selected from H or C1-C4 alkyl.
It should be understood that a wavy line in a structure indicate the connection position of the structure, for example, the wavy line in formula (D-1) indicates the connection position of D-1 linked to L.
In some embodiments, R1 is H, R2 is C1-C6 alkyl, R3 is -F.
In some embodiments, D is represented by formula (D-2) :
wherein R1, R2 and R3 are as defined above.
In some embodiments, D is represented by formula (D-3) or formula (D-4) :
In some embodiments, -L-is represented by the following structure:
-Z-E-NH-CH2-Q-L2-L1-
wherein Z is linked to Ab, L1 is linked to D;
Z is selected fromwherein m is an integer selected from 1 to 10; the carbonyl at the right end of Z is covalently linked to E;
E is a peptide residue comprising 2-10 amino acids, wherein the peptide residue is optionally substituted with one or more polyol groups, wherein the N-terminus of the peptide residue is covalently linked to Z;
Q is -O-or -S-;
L1 is absent or - (C1-C10alkylene) -;
L2 is absent, -N (R5) C (O) - (C1-C10alkylene) -*or -C (O) N (R5) - (C1-C10alkylene) -*; wherein *indicates that the terminus is covalently linked to Q; and
R5 is H or C1-C6 alkyl.
In some embodiments, E is a peptide residue consisting of 2, 3, or 4 amino acids, the amino acids are selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine and leucine, and wherein the glutamine or glutamic acid is optionally substituted with one polyol group, and -L2-L1-is - (C1-C6alkylene) -, - (C1-C6alkylene) -N (R5) C (O) - (C1-C6alkylene) -*or - (C1-C6alkylene) -C (O) N (R5) - (C1-C10alkylene) -*, wherein *indicates that said terminus is covalently linked to Q ;
R5 is H or C1-C6 alkyl.
In some embodiments, E is -Gln-Val-Ala-, -Gly-Val-Ala-, -Gln-Phe-Ala-, -Gly-Phe-Ala-or wherein R6 is H or C1-C6 alkyl, wherein these E groups are covalently linked to Z through the left N-terminus, and
-L2-L1-is - (C1-C6alkylene) -.
In some embodiments, -Z-E-NH-CH2-Q-L2-L1-is represented by the following structure
its right terminus is linked to D.
In some embodiments, the antibody-drug conjugate is represented by the following formula
Ab’- (S-L-D) p (I’ )
wherein Ab’ is as defined above for Ab; L, D and p are as defined above.
In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof has an average DAR of from 5 to 11, for example, from 7.5-8.5.
In some embodiments, the antibody-drug conjugate is represented by the following formula
wherein Ab is the bispecific antibody of the present invention; q is as defined for p in formula (I) above,
preferably, the antibody-drug conjugate has an average DAR of from 5 to 11, for example, from 7.5-8.5.
In another aspect, the present invention provides a pharmaceutical composition comprising the antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof as described above, and optionally one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immune modulators and optionally pharmaceutically acceptable excipients.
In another aspect, the present invention provides a pharmaceutical combination comprising the antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof as described above, and one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immune modulators.
In another aspect, the present invention provides a method of preventing or treating a tumor in a subject, comprising administering to the subject an effective amount of the antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof as described above, or the pharmaceutical composition as described above, or the pharmaceutical combination as described above.
In some embodiments, the tumor is a cancer, preferably, the cancer has B7-H3 and/or TROP2 at elevated level (such as at nucleic acid or protein level) compared with the corresponding tissue of a healthy subject or a healthy tissue adjacent to the cancer tissue of the patient.
In some embodiments, the cancer is selected from lung cancer (including non-small cell lung cancer and small cell lung cancer) , colon cancer, oral squamous cell carcinoma, breast cancer, melanoma, head and neck tumors, prostate cancer, esophageal cancer, cervical cancer, renal cancer, bladder cancer, ovarian cancer or pancreatic cancer.
In some embodiments, the method further comprises administering one or more therapies, such as a therapeutic approach and/or other therapeutic agents, to a patient, preferably the therapeutic approach includes radiation therapy or surgery, or the therapeutic agents include chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immune modulators.
The antibody-drug conjugate of the present invention has the following advantages, the antibody-drug conjugate:
(1) binds to target cells expressing human B7-H3 and/or TROP2 with high affinity;
(2) can enter cells through endocytosis to kill target cells;
(3) has high endocytosis efficiency;
(4) has remarkable bystander killing effect;
(5) has high antitumor effect, stronger killing effect on tumor cells and stronger inhibiting effect on the growth of tumors, particularly tumors with high expression of B7-H3 and/or TROP2; have significantly improved and even unexpected anti-tumor activity;
(6) has DAR values up to about 8, the product is more uniform.
(7) has low toxicity;
(8) has good stability, and the linker-payload used by the present invention has high stability;
(9) has good druggability.
Figure Descriptions:
Fig. 1 shows a schematic structural diagram of bispecific antibody HZ5C2.9/hRS7.13.
Fig. 2 shows the results of the binding experiments of bispecific antibody HZ5C2.9/hRS7.13 and the parental antibody to NCI-H358 cells.
Fig. 3 shows the results of the endocytosis experiments of bispecific antibody HZ5C2.9/hRS7.13 and parental antibody.
Fig. 4 shows the results of the binding experiments of the bispecific antibody ADC molecule and the parent antibody ADC molecule to cells.
Fig. 5 shows the in vitro killing effect of bispecific antibody ADC molecules and parent antibody ADC molecules on several tumor cell lines.
Fig. 6A shows the tumor inhibiton effect of bispecific antibody ADC molecules and parent antibody ADC molecules in NCI-H358 mouse graft tumor model.
Fig. 6B shows the body weight change of mice in a tumor inhibition experiment using the NCI-H358 mouse graft tumor model.
Fig. 7A shows the tumor inhibition effect of bispecific antibody ADC molecules and parent antibody ADC molecules in HCC1806 mouse graft tumor model.
Fig. 7B shows the body weight change of mice in a tumor inhibition experiment using HCC1806 mouse graft tumor model.
Fig. 8 shows a schematic of the bispecific antibody ADC molecule.
Detailed Description
Before the invention is described in detail below, it should be understood that the invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs.
I. Definition
For the purpose of explaining this specification, the following definitions will be used, and wherever appropriate, terms used in the singular may also include the plural and vice versa. It is understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
The term "about" used in combination with a numerical value is intended to encompass the numerical values in a range from a lower limit less than the specified numerical value by 5% (e.g. 4%, 3%, 2%, or 1%) to an upper limit greater than the specified numerical value by 5% (e.g. 4%, 3%, 2%, or 1%) .
The term "and/or" as used herein, means any of the options or two or more of the options.
The term "comprise" or "include" as used herein means including the elements, integers or steps described, but does not exclude any other elements, integers or steps. The term also covers the situation consisiting of /combined by the elements, integers or steps mentioned herein when the term "comprises" or "include" is used, unless otherwise specified. For example, it is also intended to cover the antibody variable region composed of the specific sequence when referring to the antibody variable region "comprises" a specific sequence.
Unless otherwise indicated, the terms "B7-H3" , "B7H3" and "CD276" are used interchangeably herein. B7-H3 is a type I transmembrane glycoprotein belonging to a member of the B7/CD28 superfamily, and is similar in sequence to the extracellular domain of PD-L1. B7-H3 has 316 amino acids and contains one putative signal peptide consisting of 28 amino acids, one extracellular region consisting of 217 amino acids, one transmembrane region and one cytoplasmic domain consisting of 45 amino acids with a molecular weight of about 45-66 kDa. In humans, the extracellular structure of B7-H3 may be an IgV-IgC-like domain (2Ig-B7-H3) or an IgV-IgC-IgV-IgC-like domain (4Ig-B7-H3) due to exon replication. The sequence of cynomolgus monkey B7-H3 has about 90%homology with its human counterpart. In some embodiments of the invention, B7-H3 is human B7-H3. In some embodiments, B7-H3 is a protein under UniProt database accession number Q5ZPR3.
The terms "anti-B7-H3 antibody" , "anti-B7-H3" , "B7-H3 antibody" , or "anti-B7-H3 antibody" , as used herein, refer to an antibody that is capable of binding B7-H3 protein with sufficient affinity. The antibodies may be used as diagnostic and/or therapeutic agents in targeting B7-H3, or to construct immunoconjugates, such as antibody drug conjugates.
The term "anti-TROP2 antibody" , "anti-TROP2" , "TROP2 antibody" or "anti-TROP2 antibody" as used herein refers to an antibody that is capable of binding to TROP2 protein with sufficient affinity. The antibodies may be used as diagnostic and/or therapeutic agents in targeting TROP2, or for the construction of immunoconjugates, such as antibody drug conjugates.
In some embodiments of the present invention, TROP2 is human TROP2. In some embodiments, TROP2 is the protein under Accession No. UniProt data Accession No. P09758.
When referring to "first" and "second" herein, it is merely to distinguish the two domains or the two chains but do not indicate the positions of the two domains in any way.
General information on the amino acid sequences or nucleotide sequences of human immunoglobulin light and heavy chains is given in Kabat, E. A. et al, Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991) .
As used herein, the amino acid positions of all variable regions of the heavy and light chains are numbered according to the Kabat numbering system described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, MD (1991) and referred to herein as "Kabat numbering" .
As used herein, when used in reference to amino acid positions in domains (e.g., constant regions, e.g., Fc regions) of antibodies other than the variable regions, numbering is according to the EU numbering system described in Kabat, E. A. et al, Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991) and referred to herein as "EU numbering" . When position numbering and/or amino acid residues are assigned to a particular antibody isotype, it is intended to apply to the corresponding position and/or amino acid residue of any other antibody isotype, as is known to those skilled in the art.
The term "antibody" is used herein in the broadest sense to refer to proteins comprising an antigen-binding site, encompassing natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) , single chain antibodies, intact antibodies, and antibody fragments.
The terms "whole antibody" , "full-length antibody" , "complete antibody" and "intact antibody" are used interchangeably herein to refer to a naturally occurring glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of 3 domains CH1, CH2 and CH3 (and optionally CH4) . Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions may be further subdivided into hypervariable regions (being Complementarity Determining Regions (CDRs) ) interspersed with relatively conserved regions (being Framework Regions (FRs) . Each VH and VL consists of three CDRs and 4 FRs, arranged from N-terminal to C-terminal in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions are not directly involved in binding of the antibody to the antigen, but exhibit multiple effector functions.
"Half-antibody" or "half-polymer" refers to a monovalent antigen-binding polypeptide. In some embodiments, the half-antibody or half-polymer comprises a VH/VL unit and optionally at least a portion of an immunoglobulin constant domain. In some embodiments, a half-antibody or a half-polymer comprises one immunoglobulin heavy chain, or antigen-binding fragment thereof, associated with one immunoglobulin light chain. In some embodiments, the half-antibody or half-polymer is monospecific, i.e., binds a single antigen or epitope. In some specific embodiments, the half-antibody binds to TROP2 and does not bind to B7-H3. In some specific embodiments, the half-antibody binds to B7-H3 and does not bind to TROP2. One skilled in the art will readily appreciate that a half-antibody may have an antigen-binding domain consisting of a single variable domain, e.g., derived from camelidae.
Herein, antibody constant regions or antibody constant domains, including CH1, CL and Fc domains and the CH2, CH3 and optionally CH4 domains that make up the Fc domains, may be selected according to the intended function of the antibody molecule. For example, the constant region may be an IgA, IgD, IgE, IgG or IgM region, especially an immunoglobulin constant domain of human IgG, e.g. a constant domain of human IgG1, IgG2, IgG3 or IgG4, preferably a constant domain of human IgG1. As another example, a Fab fragment of an antibody may comprise CH1 from IgG1 and CL constant regions. As another example, the Fc region of an antibody may comprise the CH2 and CH3 domains from IgG1. The immunoglobulin constant region may have a native sequence or a variant sequence.
The term "Fc domain" or "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" comprises two or three constant domains, namely the CH2 domain, the CH3 domain, and the optional CH4 domain. For example, in natural antibodies, the immunoglobulin Fc domain comprises the second and third constant domains (CH2 and CH3 domains) originated from the two heavy chains of IgG, IgA, and IgD class antibodies; or comprises the second, third and fourth constant domains (CH2 domain, CH3 domain and CH4 domain) originated from the two heavy chains of the IgM and IgE classes antibodies. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or heavy chain constant region is according to the EU numbering system (also known as the EU index) as described in Kabat et al, Sequences of Proteins of Immunological Interes, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Two Fc regions can be dimerized to form a dimeric Fc, and two different Fc heterodimerizations forms a heterodimeric Fc. Herein, the terms "Fc region" , "Fc portion" and "dimeric Fc (e.g., heterodimeric Fc) " do not include the heavy chain variable region VH and the light chain variable region VL and the heavy chain constant region CH1 and the light chain constant region CL of an immunoglobulin, but may in some cases include a hinge region of the heavy chain constant region at the N-terminal. In one embodiment, the human IgG heavy chain Fc region extends from Asp221 or from Cys226 or from Asp231 to the carboxy-terminus of the heavy chain.
In one embodiment, a human IgG1 Fc region polypeptide (comprising a portion of hinge region) comprises or consists of the following amino acid sequence:
In one embodiment, a human IgG1 Fc region polypeptide (comprising a portion of hinge region) comprises or consists of the following amino acid sequence, wherein E is at position 356 and M is at position 358:
In one embodiment, the Fc region is originated from an Fc region of human origin. In one embodiment, the Fc region comprises all or part of a human constant region. The antibody Fc region is directly involved in complement activation, C1q binding, C3 activation, and Fc receptor binding. In one embodiment, the Fc region is a human Fc region. In one embodiment, the Fc region belongs to the subclass human IgG 4. In one embodiment, the Fc region belongs to the subclass human IgG1.
Herein, a "heterodimeric Fc scaffold" refers to a scaffold comprising or formed from two different Fc regions by dimerization, which can be linked at its N-terminus or C-terminus to a domain (e.g., heavy and/or light chain variable regions of an antibody or antigen-binding fragment of an antibody that can bind to a target molecule, or soluble portions of a ligand or receptor that can bind to a target molecule) binding to an antigen for use in constructing multispecific antibodies, e.g., bispecific antibodies.
The term "CH1 region" refers to the portion of an antibody heavy chain polypeptide that extends from EU position 118 to EU position 220 (EU numbering system) .
In one embodiment, the CH1 domain comprises or consists of the amino acid sequence of
The term "antibody fragment" includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment. The term "antigen-binding fragment" of an antibody is a molecule distinct from a full-length antibody that comprises a portion of the full-length antibody, but is capable of binding to an antigen of the full-length antibody or competes for binding to an antigen with the full-length antibody (i.e., with the full-length antibody from which the antigen-binding fragment is derived) . Antigen-binding fragments may be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Antigen binding fragments include, but are not limited to, Fab, Fab', F (ab') 2, Fv, single chain Fv, diabodies, single domain antibodies (sdAb) , or nanobodies.
"Fab fragment" or "Fab" are used interchangeably herein to refer to an immunoglobulin fragment consisting of two polypeptide chains, comprising an immunoglobulin heavy chain variable region VH, a heavy chain constant domain CH1, a light chain variable region VL, and a light chain constant domain CL, wherein one polypeptide chain comprises, from N-terminus to C-terminus, VH and one constant region selected from CH1 and CL, and the other polypeptide chain comprises, from N-terminus to C-terminus, VL and another constant region selected from CL and CH1, wherein the VH and VL domains pair to form an antigen-binding site or an antigen-binding region. Herein, the Fab polypeptide chain comprising the heavy chain constant region CH1 is also referred to as the "Fab heavy chain" ; accordingly, the Fab polypeptide chain comprising the light chain constant region CL is also referred to as "Fab light chain" .
"Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms a structurally defined loop ( "hypervariable loop" ) and/or comprises antigen contact residues ( "antigen contact point" ) . CDRs are primarily responsible for binding to epitopes. The CDRs of the heavy and light chains are generally referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from N-terminus. The CDRs located in the variable domain of the antibody heavy chains are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located in the variable domain of the antibody light chains are referred to as LCDR1, LCDR2, and LCDR3. In a given amino acid sequence of a light chain variable region or a heavy chain variable region, the exact amino acid sequence boundaries of each CDR can be determined using any one or a combination of many well-known antibody CDR assignment systems including, e.g., Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342: 877-883; Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins" , Journal of Molecular Biology, 273, 927-948 (1997) ) , Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, U.S. Department of Health and Human Services, National Institutes of Health (1987) ) , AbM (University of Bath) , Contact (University College London) , International ImMunoGeneTics database (IMGT) (www. imgt. cines. fr/) , and North CDR definition based on the affinity propagation clustering using a large number of crystal structures. Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the schemes described above or the combination thereof. CDRs may also be determined based on the same Kabat numbering position as a reference CDR sequence (e.g., any one of the exemplary CDRs of the invention) .
In one embodiment, the CDRs of the antibodies of the invention are determined by Kabat schemes for boundaries, or by AbM schemes, or by a combination thereof.
In one embodiment of the invention, in the antigen-binding region that binds to B7-H3 of the invention, the HCDR1 of the VH is determined by the AbM scheme, HCDR2 and HCDR3 are determined by the Kabat scheme, and the LCDRs of the VL are determined by the Kabat scheme respectively.
In one embodiment of the invention, in the antigen-binding region that binds to TROP2 of the invention, the HCDR1 of the VH is determined by the AbM scheme, HCDR2 and HCDR3 are determined by the Kabat scheme, and the LCDRs of the VL are determined by the Kabat scheme respectively.
The term "hinge region" refers to the portion of an antibody heavy chain polypeptide that connects the CH1 and CH2 regions in the wild-type antibody heavy chain, e.g., the IgG1 hinge region, e.g., according to EU numbering, the sequence of D221 to P230. Other hinge region of IgG subclasses can be determined by alignment with cysteine residues in the hinge region of IgG1 subclass sequence. In some embodiments, CH1 may comprise a portion of the hinge region. In some embodiments, Fc region may comprise a portion of the hinge region.
Amino acid mutations are denoted by (original amino acid, amino acid position, mutated amino acid) . For example, when the mutation site is located in the Fc region, "T366W" means that T located at EU numbering position 366 is substituted with W. When referring to combinations of mutations, the mutations in a combination are linked by an "and" or "/" . "L234A/L235A " indicates that both mutations L234A and L235A are included. It should be noted that when describing mutations, a particular position encompasses its corresponding amino acid position on other polypeptide chains as well.
With respect to polypeptide sequences, "conservative alterations" include substitutions, deletions, or additions to the polypeptide sequence that does not substantially alter the desired functional activity of the polypeptide sequence. For example, conservative substitutions often result in the substitution of an amino acid for a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The followings list 8 groups of amino acids containing conservative substitutions for each other: 1) alanine (A) , glycine (G) ; 2) aspartic acid (D) , glutamic acid (E) ; 3) asparagine (N) , glutamine (Q) ; 4) arginine (R) , lysine (K) ; 5) isoleucine (I) , leucine (L) , methionine (M) , valine (V) ; 6) phenylalanine (F) , tyrosine (Y) , tryptophan (W) ; 7) serine (S) , threonine (T) ; and 8) cysteine (C) , methionine (M) . In some embodiments, the term "conservative sequence changes" is used to refer to amino acid modifications that do not significantly affect or alter the antigen-binding characteristics of interest of the antibody molecules of the present invention containing the amino acid sequence. For example, conservatively modified variants will retain at least 80%, 85%, 90%, 95%, 98%, 99%or more, e.g., 100-110%or more, binding affinity for the antigen of interest relative to the parent antibody.
The term "target" refers to the substance for binding against which the binding molecule is directed. The target may be an antigen, or may be a ligand or receptor. The term "antigen" refers to a molecule that elicits an immune response. Such an immune response may involve antibody production or activation of specific immune cells, or both. The skilled artisan will appreciate that any macromolecule, including substantially all proteins or peptides, may be used as an antigen. Furthermore, the antigen may be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to the portion of an antigen that specifically interacts with an antibody molecule. Where the binding molecules of the invention are directed to target binding regions derived from antibodies, "target" and "antigen" may be used interchangeably.
The term "antigen-binding region" as used herein refers to any portion of an antibody or antigen-binding fragment thereof, e.g., a multispecific antibody or bispecific antibody, that binds a particular target or antigen. The antigen-binding region may be, for example, an antibody or immunoglobulin per se or an antibody fragment. Such antigen-binding regions may or may not have tertiary structure independent of the remainder of the multispecific or bispecific antibody, and may or may not bind to their antigen/epitope as separate entities.
As used herein, the term "multispecific" antibody refers to an antibody having at least two antigen-binding regions, each of which binds to a different epitope of the same antigen or to a different epitope of different antigens. Multispecific antibodies are antibodies that have binding specificities for at least two different antigens or epitopes. In one embodiment, provided herein are bispecific antibodies having binding specificity for a first antigen and a second antigen.
Herein, the term "bispecific antibody" comprises antigen binding domains that specifically bind to two antigens or two epitopes. Unless otherwise indicated, the order of antigen binding by the bispecific antibody in the listed bispecific antibody names is arbitrary. That is, in some embodiments, the terms "anti-TROP2/B7-H3 bispecific antibody" and "anti-B7-H3/TROP2 bispecific antibody" are used interchangeably. In some embodiments, the bispecific antibody comprises two half-antibodies, wherein each half antibody comprises a single heavy chain variable region and optionally at least a portion of a heavy chain constant region and a single light chain variable region and optionally at least a portion of a light chain constant region. In some embodiments, the bispecific antibody comprises two half-antibodies, wherein each half-antibody comprises a single heavy chain variable region and a single light chain variable region and does not comprise more than one single heavy chain variable region and does not comprise more than one single light chain variable region. In some embodiments, the bispecific antibody comprises two half-antibodies, wherein each half-antibody comprises a single heavy chain variable region and a single light chain variable region, and wherein the first half-antibody binds to a first antigen/epitope and does not bind to a second antigen and the second half-antibody binds to a second antigen/epitope and does not bind to the first antigen.
When referring to a "first antigen-binding region" in a multispecific antibody or bispecific antibody, it is meant a binding region that binds to a first antigen, and is not intended to limit the number of such antigen-binding regions contained in the antibody, e.g., one or more than one first antigen-binding region may be included in a multispecific antibody or bispecific antibody. For example, a bispecific antibody comprises a first antigen-binding region and a second antigen-binding region, but may comprise one or more than one first antigen-binding region and one or more than one second antigen-binding region.
When referring to a "target-or antigen-binding region originated from an antibody" , it is meant that the binding domain constituting the target/antigen-binding region is or is derived from a binding domain of the antibody that specifically binds antigen, e.g. a fragment of the antigen-binding region that specifically binds antigen, e.g. Fab, is or is derived from a corresponding fragment of the antibody, e.g. Fab, or the heavy chain variable region and/or the light chain variable region of the antigen-binding region is or is derived from the heavy chain variable region and/or the light chain variable region of the antibody, or 1, 2, 3, 4, 5 or 6 CDRs of the antigen-binding region are CDRs of the antibody.
The term "derived from" means that the fragment in the antigen-binding region is substantially identical to the fragment of the antibody from which it is derived, but has a mutation, such as a substitution, deletion or addition, at one or more sites. In a specific embodiment, the mutation is not in a CDR of the antibody.
The multispecific or bispecific antibodies of the present invention may comprise a connector. The term "connector" as used herein refers to any molecule that enables direct attachment of different moieties of a multispecific antibody. Examples of connectors to establish covalent linkages between different moieties of the multispecific antibody include peptide connectors and non-proteinaceous polymers, including but not limited to polyethylene glycol (PEG) , polypropylene glycol, polyalkylene oxide, or copolymers of polyethylene glycol or polypropylene glycol. In some embodiments, the term "peptide connector" according to the invention refers to a sequence of amino acids, wherein said sequence links together the amino acid sequences of the various moieties of the multispecific antibody. Preferably, the peptide connector has a length sufficient to link the two entities in such a way that they maintain their conformation relative to each other so as not to interfere with the desired activity. The peptide connector may or may not comprise predominantly the following amino acid residues: Gly, Ser, Ala or Thr.
The term "effector functions" refers to those biological activities attributed to the Fc region of an immunoglobulin that vary with the isotype of the immunoglobulin. Examples of immunoglobulin effector functions include: C1q binding and Complement Dependent Cytotoxicity (CDC) , Fc receptor binding, antibody dependent cell mediated cytotoxicity (ADCC) , Antibody Dependent Cellular Phagocytosis (ADCP) , cytokine secretion, immune complex mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g., B cell receptors) , and B cell activation.
The term "…valent" antibody refers to the number of antigen binding sites present in an antibody molecule. "bivalent, " "trivalent, " and "tetravalent" antibodies refer to the presence of 2, 3, and 4 antigen binding sites, respectively, in an antibody molecule.
A "knob-into-hole (KIH) " mutation or "KIH" mutation is used herein to refer to the introduction of mutations in the first Fc-polypeptide and the second Fc-polypeptide, respectively, using the "KIH" technique to form a bulge ( "knob" ) and a complementary cavity ( "hole" ) at the interface of the first Fc-polypeptide and at the interface of the second Fc-polypeptide. It is known in the art that "knob-into-hole" techniques can engineer the interface between different chains of an antibody molecule to facilitate proper conjugation of the individual chains of the antibody molecule. Generally, this technique involves introducing a "bulge/knob" at the interface of one strand and a corresponding "cavity/hole" at the interface of the other strand to be paired with, so that the bulge can be placed in the cavity. One preferred interface comprises the CH3 domain of the heavy chain constant domain of one chain and the CH3 domain of the heavy chain constant domain of the other chain to be paired with. The bulge may be constructed by replacing the small amino acid side chain from the interface of the CH3 domain of the heavy chain constant domain of one chain with a larger side chain (e.g., tyrosine or tryptophan) . Compensatory cavities of the same or similar size to the buldge are constructed at the interface of the CH3 domains of the heavy chain constant domains of the other chain to be paired by replacing large amino acid side chains with smaller side chains (e.g., alanine or threonine) . Another alternative interface is the CL domain of the Fab fragment comprising the light chain and the CH1 domain of the heavy chain described above, which promotes the correct heterodimerization between the two chains of the Fab fragment by constructing a bulge-cavity interaction. In some embodiments, KIH mutations may further comprises cysteine mutations in the two CH3 regions, so as to form non-natural disulfide bond.
As used herein, the term "binding" or "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding site to bind to a particular antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art such as by Radioimmunoassay (RIA) or Bio-Layer Interferometry (BLI) or MSD assay or Surface Plasmon Resonance (SPR) .
"Affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can be generally represented by the dissociation constant (KD) , which is the ratio of the dissociation and association rate constants (Kdis and Kon, respectively) . Affinity can be measured by common methods known in the art. One particular method for measuring affinity is the ForteBio kinetic binding assay herein.
The calculation of sequence identity between sequences is performed as follows. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., for optimal alignment, gaps can be introduced in the first and second amino acid sequences or in one or both of nucleic acid sequences, or non-homologous sequences can be discarded for comparison purposes) . In one preferred embodiment, for comparison purposes, the length of the aligned reference sequence is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100%of the length of the reference sequence. Amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at this position.
As used herein, "immunoconjugate" is meant that the payload is attached to the antibody or antigen-binding fragment thereof by a linker so that the antibody or antigen-binding fragment thereof can act as a carrier to transport the payload to a target site in a targeted manner. The term "payload" refers to an active moiety conjugated to an antibody or antibody fragment of the invention, and may include any moiety used to attach an antibody or antibody fragment. In some embodiments, the payload can be a drug, such as a small molecule drug, a radionuclide, DNA, RNA, an enzyme, or a polypeptide, among others. In some embodiments, the immunoconjugate encompasses an Antibody Drug Conjugate (ADC) , an antibody immunostimulatory conjugate drug (ISAC) , an Antibody Oligonucleotide Conjugate (AOC) , an antibody polypeptide conjugate drug (APC) , an antibody nuclide conjugate drug (RDC) , or an antibody degrading conjugate drug (ADeC) , among others. Suitable payloads or active moieties for conjugation to the antibody include, for example, cytotoxic agents, chemotherapeutic agents, innate immune agonists (e.g., Toll-like receptor agonists (TLR) class ISAC drugs SBT6050, SBT6290, BDC-1001; STING agonist ISAC drugs XMT-2056, Treg cell regulatory ISAC drugs ADCT-301, etc. ) , immune modulators, therapeutic oligonucleotides (siRNA, PMO, etc. ) , or radionuclides, etc. In some embodiments, the immunoconjugate of the invention is an antibody drug conjugate, i.e., ADC.
As used herein, "antibody-drug conjugate (ADC) " refers to a compound/molecule obtained by linking an antibody to a drug (e.g. asmall molecule drug) via a linker.
The term "linker" refers to a structural fragment that links a drug (e.g., a small molecule drug) to an antibody moiety. It is understood that the linker, prior to attachment to the antibody or antigen-binding fragment thereof, has a functional group that can form a bond or linkage with a functional group of the antibody or antigen-binding fragment thereof.
The term "linker-payload" refers to a compound formed by linking a payload, such as a drug (e.g., a small molecule drug) , to a linker.
As used herein, the term "site-specific conjugation" refers to conjugating a drug specifically to a specific site of an antibody via a linker.
As used herein, the term "alkyl" refers to a fully saturated branched or unbranched hydrocarbon group. The alkyl group preferably contains 1 to 16 carbon atoms, for example 1 to 12 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms or 1 to 4 carbon atoms. Representative examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2-dimethylpentyl, 2, 3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like.
The term "alkylene" refers to an alkyl group as defined above, but which is divalent, i.e., has two single bonds linked to two other groups. Non-limiting examples of alkylene groups include -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH (-CH2CH3) -or -CH2CH (-CH3) -.
The term "alkenyl" refers to a straight or branched chain hydrocarbon group containing 2 to 16 carbon atoms and containing at least one double bond and no triple bond. The alkenyl group preferably contains 2 to 12 carbon atoms, 2 to 10 carbon atoms, 2 to 8 carbon atoms, 2 to 6 carbon atoms, or 2 to 4 carbon atoms. Representative examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, and the like.
The term "alkynyl" refers to a straight or branched hydrocarbon group containing 2 to 16 carbon atoms and comprising at least one triple bond. Alkynyl groups preferably contain 2-12 carbon atoms, 2-10 carbon atoms, 2-8 carbon atoms, 2-6 carbon atoms, or 2-4 carbon atoms. Representative examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like.
The term "halogen" or "halo" refers to fluoro (-F) , chloro (-Cl) , bromo (-Br) , and iodo (-I) .
The term "haloalkyl" refers to an alkyl group, as defined herein, substituted with one or more halo groups, as defined herein. The haloalkyl group may preferably be a monohaloalkyl group, a dihaloalkyl group or a polyhaloalkyl group (including a perhaloalkyl group) . The monohaloalkyl group may contain one iodo, bromo, chloro or fluoro in the alkyl group. The dihaloalkyl and polyhaloalkyl groups may contain two or more of the same halogen atoms in the alkyl group or a combination of different halo groups. Preferably, the polyhaloalkyl contains up to 12, 10 or 8 or 6 or 4 or 3 or 2 halo groups. Non-limiting examples of haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, and dichloropropyl. Perhaloalkyl refers to alkyl groups in which all hydrogen atoms are replaced by halogen atoms.
The term "haloalkenyl" refers to an alkenyl group, as defined herein, substituted with one or more halo groups as defined herein. The term "haloalkynyl" refers to an alkynyl group, as defined herein, which is substituted with one or more halo groups as defined herein. The meaning of "halo" as defined for "haloalkyl" is applicable to "haloalkenyl" and "haloalkynyl" .
The term "polyol group" refers to an alkyl group as defined above containing multiple (e.g., 2 to 10, e.g., 3, 4, 5, 6, 7 or 8) hydroxyl groups, optionally containing one or more (e.g., 2, 3 or 4) other groups (e.g., amino, carbonyl) . Non-limiting examples of "polyol groups" include, for example, 
The term "amino acid" refers to both naturally occurring and synthetic amino acids. The amino acids may be either L or D isomers. The conventional amino acids referred to herein are written following a conventional approach. See, for example, Immunology-ASynthesis (2nd Edition, E. S. Golub and D. R. Gren, eds., Sinauer Associates, Sunderland, Mass. (1991) ) which is incorporated herein by reference. And in the present disclosure, amino acids are generally represented by the single and three letter abbreviations commonly known in the art. For example, glycine may be represented by Gly, alanine by Ala, valine by Val, glutamine by Gln, glutamic acid by Glu, phenylalanine by Phe and leucine by Leu.
The term "optional" or "optionally" means that the subsequently described event or condition occurs or does not occur, and that the description includes instances where said event or condition occurs and instances where it does not. For example, when a group or structure is "optionally substituted" , the group or structure may or may not be substituted.
The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the ADC conjugates of the invention, and which is not biologically or otherwise undesirable. The ADC conjugates of the invention may exist in the form of their pharmaceutically acceptable salt, including acid addition salts and base addition salts. In the present invention, pharmaceutically acceptable non-toxic acid addition salts refer to salts formed by the ADC conjugates of the present invention with organic or inorganic acids including, but not limited to, hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, malic acid, and the like. Pharmaceutically acceptable non-toxic base addition salts mean salts formed by the ADC conjugates of the invention with organic or inorganic bases, including but not limited to alkali metal salts, such as lithium, sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; organic base salts, for example ammonium salts, formed with organic bases containing N groups.
The term "solvate" refers to an associated complex of one or more solvent molecules with an ADC antibody-drug conjugate of the invention. Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N-dimethylformamide, dimethylsulfoxide, and the like.
"Pharmaceutically acceptable" and "pharmaceutically useful" are used interchangeably herein, unless it conflicts with the context,
The term "drug to antibody ratio" or "DAR" refers to the ratio of drug moiety (D) conjugated to the Ab moiety as described herein to the Ab moiety in an ADC molecule. In some embodiments described herein, the DAR may be determined by p in formula I. For example, DAR may be from 1 to 16, e.g. 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. DAR may also be calculated as the average DAR of a population of molecules in a product, i.e., the overall ratio of drug moiety (D) conjugated to the Ab moiety described herein to the Ab moiety as measured by detection methods (e.g., by conventional methods such as mass spectrometry, ELISA assays, electrophoresis, and/or HPLC) in a product, such DAR being referred to herein as average DAR. In some embodiments, the average DAR value of a conjugate of the invention is 1 to 16, e.g., 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, e.g., 1.0-8.0, 2.0-6.0, e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or10.0, and the ranges with two of these values as endpoints. It should be understood that, in the case that an average DAR value is referred to, the ADCs according to the present invention refer to a population of ADC molecules or a mixture of ADC molecules comprising ADC molecules with the same and/or different DARs.
The term "therapeutic agent" as described herein comprises any substance effective in preventing or treating tumors (such as cancer) , including a chemotherapeutic agent, a cytokine, angiogenesis inhibitor, a cytotoxic agent, other antibodies, a small molecule drug or an immunomodulatory agent (such as an immunosuppressant) .
The term "cytotoxic agent" used in the invention refers to a substance that inhibits or prevents the cell function and/or causes cell death or destruction.
"Chemotherapeutic agents" include chemical compounds useful in treatment of cancer or immune system disease.
The term "drug" refers to an organic compound capable of modulating a biological process, in particular altering or preventing a pathological process.
The term "prodrug" refers to a chemically modified active or inactive compound that, upon administration to a subject, undergoes a physiological effect in vivo (e.g., hydrolysis, anabolism, etc. ) to become the active drug. Techniques for making and using prodrugs are well known to those skilled in the art.
"Angiogenesis inhibitor" refers to a compound that blocks or interferes to some extent with vascular development. The angiogenesis inhibitor may be, for example, a small molecule or an antibody that binds to a growth factor or a growth factor receptor involved in promoting angiogenesis.
The term "small molecule drugs" refers to organic compounds with low molecular weight that can regulate biological processes, especially change or prevent pathological processes. "Small molecule" is defined as a molecule with molecular weight less than 10kD, generally less than 2kD and preferably less than 1kD, more preferably less than 500D. Small molecules include but are not limited to inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimics and antibody mimics. As a therapeutic agent, small molecules can penetrate cells more easily than large molecules, and are less susceptible to degradation and less prone to trigger immune response.
The term "immunomodulators" as used herein refer to natural or synthetic active agents or drugs that inhibit or regulate (e.g., activate) immune response. The immune response can be humoral or cellular. Immunomodulators include immunosuppressants or immunoagonist. In some embodiments, immunemodulators of the invention include immune checkpoint inhibitors or immune checkpoint agonists.
"Anti-tumor compounds" are pharmaceutically active compounds that have an effect on tumors, including but not limited to cytotoxic or chemotherapeutic agents, such as those disclosed in WO 2021/173773, e.g., camptothecins, e.g., exatecan (topoisomerase I inhibitor, Exatecan) , Dxd (a novel topoisomerase I inhibitor, Exatecan derivative) , auristatins, e.g., monomethyl auristatin E (MMAE) , maytansinoids, e.g., small molecule microtubule inhibitor, DM1, taxanes, e.g., paclitaxel or docetaxel, anthracyclines, epothilones, mitomycins, combretastatin, vinca alkaloids, calicheamicins, duocarmycin, Tubulysins, amatoxins, bleomycin, MEK inhibitors, KSP inhibitors, and the like. It is understood that the anti-tumor compounds may be substituted with isotopes including, but not limited to, deuterium, tritium, and the like. For example, following substitution with deuterium, the carbon-deuterium bonds replace carbon-hydrogen bonds, since the former are more stable than the latter, the replacement can directly affect the certain properties of drugs such as absorption, distribution, metabolism, excretion, etc. thereby improving the efficacy, safety, and tolerability of the drugs. Thus, the "anti-tumor compound" of the present application may encompass compounds substituted with deuterium.
"Substituted with deuterium" is meant that a hydrogen in the molecule is replaced with deuterium, for example one or more hydrogens, for example 1 to 10 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) hydrogens are replaced with deuterium.
The term "effective amount" refers to the amount or dose of the antibody, ADC molecule or composition or combination of the invention, which will produce the expected effect in patients needing such treatment or prevention after being administered to patients in a single or multiple doses. Depending on the intended effect, "therapeutically effective amount" and "preventively effective amount" may be included.
"Therapeutically effective amount" refers to an amount that can effectively achieve the desired results at the required dose and for the required period of time. The therapeutically effective amount is also such an amount, where any toxic or harmful effect of the antibody, ADC or composition or combination is less than the therapeutic beneficial effect. "Therapeutically effective amount" preferably inhibits measurable parameters (such as tumor volume) by at least about 20%, more preferably by at least about 30%, or even more preferably by at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%or even 100%compared with untreated objects.
"Preventively effective amount" refers to an amount that can effectively achieve the desired prevention results at the required dose and for the required period of time. Generally, since the preventive dose is used before or at an earlier stage of the disease in the objects, the preventively effective amount will be less than the therapeutically effective amount.
The terms "host cell" , "host cell line" and "host cell culture" are used interchangeably and refer to the cells in which foreign nucleic acids are introduced, including the descendants of such cells. Host cells include "transformants" and "transformed cells" , which include primary transformed cells and offspring derived from them, regardless of the number of passages. The nucleic acid content of the descendants may not be exactly the same as that of the parent cell, but may contain mutations. The mutant descendants with the same function or biological activity screened or selected from the initially transformed cells are included herein.
The term "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and facilitates the detection of the conjugated or fused reagent. The label itself can be detectable (for example, radioisotope label or fluorescent label) or can catalyze the chemical changes of detectable substrate compounds or compositions in the case of enzymatic labeling. The term is intended to cover the direct labeling of probes or antibodies by coupling (i.e., physically connecting) detectable substances to probes or antibodies and the indirect labeling of probes or antibodies by reacting with another directly labeled reagent.
"Individuals" or "subjects" include mammals. Mammals include, but are not limited to, domestic animals (such as cattle, sheep, cats, dogs and horses) , primates (such as human and non-human primates, such as monkeys) , rabbits, and rodents (such as mice and rats) . In some embodiments, the individuals or subjects are human.
"Isolated" antibodies or other molecules are antibodies or molecules that have been separated from components of their natural environment or the environment in which they are expressed. In some embodiments, the antibody is purified to more than 95%or 99%purity, such as as determined by electrophoresis (for example, SDS-PAGE, isoelectric focusing (IEF) , capillary electrophoresis) or chromatography (for example, ion exchange or reverse phase HPLC) .
An "isolated" nucleic acid is a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.
The term "anti-tumor effect" refers to a biological effect that can be exhibited by a variety of means, including, but not limited to, for example, a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.
The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. The cancer may be in an early, intermediate or advanced stage or metastatic cancer. Cancers suitable for treatment by the molecules of the invention include, but are not limited to, such as lung cancer (including non-small cell lung cancer and small cell lung cancer) , colon cancer, oral squamous cell carcinoma, breast cancer, melanoma, head and neck tumors, prostate cancer, esophageal cancer, cervical cancer, renal cancer, bladder cancer, ovarian cancer or pancreatic cancer, including metastatic forms of these cancers.
The term "tumor" refers to the growth and proliferation of all neoplastic cells, whether malignant or benign, as well as all pre-cancerous and cancerous cells and tissues. "Tumor" encompasses both solid and hematological tumors and metastatic lesions. The terms "cancer" , "cancerous" and "tumor" are not mutually exclusive when referred to herein.
"Tumor immune escape" refers to the process by which tumors escape immune recognition and clearance. As such, as a therapeutic concept, tumor immunity is "treated" when such escape diminishes, and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage and tumor clearance.
The term "pharmaceutically acceptable excipient" refers to diluents, adjuvants (e.g., freund's adjuvant (complete and incomplete) ) , vehicles, carriers, stabilizers or the like with which the active substance is administered.
The term "pharmaceutical composition" refers to a composition that is present in a form that allows for the biological activity of the active ingredients contained therein to be effective, and that does not contain additional ingredients that have unacceptable toxicity to the subject to which the composition is administered.
The term "pharmaceutical combination or combination product" refers to non-fixed combination products or fixed combination products, including but not limited to (pharmaceutical) kits and pharmaceutical compositions. The term "unfixed combination" means that the active ingredients (for example, (i) the antibody molecule or ADC of the present invention, and (ii) other therapeutic agents) are administered to patients simultaneously, without specific time limits or at the same or different time intervals, in sequence, in separate entities, where these two or more active agents are administered to provide effective levels of prevention or treatment in patients. In some embodiments, the antibody molecule or ADC and other therapeutic agents of the invention used in the pharmaceutical combination are administered at a level not exceeding the level when they are used alone. The term "fixed combination" means that two or more active agents are administered simultaneously to patients in the form of a single entity. It is preferred to select the dose and/or time interval of two or more active agents, so that the combined use of each component can produce greater effect than the single use of any one component in the treatment of disease or disorder. Each component can take its own form of formualtion, which can be the same or different.
The term "combination therapy" refers to the administration of two or more therapeutic agents or therapeutic modes (such as radiotherapy or surgery) to treat the diseases described herein. Such administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed proportion of active ingredients. Alternatively, such administration includes the joint administration of each active ingredient in multiple or separate containers (such as tablets, capsules, powders and liquids) . The powder and/or liquid can be reconstituted or diluted to the required dose before administration. In addition, this administration also includes the use of each type of therapeutic agent at approximately the same time or at different times in a sequential manner. In either case, the treatment strategy will provide the beneficial effect of pharmaceutical combination in treating the disease or condition described herein.
As used herein, "treatment" (or "treat" or "treating" ) refers to slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.
As used herein, "prevention" (or "prevent" or "preventing" ) includes the inhibition of the onset or progression of a disease or disorder or a symptom of a particular disease or disorder. In some embodiments, subjects with family history of cancer are candidates for preventive regimens. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug prior to the onset of signs or symptoms of a cancer, particularly in subjects at risk of cancer.
The term "vector" as used herein refers to a nucleic acid molecule capable of proliferating another nucleic acid to which it is linked. The term includes vectors that serve as self-replicating nucleic acid structures as well as vectors binding to the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are called "expression vectors" herein.
"Subject/patient/individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell samples can be solid tissues, e.g., from fresh, frozen and/or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood component; body fluids such as cerebrospinal fluids, amniotic fluids, peritoneal fluids (ascites) , or interstitial fluids; cells from a subject at any time during pregnancy or development. Tissue samples may comprise compounds which are naturally not mixed with tissues, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.
Examples of tumor samples herein include, but are not limited to, tumor biopsies, fine needle aspirates, bronchial lavage, pleural fluid, sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor samples or frozen tumor samples.
II. Bispecific antibodies
In one aspect, the present invention provides a multispecific antibody that specifically binds to TROP2 and B7-H3.
In some embodiments, the multispecific antibodies of the present invention comprise a first binding specificity to TROP2 and a second binding specificity to B7-H3, and optionally other binding specificities.
In some embodiments, the multispecific antibody is a bispecific antibody.
Accordingly, the present invention relates to a bispecific antibody that specifically binds to TROP2 and B7-H3.
Accordingly, one aspect of the present invention relates to a multispecific antibody comprising
a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region specifically binds to TROP2, and/or the second antigen-binding region specifically binds to B7-H3, and optionally other antigen-binding region (s) .
Accordingly, one aspect of the present invention relates to a bispecific antibody comprising
a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region specifically binds to TROP2 and/or the second antigen-binding region specifically binds to B7-H3.
Bispecific antibodies of the invention can be prepared using bispecific antibody formats or techniques known in the art. Specific exemplary bispecific formats that may be used in the context of the present invention are described, for example, in Labrijn, et al., Bispecific antibodies: a mechanical review of the pipeline. Nature Reviews Drug Discovery, 2019, 18 (8) : 1-24. In an embodiment, the bispecific antibody format includes an IgG-like antibody (Fan et al (2015) Journal of Hematology &Oncology. 8: 130) . The most common type of IgG-like antibodies comprises two Fab regions and two Fc regions, the heavy and light chains of each Fab may be derived from a separate monoclonal antibody. In some embodiments, the bispecific antibody of the invention is an IgG-like bispecific antibody comprising as one antigen-binding region a Fab fragment that specifically binds to TROP2 and as the other antigen-binding region a Fab fragment that specifically binds to B7-H3.
The following provides a detailed description of the components of the multispecific antibodies, such as bispecific antibodies, of the present invention. Those skilled in the art will understand that, unless the context clearly indicates otherwise, any combination of any technical features of these components is within the scope of the present invention. Moreover, those skilled in the art will understand that, unless the context clearly indicates otherwise, the antibodies of the present invention (including antibodies in any form) can comprise any such combination.
II-1. An antigen-binding region that specifically binds to TROP2
In some embodiments, the first antigen-binding region applicable for the anti-TROP2/B7-H3 bispecific antibodies of the present invention may comprise or consist of an anti-TROP2 antibody or antigen binding fragment thereof (e.g. an TROP2 antibody disclosed in US9849176B2 (which is incorporated herein as a whole) , e.g. Sacituzumab hRS7) , as long as it is capable of specifically binding to TROP2, including, but not limited to, e.g. a full length antibody, a half antibody, a Fab, a Fab', a Fab'-SH, a Fv, a single chain antibody (e.g. scFv) , a (Fab') 2, a single domain antibody such as VHH, dAb (domain antibody) , a heavy chain antibody, or a linear antibody that specifically binds to TROP2, and the like.
In some embodiments, the antigen-binding region that specifically binds to TROP2 is originated from an antibody that specifically binds to TROP2, e.g., an TROP2 antibody disclosed in US9849176B2, e.g., Sacituzumab hRS7.
In some embodiments, the antigen-binding region that specifically binds to TROP2 comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds to TROP2, e.g., an TROP2 antibody disclosed in US9849176B2, e.g., Sacituzumab hRS7.
In some embodiments, the antigen-binding region that specifically binds to TROP2 comprises 1, 2, or 3 heavy chain variable region CDRs, i.e., HCDR1, HCDR2, and HCDR3, of a known antibody that specifically binds to TROP2, e.g., an TROP2 antibody disclosed in US9849176B2, e.g., Sacituzumab hRS7.
In some embodiments, the antigen-binding region that specifically binds to TROP2 comprises 1, 2, or 3 light chain variable region CDRs, i.e., LCDR1, LCDR2, and LCDR3, of a known antibody that specifically binds to TROP2, e.g., an TROP2 antibody disclosed in US9849176B2, e.g., Sacituzumab hRS7.
In some embodiments, the antigen-binding region that specifically binds to TROP2 comprises 3 heavy chain variable region CDRs and 3 light chain variable region CDRs of a known antibody that specifically binds to TROP2, e.g., an TROP2 antibody disclosed in US9849176B2, e.g., Sacituzumab hRS7.
In some embodiments, the antigen-binding region that specifically binds to TROP2 comprises the heavy chain variable region and/or the light chain variable region of a known antibody that specifically binds to TROP2, such as an TROP2 antibody disclosed in US9849176B2, e.g., Sacituzumab hRS7.
In some embodiments, the antigen-binding region that specifically binds to TROP2 comprises a Fab of a known antibody that specifically binds to TROP2 such as an TROP2 antibody disclosed in US9849176B2, e.g., Sacituzumab hRS7.
In some embodiments, the antigen-binding region that specifically binds to TROP2 comprises 3 Complementarity Determining Regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the antigen-binding region that specifically binds to TROP2 comprises 3 Complementarity Determining Regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3. In some embodiments, the antigen-binding region that specifically binds to TROP2 comprises 3 Complementarity Determining Regions (HCDRs) from the heavy chain variable region and 3 Complementarity Determining Regions (LCDRs) from the light chain variable region.
In some aspects, the antigen-binding region that specifically binds to TROP2 comprises a heavy chain variable region (VH) . In some aspects, the antigen-binding region that specifically binds to TROP2 comprises a light chain variable region (VL) . In some aspects, the antigen-binding region that specifically binds to TROP2 comprises a heavy chain variable region (VH) and a light chain variable region (VL) . In some embodiments, the heavy chain variable region comprises 3 Complementarity Determining Regions (CDRs) from a heavy chain variable region, HCDR1, HCDR2 and HCDR3. In some embodiments, the light chain variable region comprises 3 Complementarity Determining Regions (CDRs) from a light chain variable region, LCDR1, LCDR2 and LCDR3.
The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, the VH, and/or the VL, comprised by the antigen-binding region that specifically binds to TROP2 of the present invention are respectively defined herein.
In some embodiments, the heavy chain variable region of the antigen-binding region that specifically binds to TROP2 described in the present invention
(i) comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 16, or
(ii) comprises or consists of the amino acid sequence of SEQ ID NO: 16; or
(iii) comprises or consists of an amino acid sequence having one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 16, preferably said amino acid changes do not occur in the CDR regions.
In some embodiments, the light chain variable region of the antigen-binding region that specifically binds to TROP2 described in the present invention
(i) comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 22, or
(ii) comprises or consists of the amino acid sequence of SEQ ID NO: 22; or
(iii) comprises or consists of an amino acid sequence having one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 22, preferably said amino acid changes do not occur in the CDR regions.
In some embodiments, the 3 complementarity determining regions (HCDRs) from the heavy chain variable region of the antigen-binding region that specifically binds to TROP2 described in the present invention, HCDR1, HCDR2 and HCDR3 are selected from
(i) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH set forth in SEQ ID NO: 16, or
(ii) sequences which contain at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably amino acid substitution, preferably conservative substitution) in total on the three HCDR regions relative to the sequence of (i) ,
wherein the HCDRs can be determined according to any scheme for determining CDRs, such as determined respectively by the Kabat, AbM, Chothia, Contact, or IMGT schemes or combination thereof;
for example, the HCDR1 is determined by AbM scheme, the HCDR2 and HCDR3 are determined by the Kabat scheme respectively.
In some embodiments, the 3 complementarity determining regions (LCDRs) from the light chain variable region of the antigen-binding region that specifically binds to TROP2 described in the present invention, LCDR1, LCDR2, and LCDR3 are selected from
(i) the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL set forth in SEQ ID NO: 22, or
(ii) sequences which contain at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably amino acid substitution, preferably conservative substitution) in total on the three LCDR regions relative to the sequence of (i) ,
wherein the LCDRs can be determined according to any scheme for determining CDRs, such as determined respectively by the Kabat, AbM, Chothia, Contact, or IMGT schemes or combination thereof;
for example, the LCDRs1-3 are determined by the Kabat scheme respectively.
In some embodiments, the antigen-binding region that specifically binds to TROP2 of the present invention comprises 3 complementarity determining regions (HCDRs) comprised by the heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 16 and 3 complementarity determining regions (LCDRs) comprised by the light chain variable region consisting of the amino acid sequence of SEQ ID NO: 22;
wherein the HCDRs and LCDRs can be determined according to any scheme for determining CDRs, such as determined respectively by the Kabat, AbM, Chothia, Contact, or IMGT schemes or combination thereof;
for example, the HCDR1 is determined by AbM scheme, the HCDR2, HCDR3 and LCDRs1-3 are determined by the Kabat scheme respectively.
In some embodiments, the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 13 or the HCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 13.
In some embodiments, the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 14 or the HCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 14.
In some embodiments, the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 15 or the HCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 15.
In some embodiments, the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 19 or the LCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 19.
In some embodiments, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 20 or the LCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 20.
In some embodiments, the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 21 or the LCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 21.
In some particular embodiments of the invention, the antigen-binding region that specifically binds to TROP2 described in the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and/or LCDR3 as described above.
In some particular embodiments of the invention, the antigen-binding region that specifically binds to TROP2 described in the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as described above.
In some particular embodiments of the invention, the antigen-binding region that specifically binds to TROP2 described in the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 13; the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 14; the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 15; the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 19; the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 20; and the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 21.
In some particular embodiments of the invention, the antigen-binding region that specifically binds to TROP2 as described in the present invention comprises HCDR1 set forth in SEQ ID NO: 13, HCDR2 set forth in SEQ ID NO: 14, HCDR3 set forth in SEQ ID NO: 15; LCDR1 set forth in SEQ ID NO: 19, LCDR2 set forth in SEQ ID NO: 20 and LCDR3 set forth in SEQ ID NO: 21.
In some particular embodiments of the invention, the antigen-binding region that specifically binds to TROP2 as described in the present invention comprises a VH and a VL, wherein
the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity thereto, and the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity thereto.
In some particular embodiments of the invention, the antigen-binding region that specifically binds to TROP2 as described in the present invention comprises a VH and a VL, wherein the VH and VL comprise or consist of, respectively, the amino acid sequences shown below: SEQ ID NO: 16 and 22, respectively.
In some particular embodiments of the invention, the antigen-binding region that specifically binds to TROP2 as described in the present invention is an anti-TROP2 Fab.
II-2. An antigen-binding region that specifically binds to B7-H3
In some embodiments, the second antigen-binding region applicable for the anti-TROP2/B7-H3 bispecific antibodies of the present invention may comprise or consist of an anti-B7-H3 antibody or antigen binding fragment thereof, as long as it is capable of specifically binding to B7-H3, including, but not limited to, e.g. a full length antibody, a half antibody, a Fab, a Fab', a Fab'-SH, a Fv, a single chain antibody (e.g. scFv) , a (Fab') 2, a single domain antibody such as VHH, dAb (domain antibody) , a heavy chain antibody, or a linear antibody that specifically binds to B7-H3, and the like.
In some embodiments, the antigen-binding region that specifically binds to B7-H3 comprises 3 Complementarity Determining Regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the antigen-binding region that specifically binds to B7-H3 comprises 3 Complementarity Determining Regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3. In some embodiments, the antigen-binding region that specifically binds to B7-H3 comprises 3 Complementarity Determining Regions (HCDRs) from the heavy chain variable region and 3 Complementarity Determining Regions (LCDRs) from the light chain variable region.
In some aspects, the antigen-binding region that specifically binds to B7-H3 comprises a heavy chain variable region (VH) . In some aspects, the antigen-binding region that specifically binds to B7-H3 comprises a light chain variable region (VL) . In some aspects, the antigen-binding region that specifically binds to B7-H3 comprises a heavy chain variable region (VH) and a light chain variable region (VL) . In some embodiments, the heavy chain variable region comprises 3 Complementarity Determining Regions (CDRs) from a heavy chain variable region, HCDR1, HCDR2 and HCDR3. In some embodiments, the light chain variable region comprises 3 Complementarity Determining Regions (CDRs) from a light chain variable region, LCDR1, LCDR2 and LCDR3.
The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, the VH, and/or the VL, comprised by the antigen-binding region that specifically binds to B7-H3 of the present invention are respectively defined herein.
In some embodiments, the heavy chain variable region of the antigen-binding region that specifically binds to B7-H3 described in the present invention
(i) comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 4, or
(ii) comprises or consists of the amino acid sequence of SEQ ID NO: 4; or
(iii) comprises or consists of an amino acid sequence having one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 4, preferably said amino acid changes do not occur in the CDR regions.
In some embodiments, the light chain variable region of the antigen-binding region that specifically binds to B7-H3 described in the present invention
(i) comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 10, or
(ii) comprises or consists of the amino acid sequence of SEQ ID NO: 10; or
(iii) comprises or consists of an amino acid sequence having one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 10, preferably said amino acid changes do not occur in the CDR regions.
In some embodiments, the 3 complementarity determining regions (HCDRs) from the heavy chain variable region of the antigen-binding region that specifically binds to B7-H3 described in the present invention, HCDR1, HCDR2 and HCDR3 are selected from
(i) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH set forth in SEQ ID NO: 4, or
(ii) sequences which contain at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably amino acid substitution, preferably conservative substitution) in total on the three HCDR regions relative to the sequence of (i) ,
wherein the HCDRs can be determined according to any scheme for determining CDRs, such as determined respectively by the Kabat, AbM, Chothia, Contact, or IMGT schemes or combination thereof;
for example, the HCDR1 is determined by AbM scheme, the HCDR2 and HCDR3 are determined by the Kabat scheme respectively.
In some embodiments, the 3 complementarity determining regions (LCDRs) from the light chain variable region of the antigen-binding region that specifically binds to B7-H3 described in the present invention, LCDR1, LCDR2, and LCDR3 are selected from
(i) the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL set forth in SEQ ID NO: 10, or
(ii) sequences which contain at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably amino acid substitution, preferably conservative substitution) in total on the three LCDR regions relative to the sequence of (i) ,
wherein the LCDRs can be determined according to any scheme for determining CDRs, such as determined respectively by the Kabat, AbM, Chothia, Contact, or IMGT schemes or combination thereof;
for example, the LCDRs1-3 are determined by the Kabat scheme respectively.
In some embodiments, the antigen-binding region that specifically binds to B7-H3 of the present invention comprises 3 complementarity determining regions (HCDRs) comprised by the heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 4 and 3 complementarity determining regions (LCDRs) comprised by the light chain variable region consisting of the amino acid sequence of SEQ ID NO: 10;
wherein the HCDRs and LCDRs can be determined according to any scheme for determining CDRs, such as determined respectively by the Kabat, AbM, Chothia, Contact, or IMGT schemes or combination thereof;
for example, the HCDR1 is determined by AbM scheme, the HCDR2, HCDR3 and LCDRs1-3 are determined by the Kabat scheme respectively.
In some embodiments, the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1 or the HCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 1.
In some embodiments, the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2 or the HCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 2.
In some embodiments, the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3 or the HCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 3.
In some embodiments, the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 7 or the LCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 7.
In some embodiments, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 8 or the LCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 8.
In some embodiments, the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 9 or the LCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 9.
In some particular embodiments of the invention, the antigen-binding region that specifically binds to B7-H3 described in the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and/or LCDR3 as described above.
In some particular embodiments of the invention, the antigen-binding region that specifically binds to B7-H3 described in the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as described above.
In some particular embodiments of the invention, the antigen-binding region that specifically binds to B7-H3 described in the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1; the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2; the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3; the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 7; the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 8; and the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 9.
In some particular embodiments of the invention, the antigen-binding region that specifically binds to B7-H3 as described in the present invention comprises HCDR1 set forth in SEQ ID NO: 1, HCDR2 set forth in SEQ ID NO: 2, HCDR3 set forth in SEQ ID NO: 3; LCDR1 set forth in SEQ ID NO: 7, LCDR2 set forth in SEQ ID NO: 8 and LCDR3 set forth in SEQ ID NO: 9.
In some particular embodiments of the invention, the antigen-binding region that specifically binds to B7-H3 as described in the present invention comprises a VH and a VL, wherein
the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 4, and the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 10.
In some particular embodiments of the invention, the antigen-binding region that specifically binds to B7-H3 as described in the present invention comprises a VH and a VL, wherein the VH and VL comprise or consist of, respectively, the amino acid sequences shown below: SEQ ID NO: 4 and 10, respectively.
In some particular embodiments of the invention, the antigen-binding region that specifically binds to B7-H3 as described in the present invention is an anti-B7-H3 Fab.
II-3 Fab fragment
In some embodiments, the first antigen-binding region and/or the second antigen-binding region of the invention is a Fab fragment. A Fab fragment suitable for use as an antigen-binding region of the multispecific antibody such as bispecific antibody as described herein consists of two polypeptide chains comprising the antibody VH, CH1, VL and CL domains, wherein the VH is paired with VL and the CH1 is paired with CL to form the antigen-binding region.
In some embodiments, in a Fab, one chain comprises or consists of VH and CH1 (i.e., VH-CH1) from N-terminus to C-terminus, and the other chain comprises or consists of VL and CL (i.e., VL-CL) from N-terminus to C-terminus. In some embodiments, in the multispecific or bispecific antibodies of the present invention, the Fab may be linked to the N-terminus of the Fc domain of the antibody via the C-terminus of the chain comprising the VH. Preferably, the Fab comprises a VH-CH1 chain and a VL-CL chain and may be linked to an antibody Fc domain via the C-terminus of CH1 of the VH-CH1 chain. In some embodiments, the linkage is a direct linkage, or through a connector. Herein, Fab chain comprising VH-CH1 is also referred to as a Fab heavy chain, while Fab chain comprising VL-CL is also referred to as a Fab light chain.
In some embodiments, the CH1 is a CH1 from IgG1, IgG2, IgG3, or IgG4, preferably CH1 from IgG1. In some embodiments, the CH1
(i) comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 32, or
(ii) comprises or consists of the amino acid sequence of SEQ ID NO: 32; or
(iii) comprises or consists of an amino acid sequence having one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 32.
In some embodiments, the CL is a Kappa light chain constant region or a Lambda light chain constant region. In some embodiments, CL is a Kappa light chain constant region. In some embodiments, the CL
(i) comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 11;
(ii) comprises or consists of the amino acid sequence of SEQ ID NO: 11; or
(iii) comprises or consists of an amino acid sequence having one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 11.
In some embodiments, the first antigen-binding region is a Fab that specifically binds to TROP2, wherein the Fab fragment is from an anti-TROP2 antibody, and comprises the heavy chain variable region VH and the light chain variable region VL of the anti-TROP2 antibody. In some embodiments, the Fab that specifically binds to TROP2 as the first antigen-binding region comprises the 6 CDRs of the antigen-binding regions that specifically bind to TROP2 described herein. In some embodiments, the Fab that specifically binds to TROP2 as the first antigen-binding region comprises a VH or a VL of the antigen-binding region that specifically binds to TROP2 as described herein, or comprises a VH and a VL of the antigen-binding region that specifically binds to TROP2 as described herein.
In some embodiments, the second antigen-binding region is a Fab that specifically binds to B7-H3, wherein the Fab fragment is from an anti-B7-H3 antibody, and comprises the heavy chain variable region VH and the light chain variable region VL of the anti-B7-H3 antibody. In some embodiments, the Fab that specifically binds to B7-H3 as the second antigen-binding region comprises the 6 CDRs of the antigen-binding regions that specifically bind B7-H3 as described herein. In some embodiments, the Fab that specifically binds B7-H3 as the second antigen-binding region comprises a VH or a VL of the antigen-binding region that specifically binds to B7-H3 as described herein, or comprises a VH and a VL of the antigen-binding region that specifically binds to B7-H3 as described herein.
II-4 Fc region
In some embodiments, the bispecific antibody of the present invention further comprises an Fc region, wherein the Fc regions comprised may be the same or different.
In some embodiments, the antibody molecular of the present invention comprises the first Fc region and the second Fc region, wherein the first Fc region and the second Fc region are the same or different.
In some embodiments, the first Fc region and the second Fc region are different and are capable of dimerizing to form a heterodimeric Fc scaffold.
Herein, the Fc region refers to the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region, and may include native sequence Fc regions and variant Fc regions. The native sequence Fc region encompasses the naturally occurring Fc sequences of various immunoglobulins, such as the Fc regions of various Ig subclass and allotypes thereof (Gestur Vidarsson et al, IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi: 10.3389/fimmu. 2014.00520) . In some embodiments, the Fc region of the present invention comprises the antibody CH2 and CH3. In some embodiments, the antibody Fc region may also have an IgG hinge region or a partial IgG hinge region at the N-terminus, e.g., an IgG1 hinge region or a partial IgG1 hinge region, e.g., according to EU numbering, the sequence of D221 to P230. A mutation may be comprised in the hinge region.
Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region is according to the EU numbering system, also known as the EU index, as described in Kabat, E. A. et al, Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991) , NIH Publication 91-3242.
In some embodiments, the Fc region is a human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, human IgG3 Fc or human IgG4 Fc. In one embodiment, the Fc region comprises or consists of the amino acid sequence SEQ ID NO: 30 or SEQ ID NO: 31, or an amino acid sequence having at least 90%identity, e.g., 95%, 96%, 97%, 98%, 99%or more identity to said amino acid sequence.
As understood by those skilled in the art, to facilitate formation of the multispecific antibodies of the present invention as heterodimers, the Fc region comprised by the multispecific antibodies of the present invention may comprise mutations that facilitate heterodimerization of a first Fc region with a second Fc region. In one embodiment, mutations are introduced in the CH3 regions of both Fc regions.
Methods for promoting heterodimerization of Fc regions are known in the art. For example, the CH3 region of the first Fc region and the CH3 region of the second Fc region are engineered in a complementary manner such that each CH3 region (or the heavy chain comprising it) can no longer homodimerize with itself but is forced to heterodimerize with other CH3 regions that are complementarily engineered (such that the CH3 regions of the first and the second Fc regions heterodimerize and no homodimers are formed between the two first CH3 regions or the two second CH3 regions) .
Preferably, based on the Knob-in-Hole technology, the respective Knob mutations and Hole mutations are introduced in the first Fc region and the second Fc region. See, e.g., US 5, 731, 168; US 7, 695, 936; Ridgway et al, Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001) for the technique. In some embodiments, the Knob mutations and Hole mutations may further comprise mutations to cysteine residue and thus to introduce disulfide bond. Disulfide bonds can be used to stabilize the final antibody product or the ADC of the antibody.
In a particular embodiment, in the CH3 region of an Fc region, the threonine residue at position 366 is replaced with a tryptophan residue (T366W) (knob mutation) ; in the CH3 region of the other Fc region, the tyrosine residue at position 407 was replaced with a valine residue (Y407V) (hole mutation) , optionally the threonine residue at position 366 was replaced with a serine residue (T366S) and the tyrosine residue at position 407 was replaced with a valine residue (Y407V) (numbering according to the EU index) .
In some embodiments, the Fc regions may further comprise cysteine residue substitution, so that to obtain non-natural disulfide bond linkage. In some embodiments, in one Fc region, the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (in particular, the serine residue at position 354 is replaced with a cysteine residue) , while in the other Fc region, the tyrosine residue at position 349 is replaced with a cysteine residue (Y349C) (numbering according to the EU index) . Accordingly, in yet another embodiment, in the CH3 region of one Fc region, the threonine residue at position 366 is replaced with a tryptophan residue (T366W) and the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (in particular, the serine residue at position 354 is replaced with a cysteine residue) ; whereas in the CH3 region of the other Fc region, the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (hole mutation) , optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the EU index) , optionally the tyrosine residue at position 349 is replaced with a cysteine residue (Y349C) (numbering according to the EU index) .
In a particular embodiment, one Fc region comprises the amino acid substitutions T366W and the other Fc region comprises the amino acid substitutions T366S, L368A and Y407V (numbering according to the EU index) .
In a particular embodiment, one Fc region comprises the amino acid substitutions S354C and T366W and the other Fc region comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to the EU index) .
Mutations may also be introduced in the first Fc region and the second Fc region based on the Innobody technique. See, e.g., PCT/CN2021/143141 for this technique, which is incorporated herein as its entirty.
In a particular embodiment,
the first CH3 region comprises the S364R/K mutation (preferably S364R) , and optionally one or more additional mutations. In some embodiments, the second CH3 region comprises a K370S/T/A/V mutation (preferably K370S) , and optionally one or more additional mutations. In some embodiments, the first CH3 region includes the S364R/K mutation, and the second CH3 region includes the K370S/T/A/V mutation. In some embodiments, the first CH3 region comprises the S364R mutation, and the second CH3 region comprises the K370S mutation.
In some embodiments, the first CH3 region comprises S364R/K (preferably S364R) and D399K/R (preferably D399K) mutations. In some embodiments, the second CH3 region comprises a K370S/T/A/V (preferably K370S) mutation and a K409D/E (preferably K409D) mutation. In some embodiments, the first CH3 region comprises S364R/K + D399K/R and the second CH3 region comprises K370S/T/A/V + Y349T/S/A/V. In some embodiments, the first CH3 region comprises S364R + D399K and the second CH3 region comprises K370S + Y349T. In some embodiments, the first CH3 region further comprises E375N/Q (preferably E375N) and/or T350V/A (preferably T350V) . In some embodiments, the second CH3 region further comprises K409D/E (preferably K409D) , Q347D/E (preferably Q347D) and/or T350V/A (preferably T350V) .
In some embodiments, the first CH3 region comprises S364R + D399K and the second CH3 region comprises K370S + Y349T + K409D. In some embodiments, the first CH3 region further comprises E357N. In some embodiments, the second CH3 region further comprises Q347D. In some embodiments, the first CH3 region also includes E357N, and the second CH3 region also includes Q347D. In some embodiments, the first CH3 region and the second CH3 region further comprise T350V, respectively, or both comprises T350V.
Thus, in some embodiments, the first CH3 region comprises S364R + D399K and the second CH3 region comprises K370S + Y349T + K409D + Q347D. In some embodiments, the first CH3 region comprises S364R + D399K + E357N and the second CH3 region comprises K370S + Y349T + K409D + Q347D. In some embodiments, the first CH3 region comprises S364R + D399K +E357N + T350V, and the second CH3 region comprises K370S + Y349T + K409D + Q347D +T350V.
In some embodiments, the first CH3 region comprises K409E/D (preferably K409E) . In some embodiments, the second CH3 region comprises D399K/R (preferably D399K) or K370T/S/A/V (preferably K370T) . In some embodiments, the first CH3 region comprises K409E/D (preferably K409E) and the second CH3 region comprises D399K/R (preferably D399K) . In some embodiments, the first CH3 region further comprises T411R/K (preferably T411R) . In some embodiments, the second CH3 region further comprises K370T/S/A/V (preferably K370T) . In some embodiments, the CH3 region comprises K409E/D + T411R/K and the second CH3 region comprises D399K/R + K370T/S/A/V. In some embodiments, the CH3 region comprises K409E +T411R and the second CH3 region comprises D399K + K370T.
In some particular embodiments, the first and the second CH3 regions have the following combination of mutations:

Further mutations can be made on the Fc region in the antibodies of the present invention to obtain the desired properties. The mutations on the Fc regions are known in the art.
In one embodiment, the Fc region is modified in characteristics of an effector function of the Fc region (e.g., complement activation function of the Fc region) . In one embodiment, the effector function has been reduced or eliminated relative to a wild-type isotype Fc region. In one embodiment, effector function is reduced or eliminated by a method selected from the group consisting of: use of a Fc isoform which naturally has reduced or eliminated effector function or Fc region modification.
In a preferred embodiment, the Fc region has reduced effector function mediated by the Fc region, such as reduced or eliminated ADCC or ADCP or CDC effector function, e.g. comprising mutations to achieve the above.
As understood by those skilled in the art, the antibody molecules of the invention may also comprise modifications in the Fc domain that alter binding affinity to one or more Fc receptors, depending on the intended use of the the antibody molecules of the invention. In one embodiment, the Fc receptor is an Fc gamma receptor, particularly a human Fc gamma receptor. In some embodiments, the Fc region comprises a mutation that reduces binding to an Fc gamma receptor. For example, in some embodiments, the Fc region used in the invention has mutations that reduce binding to Fc gamma receptors, such as L234A/L235A. In yet another preferred embodiment, the Fc fragment may have a mutation that results in increased serum half-life, such as a mutation that improves binding of the Fc fragment to FcRn. In some embodiments, the Fc fragmetns comprises an amino acid sequence as shown in SEQ ID NO: 29, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to the amino acid sequence set forth in SEQ ID NO: 29 and comprising L234A/L235A mutations.
Thus, in a particular embodiment, the multipecific antibodies such as bispecific antibodies of the present invention comprise two Fc regions that heterodimerize, wherein one Fc-region polypeptide comprises the mutations S354C and T366W, while the other Fc-region polypeptide comprises the mutations Y349C, T366S, L368A and Y407V.
Thus, in a particular embodiment, the multipecific antibodies such as bispecific antibodies of the present invention comprise two Fc regions that heterodimerize, wherein one Fc region polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 27, while the other Fc region polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 28.
Thus, in a particular embodiment, the multipecific antibodies such as bispecific antibodies of the present invention comprise two Fc regions that heterodimerize, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to the amino acid sequence set forth in SEQ ID NO: 27, while the other Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to the amino acid sequence set forth in SEQ ID NO: 28.
Thus, in a particular embodiment, the multipecific antibodies such as bispecific antibodies of the present invention comprise two Fc regions that heterodimerize, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to the amino acid sequence set forth in SEQ ID NO: 27 and comprising the mutations S354C and T366W and optionally L234A/L235A mutations, while the other Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to the amino acid sequence set forth in SEQ ID NO: 28 and comprising the mutations Y349C, T366S, L368A and Y407V and optionally L234A/L235A mutations.
In some embodiments, the Fc region further comprises additional mutations that facilitate purification of the heterodimer.
II-5. Exemplary Bispecific antibody molecules
In some embodiments, the anti-B7-H3/TROP2 bispecific antibody of the invention has one or more of the following properties:
(1) the bispecific antibodies of the invention can specifically bind to cells expressing TROP2 and/or B7-H3, such as tumor cells;
(2) the bispecific antibody of the invention can be endocytosed by cells such as tumor cells, for example, the endocytotic activity is stronger than that of the parent monoclonal antibody.
In some embodiments, in the antibody molecule of the invention, the heavy chain variable region of the antigen binding region that specifically binds TROP2 as described herein is linked to heavy chain constant region CH, e.g., wherein the C-terminus of the heavy chain variable region is linked to the N-terminus of the heavy chain constant region CH. In some embodiments, in the antibody molecule of the invention, the light chain variable region of the antigen binding region that specifically binds TROP2 as described herein is linked to a light chain constant region CL, e.g., wherein the C-terminus of the light chain variable region is linked to the N-terminus of the light chain constant region CL. In some embodiments, in the antibody molecule of the invention, the heavy chain variable region is linked to the heavy chain constant region CH and the light chain variable region is linked to the light chain constant region CL in the antigen binding region that specifically binds TROP2 as described herein.
In some embodiments, in the antibody molecules of the invention, the heavy chain variable region of the antigen binding region that specifically binds B7-H3 as described herein is linked to heavy chain constant region CH, e.g., wherein the C-terminus of the heavy chain variable region is linked to the N-terminus of the heavy chain constant region CH. In some embodiments, in the antibody molecules of the invention, the light chain variable region of the antigen binding region that specifically binds B7-H3 as described herein is linked to a light chain constant region CL, e.g., wherein the C-terminus of the light chain variable region is linked to the N-terminus of the light chain constant region CL. In some embodiments, in the antibody molecules of the invention, the heavy chain variable region is linked to the heavy chain constant region CH and the light chain variable region is linked to the light chain constant region CL in the antigen binding region that specifically binds B7-H3 as described herein.
In some embodiments, the heavy chain constant region comprises the CH1 and the Fc region as described herein, connected via or not via a hinge region. In some embodiments, the heavy chain constant region consists of the CH1 and the Fc region.
In some preferred embodiments, the present invention provides a bispecific antibody molecule comprising one Fab fragment that specifically binds to TROP2 as described herein, one Fab fragment that specifically binds to B7-H3 as described herein, and an Fc dimer as described herein, wherein the Fab fragment that specifically binds to TROP2 and one Fc as described herein forms a half-antibody that specifically binds to TROP2 and the Fab fragment that specifically binds to B7-H3 and one Fc as described herein forms a half-antibody that specifically binds to B7-H3.
In some embodiments, the bispecific antibody is an IgG-like antibody having the configuration shown in Figure 1. In a particular embodiment, the first and second Fc regions comprise a Knob-into-Hole mutation, and optionally a mutation that reduces affinity for the Fcγ receptor, e.g., L234A/L235A mutation. In some embodiments, the bispecific antibody comprises or consists of a heavy chain 1 and a light chain 1, and a heavy chain 2 and a light chain 2, wherein heavy chain 1 and light chain 1 constitutes the first half-antibody, and heavy chain 2 and light chain 2 constitutes the second half-antibody; wherein heavy chain 1 comprises a heavy chain variable region of the first antigen binding region and a first heavy chain constant region; light chain 1 comprises a light chain variable region of the first antigen binding region and a first light chain constant region; and heavy chain 2 comprises a heavy chain variable region of the second antigen-binding region and a second heavy chain constant region; light chain 2 comprises a light chain variable region of the second antigen-binding region and a second light chain constant region.
In a specific embodiment, the bispecific antibody of the invention specifically binds TROP2 and B7-H3 and comprises or consists of:
Heavy chain 1 comprising or consisting of: from N-terminus to C-terminus, heavy chain variable region of the Fab that specifically binds TROP2 -heavy chain constant region CH1-First Fc region, wherein the heavy chain constant region CH1 is linked at its C-terminus to the N-terminus of the first Fc region with or without a connector (e.g., hinge region) ;
Light chain 1 comprising or consisting of: from N-terminus to C-terminus, a light chain variable region of the Fab that specifically binds TROP2 -light chain constant region;
Heavy chain 2 comprising or consisting of: from N-terminus to C-terminus, heavy chain variable region of the Fab that specifically binds B7-H3 -heavy chain constant region CH1-second Fc region, wherein heavy chain constant region CH1 is linked at its C-terminus to the N-terminus of the second Fc region with or without a connector (e.g., hinge region) ;
Light chain 2 comprising or consisting of: from N-terminus to C-terminus, a light chain variable region of the Fab that specifically binds B7-H3 -light chain constant region,
preferably, each domain is linked directly;
optionally, said first Fc region comprises S354C and T366W, and said second Fc region comprises mutations Y349C, T366S, L368A and Y407V, and vice versa; optionally, the first and second Fc regions further comprise mutations to reduce affinity for the Fcγ receptor, e.g., L234A/L235A mutations, respectively.
The domains of the bispecific antibody of the present invention are described herein, e.g., the Fab, the Fc, the VH and VL as described herein.
In some specific embodiments, the bispecific antibody of the invention that specifically bind to TROP2 and B7-H3 comprises or consists of:
Heavy chain 1 comprising or consisting of the amino acid sequence shown as SEQ ID NO: 18, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity thereto; Light chain 1 comprising or consisting of the amino acid sequence shown as SEQ ID NO: 23, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity thereto; Heavy chain 2 comprising or consisting of the amino acid sequence shown as SEQ ID NO: 6, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity thereto; and Light chain 2 comprising or consisting of the amino acid sequence shown as SEQ ID NO: 12, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity thereto.
II-6 Preparation method
In some embodiments, the present invention provides a method for preparing the antibody molecules of the present invention, which includes culturing the host cells under conditions suitable for expressing nucleic acids encoding the antibody molecules of the present invention, and optionally isolating the antibodies. In a certain embodiment, the method also includes recovering the antibody molecules of the present invention from the host cells.
In some embodiments, the method for preparing the antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibodies (e.g., any one and/or more polypeptide chains) or an expression vector comprising the nucleic acid, under conditions suitable for expression of the antibody or its chain, and optionally recovering the antibodies from the host cell (or host cell culture medium) .
In some embodiments, the method of preparing a bispecific antibody of the present invention comprises the steps of: a bispecific antibody of the invention is produced by culturing a host cell comprising a nucleic acid encoding each chain of the molecule under conditions suitable for expression of said each chain of the molecules.
For recombinant production of the antibody molecules of the invention, nucleic acids encoding the antibody (e.g., an antibody as described above, e.g., any one polypeptide chain and/or multiple polypeptide chains) are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids are readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of an antibody) .
In some embodiments, a nucleic acid of the invention encodes any one chain of an antibody molecule of the invention. The present invention relates to nucleic acids encoding any of the above bispecific antibodies. The polypeptide encoded by the nucleic acid is capable of exhibiting human TROP and/or B7-H3 antigen binding capacity when expressed from a suitable expression vector.
As those skilled in the art will appreciate, due to codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences. The nucleic acid sequences encoding the molecules of the present invention can be produced using well-known methods in the art, such as by solid-phase DNA synthesis de novo or by PCR amplification.
In some embodiments, the nucleic acid encoding each chain of a bispecific antibody can be in the same vector or in different vectors. In another embodiment, the nucleic acids encoding each chain of a bispecific antibody can be introduced into the same or different host cells for expression.
In some embodiments, the present invention relates to a vector comprising the nucleic acid of the present invention. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or Yeast Artificial Chromosomes (YACs) . In one embodiment, the vector is, for example, a pcDNA vector, such as pcDNA3.1.
Once an expression vector or DNA sequence has been prepared for expression, the expression vector may be transfected or introduced into a suitable host cell. A variety of techniques may be used to achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection or other conventional techniques. In the case of protoplast fusion, cells are cultured in a medium and screened for suitable activity. The methods and conditions for culturing the resulting transfected cells and for recovering the produced antibody molecules are known to those skilled in the art and can be varied or optimized based on the specific expression vector and mammalian host cell used, in accordance with this disclosure and known methods in the existing technology.
Additionally, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow for the selection of transfected host cells.
In some embodiments, the host cell comprises a nucleic acid of the invention or comprises an expression vector of the invention. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell such as a CHO cell (e.g., CHO-S, such as ExpiCHO-S) or 293 cell (e.g., 293F or HEK293 cell or Expi293 cell) ) or other cell suitable for the production of an antibody or fragment thereof. In one embodiment, the host cell is prokaryotic, e.g., a bacterium, such as E. coli.
The antibody molecules prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, and these will be apparent to those skilled in the art. The purity of the antibody molecules of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.
III. Antibody-drug conjugate
The present invention provides an antibody-drug conjugate of formula (I) :
Ab- (L-D) p (I)
or a pharmaceutically acceptable salt or solvate thereof,
wherein:
Ab is an antibody or fragment thereof (e.g., antigen-binding fragment) that specifically binds TROP2 and B7-H3 (e.g., human TROP2 and human B7-H3) ;
L is a linker;
D is a drug, preferably an anti-tumor compound; and
p is an integer selected from 1 to 16, such as an integer selected from 1-10, 1-9, 2-8, 4-10, 6-8,3-7, 4-6, 2-6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12.
It should be understood that p refers to the number of -L-P attached to the Ab in theantibody-drug conjugate molecule of formula (I).
In some embodiments, the Ab in formula (I) of the present invention is an antibody of thepresent invention, e.g., a bispecific antibody of the present invention.
In some embodiments, D in formula (I) of the present invention may be any anti-tumorcompound, and is not particularly limited, as long as it has an anti-tumor effect and has a substituent,or a structural moiety, which can be linked to a linker structure. For example, the anti-tumorcompound may be a pharmaceutically active compound that acts on a tumor. In the case of anantitumor compound, it is preferable that a part or whole of the linker may be cleaved in a tumorcell to release the antitumor compound, thereby exhibiting an antitumor effect. When the linker iscleaved at the linker to the drug, the antitumor compound is released in an unmodified or modifiedstructure and exhibits its antitumor effect.
In some embodiments, the anti-tumor compound can be, for example, a cytotoxic orchemotherapeutic agent, e.g., camptothecins, e.g., exatecan (topoisomerase I inhibitor, Exatecan),Dxd (a novel topoisomerase I inhibitor, Exatecan derivative), auristatins, e.g., monomethylauristatin E (MMAE), maytansinoids, e.g., small molecule microtubule inhibitor, DM1, taxanes,e.g., paclitaxel or docetaxel, anthracyclines, epothilones, mitomycins, combretastatin, vincaalkaloids, calicheamicins, duocarmycin, Tubulysins, amatoxins, bleomycin, MEK inhibitors, KSPinhibitors, and the like.
In some embodiments, D is represented by formula (D-1):
wherein R1 is selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl,C2-C6 haloalkenyl and C2-C6 haloalkynyl;
R2 is selected from H, halo, C1-C6 alkyl, C1-C6 haloalkyl, -OR4和-SR4;R3 is selected from H,halo, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR4; or R2 and R3 together form -O (CH2)nO- or-O (CF2)nO-, wherein n is 1 or 2;
R4 is selected from H or C1-C4 alkyl.
In some embodiments, R1 is H, R2 is C1-C6 alkyl, R3 is -F.
In some embodiments, D is represented by formula (D-2):
wherein R1, R2 and R3 are as defined above.
In some embodiments, D is represented by formula (D-3) :
In some embodiments, D is represented by formula (D-4) :
In some embodiments, -L-is represented by the following structure: -Z-E-NH-CH2-Q-L2-L1-wherein Z is linked to Ab, L1 is linked to D;
Z is selected fromwherein m is an integer selected from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7 or 8, preferably is 5; the carbonyl at the right end of Z is covalently linked to E;
E is a peptide residue comprising 2-10 amino acids, wherein the peptide residue is optionally substituted with one or more polyol groups, wherein the N-terminus of the peptide is covalently linked to Z;
Q is -O-or -S-;
L1 is absent or - (C1-C10alkylene) -;
L2 is absent, -N (R5) C (O) - (C1-C10alkylene) -*or -C (O) N (R5) - (C1-C10alkylene) -*; wherein *indicates that said terminus is covalently linked to Q; and
R5 is H or C1-C6 alkyl.
In some embodiments, E is a peptide residue consisting of 2, 3, or 4 amino acids.
In some embodiments, the amino acids are selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine and leucine, and wherein the glutamine or glutamic acid is optionally substituted with one polyol group.
In some embodiments, the polyol group is
In some embodiments, E is -Gln-Val-Ala-, -Gly-Val-Ala-, -Gln-Phe-Ala-, -Gly-Phe-Ala-or wherein R6 is H or C1-C6 alkyl, wherein these E groups are covalently linked to Z through the left N-terminus.
In some embodiments, -L2-L1-is - (C1-C6alkylene) -, - (C1-C6alkylene) -N (R5) C (O) - (C1-C6alkylene) -*or - (C1-C6alkylene) -C (O) N (R5) - (C1-C10alkylene) -*, wherein *indicates that the terminus is covalently linked to Q;
R5 is H or C1-C6 alkyl.
In some embodiments, -L2-L1-is - (C1-C6alkylene) -.
In some embodiments, -Z-E-NH-CH2-Q-L2-L1-is represented by the following structure
It should be understood that the right terminus of the structure is linked to the D moiety.
In some embodiments, the payload (e.g., drug) of the immunoconjugate of the invention is attached to the modified antibody or antibody fragment through the thiol group of the free cysteine of the antibody or antigen binding fragment thereof (optionally via a linker) . In some embodiments, the payload (e.g., drug) of the immunoconjugate of the invention is linked to the sulfhydryl group of the cysteine through a cleavable or non-cleavable linker.
In some embodiments, Ab is linked to L through a sulfur atom on its sulfhydryl group, the antibody-drug conjugate of formula (I) may be represented by the following formula:
Ab’ - (S-L-D) p (I’ )
wherein Ab’ is as defined above for Ab; L, D and p are as defined above.
In some embodiments, the antibody-drug conjugate has an average DAR of from 5 to 11 or from 7.5 to 8.5.
In some embodiments, the antibody-drug conjugate is selected from
wherein Ab is the bispecific antibody of the present invention, preferably HZ5C2.9/hRS7.13; q is as defined for p in formula (I) above,
preferably, the antibody-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof has an average DAR of, for example, from 5 to 11, from 6 to 10, from 7 to 9 or from 7.5 to 8.5.
It should be understood that the S atom connected to Ab in the above ADC comes from the antibody Ab. Ab opens the disulfide bond under the action of a reducing agent such as TCEP to generate a thiol (-SH) , which is then connected to the maleimide moiety of the linker.
IV. Preparation method of ADC of the present invention
In another aspect, the invention provides a method of using the antibodies of the present invention to prepare ADCs. The "ADC" in the present invention is defined as an antibody coupled to an active substance (D) having biological and/or pharmaceutical activity via a linker (L) . The method comprises coupling an antibody (Ab) of the present invention to one or more active substances D via one or more linkers (L) as defined herein.
In some embodiments, the method comprises preparing an Ab for an ADC comprising culturing a host cell comprising a nucleic acid encoding the Ab (e.g., any one and/or more polypeptide chains) or an expression vector comprising the nucleic acid, under conditions suitable for expression of the Ab or its chain, and optionally recovering the Ab from the host cell (or host cell culture medium) .
In some embodiments, the method comprises the steps of:
(a) adding the antibody Ab into a buffer solution, adding a reducing agent, and then incubating;
(b) adding a linker-payload to the reaction solution of step (a) for coupling to obtain a crude product; and
(c) optionally purifying the crude product to obtain the antibody drug conjugate of the present invention;
wherein Ab is as defined above.
It is understood that the linker-payload is reacted with the Ab to provide the-L-D moiety in the compound of formula I, and when -L-D is clearly defined, the structure of the linker-payload can be determined or selected.
In some embodiments, the buffer solution of step a) is a PBS buffer, preferably at a pH of 5.0-9.0, e.g. 6.0-8.0.
In some embodiments, the reducing agent of step a) is TCEP.
In some embodiments, the linker-payload has the structure: Z’ -E-NH-CH2-Q-L2-L1-D, wherein E, Q, L2, L1, D are as defined above, Z'isand m is as defined above.
In some embodiments, the steps are performed under the specific reaction conditions disclosed in the examples.
It should be noted that the present invention also contemplates the embodiments in which ranges or specific values for the reaction conditions vary by 100%, 80%, 60%, 40%, 20%, or 10%compared with ranges or specific values for the specific reaction conditions disclosed in the examples.
In some embodiments, the ADC may be prepared according to the schematic shown in Fig. 8. The resulting ADC is also contemplated by the present invention.
V. Pharmaceutical composition
In some embodiments, the present invention provides a composition comprising any of the bispecific antibody molecules and/or the ADC molecules described herein, or a pharmaceutically acceptable salt thereof, preferably the composition is a pharmaceutical composition or pharmaceutical formulation. In one embodiment, the composition further comprises a pharmaceutically acceptable excipient. In one embodiment, a composition, e.g., a pharmaceutical composition, comprises a combination of a bispecific antibody molecules and/or an ADC molecule of the invention, and one or more other therapeutic agents.
The present invention also includes compositions (including pharmaceutical compositions) comprising a salt of a bispecific antibody molecules or fragment thereof of the present invention. The present invention also includes compositions (including pharmaceutical compositions) comprising the ADC molecules of the invention, or pharmaceutically acceptable salts thereof. These compositions may also contain suitable pharmaceutically acceptable excipients such as pharmaceutically acceptable carriers, pharmaceutically acceptable vehicles, including buffers, as are known in the art.
As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion medium, isotonic agents and absorption delaying agents, and the like that are physiologically compatible.
For the use of pharmaceutically acceptable excipients and their use, see also "Handbook of Pharmaceutical Excipients" , eighth edition, R.C. Rowe, P.J. Seskey and S. C. Owen, Pharmaceutical Press, London, Chicago.
The compositions of the present invention may be in a variety of forms. Such forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions) , powders or suspensions, liposomal formulations, and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.
Medicaments comprising the bispecific antibody molecules or ADCs described herein may be prepared by mixing the bispecific antibody molecules or ADC molecules of the present invention having the desired purity with one or more optional pharmaceutically acceptable excipients, preferably in the form of lyophilized formulations or aqueous solutions.
The pharmaceutical compositions or formulations of the present invention may also contain more than one active ingredient as required for the particular indication being treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, it may be desirable to further provide other therapeutic agents, including chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immune modulators (e.g., immune checkpoint inhibitors or agonists) , and the like. The active ingredients are suitably present in combination in an amount effective for the intended use.
Sustained release formulations can be prepared. Appropriate examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody or ADC molecule, which matrices are in the form of shaped articles, e.g., films, or microcapsules.
VI. Pharmaceutical combination and Kit
In some embodiments, the invention further provides a pharmaceutical combination or a pharmaceutical combination product, which comprises the bispecific antibody molecules and/or ADC of the present invention, and one or more other therapeutic agents (such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immune modulators (e.g., immune checkpoint inhibitors or agonists) etc. ) .
Another object of the present invention is to provide a kit comprising the pharmaceutical combination of the invention, preferably in the form of drug dose unit. Therefore, the dose unit can be provided according to the regimen or interval of the administration.
In one embodiment, the kit of the present invention comprises:
- a first container comprising a pharmaceutical composition containing the bispecific antibody molecules or ADC molecules of the present invention;
- a second container comprising a pharmaceutical composition containing other therapeutic agent (s) .
VII. Use and Method
In one aspect, the present inventin provides a method of preventing or treating a disease in a subject, comprising administering to the subject an effective amount of the bispecific antibody molecule, the ADC molecule, pharmaceutical composition, pharmaceutical combination or kit of the present invention.
In some embodiment, the disease is B7-H3 and/or TROP2 related disease. In some embodiments, the disease is related to aberrant expression or aberrant activity of B7-H3 and/or TROP2.
In some embodiments, the disease is a tumor, e.g., cancer.
In some embodiments, the tumor, e.g., cancer, is B7-H3 positive, e.g., it comprises tumor cells that express B7-H3. In some embodiments, the patient's tumor comprises tumor cells that express B7-H3. In some embodiments, the patient's tumor cells express B7-H3, e.g., moderately express B7-H3, preferably highly express B7-H3. In some embodiments, the tumor (e.g., cancer) patient has B7-H3 in the tumor tissue (e.g., at an elevated level, e.g., at a nucleic acid or protein level or activity) , e.g., as compared with the B7-H3 level in the same tissue of a healthy subject or in a healthy tissue adjacent to the tumor tissue of the patient. In some embodiments, the patient has B7-H3 in the tumor cells (e.g., at an elevated level, e.g., a nucleic acid or protein level or activity) , e.g., as compared with the B7-H3 level in the same cells of a healthy subject or in healthy cells adjacent to the patient's tumor cells.
In some embodiments, the tumor, e.g., cancer, is TROP2 positive, e.g., it comprises tumor cells that express TROP2. In some embodiments, the patient's tumor comprises tumor cells that express TROP2. In some embodiments, the patient's tumor cells express TROP2, e.g., moderately express TROP2, preferably highly express TROP2. In some embodiments, the tumor (e.g., cancer) patient has TROP2 in the tumor tissue (e.g., at an elevated level, e.g., at a nucleic acid or protein level or activity) , e.g., as compared with the TROP2 level in the same tissue of a healthy subject or in a healthy tissue adjacent to the tumor tissue of the patient. In some embodiments, the patient has TROP2 in the tumor cells (e.g., at an elevated level, e.g., a nucleic acid or protein level or activity) , e.g., as compared with the TROP2 level in the same cells of a healthy subject or in healthy cells adjacent to the patient's tumor cells.
In some embodiments, the tumor, e.g., cancer, is B7-H3 positive and TROP2 positive, e.g., it comprises tumor cells that express B7-H3 and TROP2. In some embodiments, the patient's tumor comprises tumor cells that express B7-H3 and TROP2. In some embodiments, the patient's tumor cells express B7-H3 and TROP2, e.g., moderately or highly express B7-H3 and TROP2. In some embodiments, the tumor (e.g., cancer) patient has B7-H3 and TROP2 in the tumor tissue (e.g., at an elevated level, e.g., at a nucleic acid or protein level or activity) , e.g., as compared with the B7-H3 and TROP2 level in the same tissue of a healthy subject or in a healthy tissue adjacent to the tumor tissue of the patient. In some embodiments, the patient has B7-H3 and TROP2 in the tumor cells (e.g., at an elevated level, e.g., a nucleic acid or protein level or activity) , e.g., as compared with the B7-H3 and TROP2 level in the same cells of a healthy subject or in healthy cells adjacent to the patient's tumor cell`s.
In some embodiments, the tumors, such as cancers, include solid tumors and hematological tumors as well as metastatic lesions. In one embodiment, examples of solid tumors include malignant tumors. The cancer may be in an early, intermediate or advanced stage or metastatic cancer.
In a specific embodiment, the ADC molecules of the present invenntion are capable of killing tumor cells, and/or inhibiting tumor cell proliferation, for example, the tumor cell are tumor cells expressing B7-H3 and/or TROP2, e.g., cells of the following cancers: lung cancer including non-small cell lung cancer and small cell lung cancer, colon cancer, oral squamous cell carcinoma, breast cancer, melanoma, head and neck tumors, prostate cancer, esophageal cancer, cervical cancer, renal cancer, bladder cancer, ovarian cancer or pancreatic cancer.
In some embodiments, the tumor is tumor immune escape.
In some embodiments, the tumor is a cancer, such as lung cancer including non-small cell lung cancer and small cell lung cancer, colon cancer, oral squamous cell carcinoma, breast cancer, melanoma, head and neck tumors, prostate cancer, esophageal cancer, cervical cancer, renal cancer, bladder cancer, ovarian cancer or pancreatic cancer.
The subject can be a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., a subject having or at risk of having a disease as described herein) . In one embodiment, the subject has or is at risk of having a disease described herein (e.g., cancer) . In some embodiments, the subject receives or has received other treatment, such as chemotherapy treatment and/or radiation therapy. In some embodiments, the subject has previously received or is receiving immunotherapy.
In other aspects, the present invention provides the use of the bispecific antibody molecule, ADC molecule or pharmaceutical composition or pharmaceutical combination or kit as described above in the manufacture or preparation of a medicament for use as described herein, for example for the prevention or treatment of a related disease or condition mentioned herein.
In other aspects, the present invention provides the bispecific antibody molecule, ADC molecules or pharmaceutical compositions or pharmaceutical combinations or kits as described above for use in therapy, e.g. for use as described herein, e.g. for the prevention or treatment of a related disease or disorder mentioned herein.
In some embodiments, the bispecific antibody molecule, ADC molecule or pharmaceutical composition or pharmaceutical combination or kit of the present invention delays the onset of a disease or condition and/or symptoms associated with a disease or condition.
In some embodiments, the bispecific antibody molecule, ADC molecules or pharmaceutical compositions of the present invention can also be administered in combination with one or more other therapies, e.g., therapeutic modalities and/or other therapeutic agents, for uses described herein, e.g., for the prevention and/or treatment of related diseases or conditions mentioned herein.
In some embodiments, the treatment modality comprises surgery; radiotherapy, localized irradiation, focused irradiation, or the like.
In some embodiments, the therapeutic agent is selected from a chemotherapeutic agent, an angiogenesis inhibitor, a cytokine, a cytotoxic agent, another antibody, a small molecule drug, or an immunomodulatory agent (e.g., an immune checkpoint inhibitor or agonist) .
Exemplary other antibodies include antibodies that specifically bind to immune checkpoints.
The combination therapies of the present invention encompass both combined administration (e.g., two or more therapeutic agents contained in the same formulation or separate formulations) , and separate administration, in which administration of the ADC molecules of the present invention can occur prior to, simultaneously with, and/or after administration of the other therapeutic agents and/or active agents.
The route of administration of the pharmaceutical composition is according to known methods, e.g., orally, by intravenous injection, intraperitoneally, intracerebrally (intraparenchymal) , intracerebroventricularly, intramuscularly, intraocularly, intraarterially, intraportally or intralesionally; by a sustained release system or by an implanted device. In certain embodiments, the composition may be administered by bolus injection or by continuous infusion or by an implanted device.
The composition may also be administered topically via an implant membrane, sponge, or another suitable material onto which the desired molecule is absorbed or encapsulated. In certain embodiments, when an implant device is used, the device may be implanted into any suitable tissue or organ and the desired molecule may be delivered via diffusion, timed-release bolus, or continuous administration.
These and other aspects and embodiments of the application are described in the drawings and detailed description below and are illustrated in the following examples. Any or all of the features discussed above and throughout this disclosure may be combined in various embodiments of the present application. The following examples further illustrate the invention. However, it is to be understood that the examples are described by way of illustration and not limitation, and that various modifications may be made by those skilled in the art.
Examples
Example 1: Expression and purification of the recombinant humanized anti TROP2/B7-H3 bispecific antibody
The antibodies of the present invention can be expressed and purified as follows. The optimal predetermined ratio of heavy chain to light chain vectors can be used, or a single vector system that encodes both the heavy and light chains can be used to transiently or stably transfect suitable host cells, such as HEK 293 (other host cells, such as CHO cells, can also be used) , with an expression system for secretion of antibodies.
Specifically, one or more DNA molecules encoding the following amino acid sequences can be used: a first heavy chain with the amino acid sequence SEQ ID NO: 6, a first light chain with SEQ ID NO: 12, a second heavy chain with SEQ ID NO: 18, and a second light chain with SEQ ID NO: 23, and are transiently or stably transfect with an expression system for secretion of antibodies to prepare the bispecific antibody HZ5C2.9/hRS7.13 of the present invention. In which, the first heavy chain with the amino acid sequence SEQ ID NO: 6 and the first light chain with SEQ ID NO: 12 together form the first half-antibody, which specifically binds to B7-H3 or its extracellular domain; and the second heavy chain with the amino acid sequence SEQ ID NO: 18 and the second light chain with SEQ ID NO: 23 together form the second half-antibody, which specifically binds to Trop2 or its extracellular domain; and the first half-antibody and the second half-antibody together form the complete bispecific antibody HZ5C2.9/hRS7.13 of the present invention.
Figure 1 shows a schematic diagram of the structure of the aforementioned antibody.
One of the various common techniques can be used to purify the antibody. For example, the culture medium can be conveniently applied to a MabSelect column (GE Healthcare) or KappaSelect column (GE Healthcare) that has been equilibrated with a compatible buffer such as phosphate-buffered saline (pH 7.4) . The column is washed to remove non-specifically bound components. The bound antibody can be eluted, for example, by a pH gradient (e.g., from 20 mM Tris buffer pH 7.0 to 10 mM sodium citrate buffer pH 3.0, or from phosphate-buffered saline pH 7.4 to 100 mM glycine buffer pH 3.0) . The antibody fractions are detected, for example, by UV absorption or SDS-PAGE, and then the antibody fractions are pooled. Further purification can be optionally carried out according to the intended use. The purified antibody can be concentrated and/or sterile filtered using conventional techniques. Soluble aggregates and polymers can be effectively removed using conventional techniques, including size exclusion, hydrophobic interaction, ion exchange, multi-mode or hydroxyapatite chromatography, etc. The purified antibody can be immediately frozen at below -30℃ or lyophilized.
Expression and Purification of Specific Bispecific Antibodies
The heavy chain sequence of the TROP2-Arm and the light chain sequence of the TROP2-Arm, as well as the heavy chain sequence of the B7-H3-Arm and the light chain sequence of the B7-H3-Arm, were inserted into the vector pcDNA3.1 (Invitrogen, V790-20) , respectively obtaining the heavy chain plasmid and light chain plasmid of the anti-TROP2 end, and the heavy chain plasmid and light chain plasmid of the anti-B7-H3 end.
The heavy chain plasmid and light chain plasmid of the anti-TROP2 end, and the heavy chain plasmid and light chain plasmid of the anti-B7-H3 end were transiently transfected into ExpiCHO (Invitrogen, A29133) cells, respectively. After 7 days, the cell fermentation broth was harvested, filtered and clarified, and captured using a Hitrap Mabselect Sure chromatography column (GE Healthcare, 11-0034-95) to obtain the TROP2 antibody parent and B7-H3 antibody parent.
After detecting the concentration by the A280 method, the antibody parents were mixed in a molar ratio of 1: 1, and an appropriate amount of reducing agent GSH was added to react overnight at room temperature. The reducing agent was removed by ultrafiltration to terminate the reaction. MonoS cation exchange chromatography column (GE Healthcare, 17-5168-01) was used for fine purification, with the anti-B7-H3 parent liquid being 20 mM sodium phosphate buffer (pH 6.6) and the anti-TROP2 parent liquid being 20 mM sodium phosphate buffer containing 1M sodium chloride (pH 6.6) , with an elution gradient of 0-50% (30 column volumes) . The eluted protein solution was ultrafiltered and exchanged into PBS (Gibco, 70011-044) , and the purity was detected by SEC-HPLC.
Expression and Purification of Monospecific Antibodies
First, the expression vector was constructed by placing the heavy chain variable region and light chain variable region of the anti-B7-H3 humanized antibody HZ5C2.9, anti-TROP2 antibody hRS7.13, and control antibody (negative control antibody IgG) (see sequence list information) at the N-terminus of the human IgG1 heavy chain constant region (SEQ ID NO: 24) and light chain kappa constant region (SEQ ID NO: 11) respectively. Then, it was constructed into the pcDNA3.1 expression vector with an N-terminal signal peptide to obtain the heavy and light chain expression vectors. HEK293 cells (Invitrogen) were passaged according to the required transfection volume, and the cell density was adjusted to 1.5×106 cells/ml the day before transfection. On the day of transfection, the cell density was about 3×106 cells/ml. A final volume of 1/10 (v/v) of Opti-MEM medium (Gibco catalog number: 31985-070) was taken as the transfection buffer, added with the constructed expression vector, mixed well, filtered with a 0.22μm filter head, and set aside. An appropriate amount of polyethyleneimine (PEI) (Polysciences, 23966) was added to the above plasmid (the mass ratio of plasmid to PEI was 1: 3) , mixed and incubated at room temperature for 10 minutes to obtain a DNA/PEI mixture. The DNA/PEI mixture was gently poured into HEK293 cells and mixed, cultured at 37℃, 8%CO2 for 24 hours, then supplemented with VPA (Sigma, catalog number: P4543-100G) at a final concentration of 2mM, and 2% (v/v) of Feed solution (1g/L Phytone Peptone + 1g/L Difco Select Phytone) , and continue to culture for 6 days. After cell culture, the cell culture broth was centrifuged at 13000rpm for 20 minutes, the supernatant was collected, and the supernatant was purified according to the manufacturer's instructions using a pre-packed column Hitrap Mabselect Sure (GE, 11-0034-95) , and the concentration was determined. 100μg of purified protein was takend, adjusted to the concentration to 1mg/mL, and determined for the protein purity with a gel filtration chromatography column SW3000 (TOSOH catalog number: 18675) .
Example 2: Determination of the Binding Kinetics of the Bispecific Antibody of the Invention with Antigen by Biomolecular Thin-Film Interferometry
The equilibrium dissociation constant of the binding of the antibodies of the invention with human B7-H3 and human Trop2 were determined using biomolecular thin-film interferometry (ForteBio) . The ForteBio affinity assay was performed according to established methods (Estep, P et al., High throughput solution Based measurement of antibody antigen affinity and epitope binning. MAbs, 2013.5 (2) : pp. 270-8) .
In brief, the sensor was equilibrated in the analysis buffer for 30 minutes offline, then a baseline was established by online detection for 60 seconds, and the purified antibodies obtained as described above were loaded online onto the AHQ sensor (ForteBio) for ForteBio affinity measurement. The sensor loaded with antibodies was then exposed to human B7-H3 protein (Sinobiological, 29849-H08H) or human Trop2 protein (Sinobiological, 90893-C08H) , after which the sensor was transferred to the analysis buffer for dissociation rate measurement. The KD values were analyzed using ForteBio analysis software.
Table 1. ForteBio detection of the affinity constant (equilibrium dissociation constant) of the antibodies binding to human B7-H3.
Table 2. ForteBio detection of the affinity constant (equilibrium dissociation constant) of the antibodies binding to human Trop2.
Example 3: Binding of Trop2/B7-H3 bispecific antibody to tumor cell lines
The binding of the Trop2/B7-H3 bispecific antibody HZ5C2.9/hRS7.13 and the parental mAbs (HZ5C2.9 and hRS7.13) to the human lung cancer cell line NCI-H358 (Cobioer, CBP60136) was determined by flow cytometry (FACS) . Specifically, the cells to be tested were counted and diluted to a concentration of 2×106 cells/ml, and 50 μl/well was added to a U-bottom 96-well plate. After centrifugation at 500 g for 5 minutes, the cell culture medium was removed. The Trop2/B7-H3 bispecific antibody HZ5C2.9/hRS7.13 and the parental mAb were added to the U-bottom plate and the cells were resuspended, with 50 μl of antibody starting at a concentration of 140 nM and then diluted threefold in a series, for a total of 10 concentration points for each well. The plate was incubated on ice for 30 minutes. After centrifugation at 500 g for 5 minutes to remove the supernatant, the cells were washed once with PBS. Then, 100 μl of goat anti-human IgG PE-labeled secondary antibody (SouthernBiotech, 51380) was added to each well. The plate was incubated in the dark on ice for 30 minutes. After centrifugation at 500 g for 5 minutes to remove the supernatant, the cells were washed once with PBS. The cells were resuspended with 50 μl of 1×PBS, and the FACS analysis was performed. The experimental data were analyzed using GraphPad Prism software to obtain Figure 2. As shown in Figure 2, the binding activity (EC50) of HZ5C2.9/hRS7.13 is comparable to that of the parental mAbs, and the total amount of binding (MFI) is higher.
Example 4: Endocytosis assay of Trop2/B7-H3 bispecific antibody
NCI-H358 cells were trypsinized, and the cell density was adjusted to 4×104/ml; 50 μl/well was added to an opaque white 96-well plate and incubated overnight in a 37℃ CO2 incubator; the test antibodies were diluted to 4 nM with complete culture medium, and this was used as the initial concentration with a gradient dilution of 1/3 ratio; Fab-ZAP (Advanced Targeting Systems, IT-51-250) was diluted to 40 nM with complete culture medium, and this was used as the initial concentration with a gradient dilution of 1/3 ratio; an equal volume of antibody and Fab-ZAP was mixed at a ratio of 1: 1 and incubated at room temperature for 20 to 30 minutes; after incubation, 50 μl/well was added to the cells, mixed well, and then continued for incubation at 37℃; on Day 4, the plate was removed, and 100 μl of CellTiter-Glo detection reagent was added to each well, shaken at 150 rpm for 2 minutes, and after standing for 10 minutes, the luminescence value was measured using a multifunctional plate reader. The experimental data were analyzed using GraphPad Prism software to obtain Figure 3. As shown in Figure 3, the endocytosis activity (IC50) of the bispecific antibody HZ5C2.9/hRS7.13 is stronger than that of the parental mAbs.
Example 5: Synthesis of ADC molecules coupling with NT3
Based on the Trop2/B7-H3 bispecific antibody and the parent mAb, ADC coupling with small molecular compounds was further designed and synthesized.
5.1 Synthesis of Compound NT3
Compound NT3 was prepared according to the procedure disclosed in example 4 of WO2021173773A1. The physico-chemical data, including 1H NMR and mass spectral data, were in consistent with those disclosed in WO2021173773A1.
5.2 Synthesis of HZ5C2.9/hRS7.13-NT3
wherein Ab is antibody HZ5C2.9/hRS7.13; q is for example an integer selected from 4 to 10, for example 8, and HZ5C2.9/hRS7.13-NT3 obtained had an average DAR of 7.98 as determined in the experiments described below.
The specific procedures were as follows:
preparation of HZ5C2.9/hRS7.13-NT3
(a) The antibody HZ5C2.9/hRS7.13 prepared according to the method of Example 1 was dissolved in PBS buffer (thermofisher, 10010023) . Reducing agent solution (TCEP, Aldrich, Catalog Number 646547, dissolved in water) was added and the reaction mixture was placed at room temperature for 2-4 hours, wherein
(i) the optimal concentration of HZ5C2.9/hRS7.13 was 2-10 mg/mL,
(ii) the optimal molar ratio of TCEP/Ab was 10.0-20.0,
(iii) the optimal temperature of the reaction was 20-37 ℃,
(iv) the optimum pH for the reaction was between 6.0 and 8.0.
(b) An excess of linker-toxin (NT3, dissolved in DMSO) was added, reacted with the reduced antibody in step (a) , and the reaction mixture was placed at room temperature for 1-2 hours, wherein
(i) the optimal molar ratio of NT3/Ab was 10.0-16.0,
(ii) the optimal temperature for the reaction was 20-37 ℃, and the ADC crude product was obtained.
(c) The crude ADC product was desalted via spin, purified by ultrafilteration or dialyzation to obtain the final ADC product HZ5C2.9/hRS7.13-NT3.
(d) DAR value was calculated using RP-HPLC from the peak areas of the individual peaks at UV 280nm. After calculation, the HZ5C2.9/hRS7.13-NT3 had an average DAR of 7.98.
(e) The purity of the ADC was obtained by SEC-HPLC based on the ratio of the peak areas of monomer peak, aggregate, oligomer at UV 280nm. After calculation, the purity was 98.96%for HZ5C2.9/hRS7.13-NT3.
5.3 Synthesis of HZ5C2.9-NT3
The preparation process was similar to that of HZ5C2.9/hRS7.13-NT3, except that the bispecific antibody was replaced by the parent B7-H3 mAb HZ5C2.9. Using the same analytical method as HZ5C2.9/hRS7.13-NT3, the average DAR was determined to be 7.85 and the purity was 97.27%.
5.4 Synthesis of hRS7.13-NT3
The preparation process was similar to that of HZ5C2.9/hRS7.13-NT3, except that the bispecific antibody was replaced by the parent Trop2 mAb hRS7.13. Using the same analytical method as HZ5C2.9/hRS7.13-NT3, the average DAR was determined to be 7.85 and the purity was 97.14%.
5.5 Synthesis of Control IgG1-NT3
The preparation process was similar to that of HZ5C2.9/hRS7.13-NT3, except that the bispecific antibody was changed to the control antibody IgG. Using the same analytical method as HZ5C2.9/hRS7.13-NT3, the average DAR was determined to be 7.94 and the purity was 99.35%.
Example 6: Cell binding assay for Trop2/B7-H3 bispecific antibody ADC molecules
In order to determine whether the binding property of the Trop2/B7-H3 bispecific antibody to target cells can be changed by small molecule coupling, the binding activity of HZ5C2.9/hRS7.13-NT3 to the targets was determined by flow cytometry using a human lung cancer cell line NCI-H322 (Cobioer, CBP60134) and a Trop2&B7-H3 negative cell KM12 (Meisen, CTCC-001-0684) . The process was the same as Example 3.
The results were shown in Fig. 4, HZ5C2.9/hRS7.13-NT3 and HZ5C2.9/hRS7.13 had similar binding activity in NCI-H322, and did not bind to non-target cell KM12, indicating that the binding of the antibody depended on the expression specificity of the target, and the binding of the antibody was not influenced by the coupling of the small molecule NT1 (payload) .
Example 7 In vitro cell killing effect of Trop2/B7-H3 bispecific antibody ADC molecules
The effects of ADC molecules on cell viability were tested using the Cell Titer-Glo (Promega, G9242) assay kit in multiple tumor Cell lines co-expressing Trop2&B7-H3: NCI-H358, NCI-H322, SUM149PT (Meisen, CTCC-001-0949) , SCC25 (Cobioer, CBP61033) and normal cell MCF10A (Cobioer, CBP60419) .
After digestion of the used cells with Trypsin, the density was adjusted, the cells were plated evenly in 96-well plates (table 3) and samples with gradient dilution concentration (Trop2/B7-H3 bispecific antibody ADC molecule and the parent mAb ADC) were added, and placed in an incubator at 37 ℃ for 5 days. After the 5 days, 100 μl Cell Titer Glo detection reagent was added to each well, incubated at room temperature for 30 min, and detected using a microplate reader. Relative cell viability was calculated (relative cell viability = experimental wells /control wells*100%) and curves were fitted with Graph Pad Prism 8.0.
As shown in Fig. 5, the killing effects of the Trop2/B7-H3 bispecific antibody ADC molecule on a plurality of tumor cell lines were stronger than that of the parent mAb ADC. In normal cells MCF10A, Trop2 mAb ADC hRS7.13-NT3 has stronger cytotoxicity, while the cytotoxicity of bispecific ADC HZ5C2.9/hRS7.13-NT3 was significantly weaker than that of hRS7.13-NT3. As can be seen, the bispecific ADC had a tumor inhibition effect better than that of the parent mAb ADC, and can siginificantly weaken the on-target toxicity of the Trop2 ADC to normal tissues.
Table 3 cell plating Density
Example 8: Antitumor effect of Trop2/B7-H3 bispecific antibody ADC molecules in NCI-H358 mouse graft tumor model
To demonstrate the in vivo efficacy of Trop2/B7-H3 bispecific ADC molecules, CB17-SCID mice were inoculated with NCI-H358 cells to determine the anti-tumor efficacy of the molecules of the present invention. Female mice of SPF grade (14-17 g, from Vital River Laboratory Animal Technology Co., Ltd., Beijing) were used for the experiments.
NCI-H358 cells were routinely subcultured for subsequent in vivo experiments. Cells were harvested by centrifugation, and NCI-H358 cells were dispersed with PBS (1X) to prepare a cell suspension having a cell concentration of 2.5x107 cells/ml. A model of NCI-H358 tumor-bearing mice was established by inoculating 0.2 ml of the cell suspension subcutaneously on day 0 into the right abdominal region of CB17-SCID mice. All mice were randomized into 5 groups (5 mice per group) on Day 7 after tumor cell inoculation. Administration doses and mode were shown in table 4. Administration was performed on day 7 after inoculation, and tumor volume and body weight were monitored twice per week, as shown in Fig. 6A, 6B. The monitoring continued until Day 50.
The relative tumor growth inhibition rate (TGI%) was calculated on Day 50 after inoculation and the equation was as follows:
TGI%=100%* (control group tumor volume -treatment group tumor volume) / (control group tumor volume -control group tumor volume prior to administration) .
Tumor volume determination: the maximum major axis (L) and maximum broad axis (W) of the tumor was measured using a vernier caliper, tumor volume was calculated as follows:
V = L*W2/2. Body weight was measured using an electronic balance.
Table 4 Experimental design table
The tumor growth inhibition rate results were shown in table 5 and Fig. 6A: on Day 50 after inoculation, the single-dose of HZ5C2.9/hRS7.13-NT3 reached 93%tumor growth inhibition rate, which was better than 29%and 16%of the tumor growth inhibition rates of HZ5C2.9-NT3 and hRS7.13-NT3. Meanwhile, the body weights of the mice were detected, and the results were shown in Fig 6B, the body weights of the mice had no significant difference.
Table 5 Tumor growth inhibition rate On Day 50
Example 9: Antitumor effect of Trop2/B7-H3 bispecific antibody ADC molecules in HCC1806 mouse graft tumor model
To demonstrate the in vivo efficacy of Trop2/B7-H3 bispecific ADC molecules, CB17-SCID mice were inoculated with HCC1806 (HCC 1806 human breast cancer cells, from ATCC, Cat. No. CBP60373, Nanjing Cobioer Biotech Co., Ltd. ) cells to determine the antitumor efficacy of the molecules of the present invention. Female mice of SPF grade (14-17 g, from Vital River Laboratory Animal Technology Co., Ltd., Beijing) were used for the experiments.
HCC1806 cells were routinely subcultured for subsequent in vivo experiments. Cells were harvested by centrifugation, and HCC1806 cells were dispersed in PBS (1X) to prepare a cell suspension with a cell concentration of 2.5X107 cells/ml. HCC1806 tumor-bearing mouse model was established by subcutaneous inoculation of 0.2 ml of cell suspension into the right abdominal region of CB17-SCID mice on day 0. All mice were randomized into 5 groups (5 mice per group) on Day 7 after tumor cell inoculation. Administration doses and mode were shown in table 6, Administration was performed on day 7 after inoculation, and tumor volume and body weight were monitored twice per week, as shown in Fig. 7A, 7B. The monitoring continued until Day 26.
Relative tumor growth inhibition rate (TGI%) was calculated on Day 26 after inoculation, and the equation was as follows:
TGI%=100%* (control group tumor volume -treatment group tumor volume) / (control group tumor volume -control group tumor volume prior to administration) .
Tumor volume determination: the maximum major axis (L) and maximum broad axis (W) of the tumor was measured using a vernier caliper, tumor volume was calculated as follows:
V=L*W2/2. Body weight was measured using an electronic balance.
Table 6 Experimental design table
The tumor growth inhibition rate results were shown in table 7 and Fig. 7A: on Day 26 after inoculation, the single administration of HZ5C2.9/hRS7.13-NT3 achieved a tumor growth inhibition rate of 96%, which was superior to 49%and 46%of the tumor growth inhibition rates of HZ5C2.9-NT3 and hRS7.13-NT3. Meanwhile, the body weights of the mice were detected, and the results were shown in Fig 7B. The body weights of the mice had no significant difference.
Table 7 Tumor growth inhibition rate On Day 26

Sequence information



Claims (41)

  1. An antibody-drug conjugate represented by formula (I) :
    Ab- (L-D) p (I)
    or a pharmaceutically acceptable salt or solvate thereof,
    wherein:
    Ab is an antibody or fragment thereof (e.g., antigen-binding fragment) that specifically binds TROP2 and B7-H3 (e.g., human TROP2 and human B7-H3) ;
    L is a linker;
    D is a drug, preferably an anti-tumor compound; and
    p is an integer selected from 1 to 16, such as 6, 7, 8, 9 or 10,
    wherein Ab in formula (I) is a bispecific antibody that specifically binds to B7-H3 and TROP2 comprising a first antigen-binding region that specifically binds to TROP2 and a second antigen-binding region that specifically binds to B7-H3, wherein the second antigen-binding region that specifically binds to B7-H3 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein
    HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 7; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 8; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 9.
  2. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 1, wherein the second antigen-binding region that specifically binds B7-H3 comprises a VH and a VL, wherein the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity the amino acid sequence set forth in SEQ ID NO: 4, and/or said VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity the amino acid sequence set forth in SEQ ID NO: 10.
  3. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 1 or claim 2, wherein the second antigen-binding region that specifically binds B7-H3 comprises a VH and a VL, wherein the VH and VL comprise or consist of, respectively, the amino acid sequences shown below: SEQ ID NO: 4 and 10, respectively.
  4. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 3, wherein the first antigen-binding region that specifically binds TROP2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein
    HCDR1 comprises or consists of the amino acid sequence of SEQ ID No: 13; HCDR2 comprises or consists of the amino acid sequence of SEQ ID No: 14; HCDR3 comprises or consists of the amino acid sequence of SEQ ID No: 15; LCDR1 comprises or consists of the amino acid sequence of SEQ ID No: 19; LCDR2 comprises or consists of the amino acid sequence of SEQ ID No: 20; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID No: 21.
  5. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 4, wherein the first antigen-binding region that specifically binds TROP2 comprises a VH and a VL, wherein the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity thereto, and/or said VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity thereto.
  6. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 5, wherein the first antigen-binding region that specifically binds TROP2 comprises a VH and a VL, wherein the VH and VL comprise or consist of, respectively, the amino acid sequences shown below: SEQ ID NO: 16 and 22, respectively.
  7. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 6, wherein the bispecific antibody comprises a first Fc region and a second Fc region, wherein the first Fc region and the second Fc region are the same or different.
  8. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 7, wherein the first Fc region and second Fc regions are, respectively, human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc, e.g., comprise or consist of the amino acid sequence of SEQ ID NO: 30 or 31 or an amino acid sequence having at least 90%, e.g., 95%, 96%, 97%, 98%or 99%or more identity thereto.
  9. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 7 or 8, wherein the first and second Fc regions of the bispecific antibody have introduced therein mutations that promote heterodimerization of the first and second Fc regions.
  10. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 9, wherein the mutation of the Fc region is introduced based on the Knob-into-Hole technique, wherein the corresponding Knob mutation and Hole mutation are introduced in the first Fc region and the second Fc region, respectively.
  11. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 10, wherein one Fc region comprises the amino acid substitutions S354C and T366W and the other Fc region comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to the EU index) , e.g., wherein the S354C and Y349C form a non-natural disulfide bond.
  12. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 9, wherein the mutation of the Fc region is introduced based on the Innobody technology.
  13. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of claims 7 to 12, wherein the Fc region further comprises a mutation that reduces binding to the Fcγ receptor, such as the L234A/L235A mutation.
  14. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of claims 7 to 13, wherein one Fc region polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 27 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to the amino acid sequence set forth in SEQ ID No: 27, and the other Fc region polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 28 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to the amino acid sequence set forth in SEQ ID No: 28.
  15. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of claims 7 to 14, wherein in the bispefic antibody, the first antigen-binding region is a Fab fragment, and/or the second antigen-binding region is a Fab.
  16. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 15, wherein the Fab, which is the first antigen binding region or the second antigen binding region, comprises CH1, wherein the CH1 is CH1 from IgG1, IgG2, IgG3, or IgG4, preferably CH1 from IgG1.
  17. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 16, wherein the CH1
    (i) comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 32, or
    (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 32.
  18. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of claims 15 to 17, wherein the Fab, which is the first antigen binding region or the second antigen binding region, comprises a light chain constant region, wherein the light chain constant region is a Kappa light chain constant region or a Lambda light chain constant region.
  19. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 18, wherein the Kappa light chain constant region
    (i) comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity to the amino acid sequence of SEQ ID NO: 11, or
    (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 11.
  20. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of claims 15 to 19, wherein the first antigen binding region that specifically binds TROP2 comprises a first Fab linked at its C-terminus of CH1 to the N-terminus of the first Fc region (with or without a connector, e.g., a hinge region) and the second antigen binding region that specifically binds B7-H3 comprises a second Fab linked at its C-terminus of CH1 to the N-terminus of the second Fc region (with or without a connector, e.g., a hinge region) .
  21. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 20, wherein the bispecific antibody is an IgG-like antibody having the configuration shown in Figure 1.
  22. The antibody-drug conjugate, or pharmaceutically acceptable salt or solvate thereof according to claim 20 or 21, wherein the bispecific antibody comprises a first Fab as a first antigen-binding region that specifically binds TROP2 and a second Fab as a second antigen-binding region that specifically binds B7-H3, wherein the bispecific antibody comprises or consists of
    Heavy chain 1: from N-terminus to C-terminus comprises or consists of: heavy chain variable region of the Fab that specifically binds TROP2 -heavy chain constant region CH1-First Fc region, wherein heavy chain constant region CH1 is linked at its C-terminus to the N-terminus of the first Fc region with or without a connector (e.g., hinge region) ;
    Light chain 1: from N-terminus to C-terminus comprises or consists of: a light chain variable region of the Fab that specifically binds TROP2 -light chain constant region;
    Heavy chain 2: from N-terminus to C-terminus comprises or consists of: heavy chain variable region of the Fab that specifically binds B7-H3 -heavy chain constant region CH1-second Fc region, wherein heavy chain constant region CH1 is linked at its C-terminus to the N-terminus of the second Fc region with or without a connector (e.g., hinge region) ;
    Light chain 2: from N-terminus to C-terminus comprises or consists of: a light chain variable region of the Fab that specifically binds B7-H3 -light chain constant region,
    preferably, each domain is linked directly;
    optionally, said first Fc region comprises mutations Y349C, T366S, L368A and Y407V, and said second Fc region comprises mutations S354C and T366W, and vice versa ; optionally, the first and second Fc regions further comprise L234A/L235A mutations, respectively.
  23. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 22, wherein Heavy chain 1 comprising or consisting of the amino acid sequence shown as SEQ ID NO: 18, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity thereto; Light chain 1 comprising or consisting of the amino acid sequence shown as SEQ ID NO: 23, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity thereto; Heavy chain 2 comprising or consisting of the amino acid sequence shown as SEQ ID NO: 6, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity thereto; ; and Light chain 2 comprising or consisting of the amino acid sequence shown as SEQ ID NO: 12, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identity thereto.
  24. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to anyone of claims 1 to 23, wherein the anti-tumor compound is a cytotoxic agent, such as camptothecins, auristatins, maytansinoids, taxanes, anthracyclines, vinca alkaloids, MEK inhibitors, or KSP inhibitors.
  25. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 24, wherein D is represented by formula (D-1) :
    wherein R1 is selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C2-C6 haloalkenyl and C2-C6 haloalkynyl;
    R2 is selected from H, halo, C1-C6 alkyl, C1-C6 haloalkyl, -OR4和-SR4; R3 is selected from H, halo, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR4; or R2 and R3 together form -O (CH2) nO-or -O (CF2) nO-, wherein n is 1 or 2;
    R4 is selected from H or C1-C4 alkyl.
  26. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 25, wherein R1 is H, R2 is C1-C6 alkyl, and R3 is -F.
  27. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 25, wherein D is represented by formula (D-2) :
    wherein R1, R2 and R3 are as defined in claim 25 or 26.
  28. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 25, wherein D is represented by formula (D-3) or formula (D-4) :
  29. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to anyone of claims 1 to 28, wherein -L-is represented by the following structure:
    -Z-E-NH-CH2-Q-L2-L1-
    wherein Z is linked to Ab, L1 is linked to D;
    Z is selected fromwherein m is an integer selected from 1 to 10; the carbonyl at the right end of Z is covalently linked to E;
    E is a peptide residue comprising 2-10 amino acids, wherein the peptide residue is optionally substituted with one or more polyol groups, wherein the N-terminus of the peptide residue is covalently linked to Z;
    Q is -O-or -S-;
    L1 is absent or - (C1-C10alkylene) -;
    L2 is absent, -N (R5) C (O) - (C1-C10alkylene) -*or -C (O) N (R5) - (C1-C10alkylene) -*; wherein *indicates that the terminus is covalently linked to Q; and
    R5 is H or C1-C6 alkyl.
  30. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 29,
    wherein E is a peptide residue consisting of 2, 3, or 4 amino acids, the amino acids are selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine and leucine, and wherein the glutamine or glutamic acid is optionally substituted with one polyol group; and
    -L2-L1-is - (C1-C6alkylene) -, - (C1-C6alkylene) -N (R5) C (O) - (C1-C6alkylene) -*or - (C1-C6alkylene) -C (O) N (R5) - (C1-C10alkylene) -*, wherein *indicates that the terminus is covalently linked to Q ;
    R5 is H or C1-C6 alkyl.
  31. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 30,
    wherein, E is -Gln-Val-Ala-, -Gly-Val-Ala-, -Gln-Phe-Ala-, -Gly-Phe-Ala-or
    wherein R6 is H or C1-C6 alkyl, wherein these E groups are covalently linked to Z through the left N-terminus; and
    -L2-L1-is - (C1-C6alkylene) -.
  32. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 31,
    wherein -Z-E-NH-CH2-Q-L2-L1-is represented by the following structure
    its right terminus is linked to D.
  33. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 32, wherein the antibody-drug conjugate of formula (I) is represented by the following structure:
    Ab’- (S-L-D) p (I’)
    wherein Ab’ is as defined for Ab in one of claims 1 to 23; L, D and p are as defined in one of claims 1 to 32.
  34. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 33, which has an average DAR of from 5 to 11, for example, from 7.5-8.5.
  35. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 1, wherein the antibody-drug conjugate is represented by the following structure:
    wherein Ab is a bispecific antibody that specifically binds TROP2 and B7-H3, said bispecific antibody comprises or consists of heavy chain 1, light chain 1, heavy chain 2 and light chain 2, wherein
    heavy chain 1 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 18; light chain 1 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 23; heavy chain 2 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 6; and light chain 2 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 12;
    q is as defined for p in claim 1,
    preferably, the antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof has an average DAR of from 5 to 11 or from 7.5 to 8.5.
  36. A pharmaceutical composition, comprising the antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 35, and optionally one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immune modulators and optionally pharmaceutically acceptable excipients.
  37. A pharmaceutical combination comprising the antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 35, and one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immune modulators.
  38. A method of preventing or treating a tumor in a subject, comprising administering to the subject an effective amount of the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 35, or the pharmaceutical composition of claim 36, or the pharmaceutical combination of claim 37.
  39. The method of claim 38, wherein the tumor is a cancer, preferably, the cancer has B7-H3 and/or TROP2 at elevated level (such as at nucleic acid or protein level) compared with the corresponding tissue of a healthy subject or a healthy tissue adjacent to the tumor tissue of the patient.
  40. The method of claim 39, wherein the cancer is selected from lung cancer, such as non-small cell lung cancer and small cell lung cancer, colon cancer, oral squamous cell carcinoma, breast cancer, melanoma, head and neck tumors, prostate cancer, esophageal cancer, cervical cancer, renal cancer, bladder cancer, ovarian cancer or pancreatic cancer.
  41. A method of any one of claims 38 to 40, wherein the method further comprises administering to the subject one or more therapies, such as a therapeutic approach and/or other therapeutic agents, preferably the therapeutic approach includes radiation therapy or surgery, or the therapeutic agents include chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immune modulators.
PCT/CN2024/140994 2023-12-22 2024-12-20 Antibody drug conjugates targeting b7-h3 and trop2 and the use thereof Pending WO2025131063A1 (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180147292A1 (en) * 2015-06-29 2018-05-31 Daiichi Sankyo Company, Limited Method for selectively manufacturing antibody-drug conjugate
WO2021173773A1 (en) * 2020-02-25 2021-09-02 Mediboston, Inc. Camptothecin derivatives and conjugates thereof
WO2022126569A1 (en) * 2020-12-18 2022-06-23 上海复旦张江生物医药股份有限公司 B7-h3 targeting antibody-drug conjugate, and preparation method therefor and use thereof
CN116120460A (en) * 2021-11-15 2023-05-16 成都百利多特生物药业有限责任公司 Bispecific antibody-camptothecins drug conjugate and medical application thereof
EP4265275A1 (en) * 2020-12-18 2023-10-25 Shanghai Fudan-Zhangjiang Bio-Pharmaceutical Co., Ltd. Trop2 targeting antibody-drug conjugate, and preparation method and use therefor

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180147292A1 (en) * 2015-06-29 2018-05-31 Daiichi Sankyo Company, Limited Method for selectively manufacturing antibody-drug conjugate
WO2021173773A1 (en) * 2020-02-25 2021-09-02 Mediboston, Inc. Camptothecin derivatives and conjugates thereof
WO2022126569A1 (en) * 2020-12-18 2022-06-23 上海复旦张江生物医药股份有限公司 B7-h3 targeting antibody-drug conjugate, and preparation method therefor and use thereof
EP4265275A1 (en) * 2020-12-18 2023-10-25 Shanghai Fudan-Zhangjiang Bio-Pharmaceutical Co., Ltd. Trop2 targeting antibody-drug conjugate, and preparation method and use therefor
CN116120460A (en) * 2021-11-15 2023-05-16 成都百利多特生物药业有限责任公司 Bispecific antibody-camptothecins drug conjugate and medical application thereof

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