WO2022268168A1 - 靶向lag-3和pd-l1的新型双特异抗体及其应用 - Google Patents

靶向lag-3和pd-l1的新型双特异抗体及其应用 Download PDF

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WO2022268168A1
WO2022268168A1 PCT/CN2022/100816 CN2022100816W WO2022268168A1 WO 2022268168 A1 WO2022268168 A1 WO 2022268168A1 CN 2022100816 W CN2022100816 W CN 2022100816W WO 2022268168 A1 WO2022268168 A1 WO 2022268168A1
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amino acid
binding molecule
seq
acid sequence
bispecific binding
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French (fr)
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张畅
王荣娟
焦莎莎
曾大地
张姣
杨莹莹
王双
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迈威(上海)生物科技股份有限公司
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]

Definitions

  • the present invention belongs to the field of antibody engineering. Specifically, the present invention relates to novel artificially designed bispecific antibody molecules, especially novel antibodies and antibody fragments that specifically bind to LAG-3 and PD-L1 at the same time, as well as novel antibodies and antibody fragments containing the antibodies or The composition of antibody fragments also relates to the use of antibodies binding to LAG-3 and PD-L1 in the treatment of diseases such as tumors.
  • LAG-3 (Lymphocyte-activation-gene-3) is an important immune checkpoint protein, belonging to type I transmembrane protein, encoded by LAG-3 gene, mainly expressed in activated T cells, NK cells, and plasma cell-like trees Cell surfaces such as dendrites. LAG-3 regulates T cell function by interacting with its ligand.
  • LAG-3 ligands have been discovered: MHC-II, liver sinusoidal endothelial cell lectin (LSECtin), galectin-3 (galectin3), ⁇ -synaptic Nucleoprotein fibrils ( ⁇ -synuclein fibrils), and fibrin-like protein 1 (fibrinogen-like protein 1, FGL1), the main ligand of which is MHC II.
  • LAG-3 and its ligands mediate negative signals, regulate the proliferation and function of T cells, and maintain the homeostasis of T cells in the body.
  • PD-1 and its ligand PD-L1 are important targets of tumor immunity.
  • PD-1 and PD-L1 are a pair of immunosuppressive molecules, which are important components of the immune system to prevent autoimmune hyperactivity. The activation of their pathways can inhibit tumor immune response and induce tumor-specific T cell apoptosis, which is closely related to tumor development. close relationship.
  • the full name of PD-L1 is programmed death ligand-1, which can bind to the receptor PD-1 on the surface of T cells to exert an immunosuppressive effect.
  • PD-L1 is an inhibitory immune checkpoint molecule expressed on the surface of various malignant tumor cells such as melanoma, non-small cell lung cancer, renal cell carcinoma, and head and neck squamous cell carcinoma.
  • PD-L1 After PD-L1 binds to the immunosuppressive receptor PD-1 on the surface of T cells, it can induce apoptosis, incapacity, and exhaustion of T cells, thereby inhibiting the activation, proliferation, and anti-tumor functions of tumor antigen-specific T cells, and achieving tumor immunity. immune escape.
  • PD-1/PD-L1 blocking antibody can relieve the immunosuppressive effect of PD-L1, enhance the recognition and killing of tumor cells by immune cells T cells in the body, so as to achieve the effect of killing tumors.
  • bispecific antibodies have various applications, it is necessary to study treatments for various diseases based on bispecific antibodies.
  • an object of the present invention is to propose a bispecific antibody targeting LAG-3 and PD-L1 and its application.
  • the anti-PD-L1 nanobody is connected to the C-terminal of the heavy chain of the antibody through a linker.
  • the method of linking the nanobody to the C-terminal of the heavy chain of the antibody through the above-mentioned linker not only reduces the effector function of Fc, avoids the elimination of activated T cells, but also makes the structure of the bispecific antibody more stable and the assembly efficiency of recombinant expression is higher.
  • the bispecific antibodies hz7F10-hzF2 and hz7F10-hzB6 targeting LAG-3 and PD-L1 prepared by the present invention can simultaneously inhibit PD-1/PD-L1 signaling pathway and LAG-3/MHCII,
  • the LAG3/ signaling pathway realizes the bridging of LAG-3-positive and PD-L1-positive cells, and gathers deinhibited T cells around tumor cells expressing PD-L1, thereby effectively inhibiting tumor growth.
  • the present invention provides a bispecific binding molecule.
  • the bispecific binding molecule includes: a first antigen-binding portion, the first antigen-binding portion includes: a first peptide segment, and the first peptide segment includes a first heavy chain variable region and The first light chain variable region, wherein, the complementarity determining region (CDR) of the first heavy chain variable region includes H1-CDR1, H1-CDR2 and H1-CDR3, and the CDR of the first light chain variable region including L1-CDR1, L1-CDR2 and L1-CDR3; a second peptide segment, the second peptide segment comprising a second heavy chain variable region and a second light chain variable region, wherein the second heavy chain can be
  • the CDRs of the variable region include H2-CDR1, H2-CDR2, and H2-CDR3, the CDRs of the second light chain variable region include L2-CDR1, L2-CDR2, and L2-CDR3, and the second
  • the first antigen-binding portion specifically binds to programmed cell death-ligand 1 (PD-L1)
  • the second antigen-binding portion specifically binds to lymphocyte activation gene-3 (LAG3 ).
  • the first antigen-binding part comprises an Fc domain
  • the Fc domain is IgG, especially an IgG1 Fc domain.
  • the Fc domain comprises point mutations L234A and L235A.
  • the H1-CDR1 comprises the amino acid sequence described in SEQ ID NO: 17, the H1-CDR2 comprises the amino acid sequence described in SEQ ID NO: 18 and the H1-CDR3 comprises the amino acid sequence described in SEQ ID NO : the amino acid sequence described in 19, and the L1-CDR1 comprises the amino acid sequence described in SEQ ID NO: 14, L1-CDR2 comprises the amino acid sequence described in SEQ ID NO: 15 and L1-CDR3 comprises the amino acid sequence described in SEQ ID NO: 16 The amino acid sequence described.
  • the amino acid sequences of the H2-CDR1, H2-CDR2, and H2-CDR3 are identical to the amino acid sequences of the H1-CDR1, H1-CDR2, and H1-CDR3, respectively, and the L2-CDR1,
  • the amino acid sequences of L2-CDR2 and L2-CDR3 are identical to the amino acid sequences of L1-CDR1, L1-CDR2 and L1-CDR3, respectively.
  • the H3-CDR1, H3-CDR2 and H3-CDR3 are selected from one of the following groups: (a) the H3-CDR1 comprises the amino acid sequence described in SEQ ID NO: 20, and the H3 -CDR2 comprises the amino acid sequence described in SEQ ID NO: 21 and the H3-CDR3 comprises the amino acid sequence described in SEQ ID NO: 22; (b) the H3-CDR1 comprises the amino acid sequence described in SEQ ID NO: 23 , the H3-CDR2 comprises the amino acid sequence described in SEQ ID NO: 24 and the H3-CDR3 comprises the amino acid sequence described in SEQ ID NO: 25.
  • the first heavy chain variable region includes the amino acid sequence described in SEQ ID NO: 3, and the first light chain variable region includes the amino acid sequence described in SEQ ID NO: 1.
  • the first peptide segment further includes a first light chain constant region and a first heavy chain constant region
  • the second peptide segment further includes a second light chain constant region and a second heavy chain constant region Area
  • both the first light chain constant region and the second light chain constant region include the amino acid sequence described in SEQ ID NO:2.
  • both the first heavy chain constant region and the second heavy chain constant region include the amino acid sequence described in SEQ ID NO:4.
  • the third heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 7 or 8.
  • the polypeptide complex further includes: a fourth peptide segment, the fourth peptide segment includes the fourth heavy chain variable region, and is connected to the C-terminus of the second peptide segment through the linker connected.
  • the fourth peptide is a Nanobody and has the same CDR sequence as the third peptide.
  • the fourth heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 7 or 8.
  • the linker has the amino acid sequence shown in (G4S) 2-4 or SEQ ID NO:10.
  • the heavy chain of the bispecific binding molecule has the amino acid sequence shown in SEQ ID NO: 11 or 12.
  • the light chain of the bispecific binding molecule has the amino acid sequence shown in SEQ ID NO:13.
  • the invention provides a bispecific binding molecule.
  • the bispecific binding molecule comprises two antibody heavy chains and two antibody light chains, the variable regions of the two antibody heavy chains interact with the variable regions of the two antibody light chains to form Two first antigen-binding parts; at least one of the two antibody heavy chains is connected to a second antigen-binding part through a linker at the C-terminus, and the second antigen-binding part comprises a Nanobody.
  • the heavy chain of the antibody includes in sequence from the N-terminal to the C-terminal: the heavy chain variable region of the first antigen-binding part, the CH1 region, the CH2 region, the CH3 region, and an optional linker + the second Second antigen binding part.
  • the two antibody heavy chains include point mutations L234A and L235A in the Fc segment, and the two antibody heavy chains are the same or different;
  • one of the antibody heavy chains has the second antigen-binding portion, and the other antibody heavy chain does not have the second antigen-binding portion; or the two antibody heavy chains have the second antigen-binding portion with different sequences.
  • the light chain of the antibody comprises in sequence from the N-terminus to the C-terminus: the light chain variable region of the first antigen-binding part, and VH.
  • the linker is selected from (G4S)2-4, GGGGSPGGGSPGGGS (SEQ ID NO: 10).
  • the first antigen-binding part specifically binds to LAG-3
  • the light chain variable region contained in the first antigen-binding part has the amino acid sequence shown in SEQ ID NO: 1
  • the heavy chain included The chain variable region has the amino acid sequence shown in SEQ ID NO:3.
  • the second antigen-binding part specifically binds to PD-L1, and the amino acid sequence contained in the Nanobody of the second antigen-binding part is selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 8. Group.
  • the antibody heavy chain has an amino acid sequence selected from the group consisting of SEQ ID NO:11 and SEQ ID NO:12.
  • the light chain of the antibody has the amino acid sequence shown in SEQ ID NO: 13.
  • the present invention provides a composition comprising the bispecific binding molecule according to any one of the foregoing inventions and optional pharmaceutically acceptable excipients.
  • the composition further includes biotherapeutic agents, chemotherapeutic agents, natural active ingredients and the like.
  • the dosage form of the composition is water, injection, or powder injection.
  • the present invention provides a polynucleotide.
  • the polynucleotide encodes the antibody heavy chain and/or the antibody light chain in any one of the aforementioned bispecific binding molecules.
  • the present invention also provides polynucleotide combinations.
  • the polynucleotide combination includes a polynucleotide encoding the heavy chain of the antibody and a polynucleotide encoding the light chain of the antibody.
  • the present invention provides a nucleic acid construct.
  • the nucleic acid construct comprises the aforementioned polynucleotide.
  • the present invention also provides nucleic acid construct combinations.
  • the nucleic acid construct combination includes a nucleic acid construct comprising a polynucleotide encoding the heavy chain of the antibody and a nucleic acid construct comprising a polynucleotide encoding the light chain of the antibody.
  • the present invention provides a host cell.
  • the host cell comprises the aforementioned polynucleotide or combination of polynucleotides, or comprises the aforementioned nucleic acid construct or combination of nucleic acid constructs.
  • the present invention provides a method for preparing any one of the aforementioned bispecific binding molecules. According to an embodiment of the present invention, the method includes the following steps:
  • the bispecific binding molecule is isolated and purified from cell culture.
  • the present invention provides any one of the aforementioned bispecific binding molecules, compositions, polynucleotides or polynucleotide combinations, nucleic acid constructs, nucleic acid construct combinations, or host cells that bind and inhibit LAG3 and PD-1 Functional applications, wherein the LAG3 and PD-1 are derived from humans or cynomolgus monkeys.
  • the present invention provides any of the aforementioned bispecific binding molecules, compositions, polynucleotides or combinations of polynucleotides, nucleic acid constructs or combinations of nucleic acid constructs, or host cells that inhibit or block LAG-3 /MHCII, LAG-3/FGL1, and/or PD-1/PD-L1 signaling pathway applications.
  • the present invention provides any one of the aforementioned bispecific binding molecules, compositions, polynucleotides or combinations of polynucleotides, nucleic acid constructs or combinations of nucleic acid constructs, or host cells in bridging LAG-3 positive cells and Application in PD-L1 positive cells.
  • the present invention provides any of the aforementioned bispecific binding molecules, compositions, polynucleotides or combinations of polynucleotides, nucleic acid constructs or combinations of nucleic acid constructs, or host cells for the preparation of therapeutic LAG-3 and /or use in medicines for diseases related to PD-L1 signaling pathway abnormalities.
  • the diseases related to the abnormality of LAG-3 and/or PD-L1 signaling pathway include dysproliferative diseases or immune-related diseases.
  • abnormal proliferative diseases include tumors, cysts, hyperplasia, etc.
  • immune-related diseases include inflammation, immunodeficiency, immune tolerance, allergy, etc.
  • the tumor is colon cancer or lung cancer.
  • the present invention provides a method of treating an individual suffering from a dysproliferative disease or an immune-related disease.
  • the method comprises administering the aforementioned bispecific binding molecule or the aforementioned composition to the individual.
  • the invention provides a method of inhibiting the growth of tumor cells in a subject.
  • the method comprises administering to the individual an effective dose of the aforementioned bispecific binding molecule or the aforementioned composition to inhibit the growth of the tumor cells.
  • the term "specific binding” refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its antigen.
  • immunological binding refers to a specific binding reaction that occurs between an antibody molecule and an antigen for which the antibody is specific.
  • the strength or affinity of the immune binding interaction can be expressed by the equilibrium dissociation constant (KD) of the interaction, wherein the smaller the KD value, the higher the affinity.
  • KD equilibrium dissociation constant
  • the immunological binding properties between two molecules can be quantified using methods well known in the art. One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation.
  • association rate constant Ka or Kon
  • dissociation rate constant Kd or Koff
  • concentration a ratio of Kd/Ka is equal to the dissociation constant KD, see Davies DR et al., 1990, Annual Rev Biochem., 59:439-473.
  • KD, Ka and Kd values can be measured by any effective method.
  • the dissociation constant is measured using bioluminescence interferometry.
  • the dissociation constant can be measured using surface plasmon resonance techniques (eg Biacore) or KinExa.
  • antibody herein is intended to include full-length antibodies and any antigen-binding fragment (ie, antigen-binding portion) or single chains thereof.
  • Full-length antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains linked by disulfide bonds.
  • Each heavy chain is composed of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain is composed of a light chain variable region (abbreviated as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • the VH and VL regions can also be divided into hypervariable regions called complementarity determining regions (CDRs), which are separated by more conserved framework region (FR) regions.
  • CDRs complementarity determining regions
  • FR conserved framework region
  • Each VH and VL consists of three CDRs and four FRs, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminal to the carboxyl terminal.
  • the variable regions of the heavy and light chains contain the binding domains that interact with the antigen.
  • the constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • monoclonal antibody or “monoclonal antibody” or “monoclonal antibody composition” refers to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.
  • the "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. It has been demonstrated that the antigen-binding function of antibodies can be performed by fragments of full-length antibodies.
  • binding fragments contained in the "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1; (ii) a F(ab')2 fragment, comprising the hinge region II; Bivalent fragments of two Fab fragments connected by sulfur bridge; (iii) Fd fragment composed of VH and CH1; (iv) Fv fragment composed of antibody single arm VL and VH; (v) dAb fragment composed of VH ( Ward et al., (1989) Nature 341:544-546); (vi) isolated complementarity determining regions (CDRs); and (vii) a Nanobody, a nanobody comprising a single variable domain and two constant domains Heavy chain variable region.
  • the two domains VL and VH of the Fv fragment are encoded by different genes, they can be linked by recombinant methods via a synthetic linker that makes the two a single protein chain, where the VL and VH regions pair to form a monovalent molecule (called Single-chain Fc (scFv); see eg Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
  • Single chain antibodies are also intended to be included within the meaning of the term.
  • These antibody fragments can be obtained by common techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as whole antibodies. Examples of antigen-binding fragments of the invention include, for example but not limited to, Fab, Fab', F(ab') 2 , Fv fragments, single chain Fv (scFv) fragments and single domain fragments.
  • the Fab fragment contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy-terminus of the CH1 domain of the heavy chain, including one or more cysteines from the antibody hinge region.
  • Fab' fragments are generated by cleavage of disulfide bonds at the hinge cysteines of F(ab')2 pepsin digests. Additional chemical couplings of antibody fragments are known to those of ordinary skill in the art.
  • Fab and F(ab')2 fragments lack the fragment crystallizable (Fc) region of intact antibodies, are more rapidly cleared from the animal's circulation, and may have less nonspecific tissue binding than intact antibodies (see, e.g., Wahl et al. Man, 1983, J. Nucl. Med. 24:316).
  • Fc region refers to the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of the hinge region, the CH2 domain and the CH3 domain, which mediate the binding of the immunoglobulin to host tissues or factors , including binding to Fc receptors located on various cells of the immune system (eg, effector cells) or binding to the first component of the classical complement system (eg, C1q), including native sequence Fc regions and variant Fc regions.
  • the human IgG heavy chain Fc region is the segment from its amino acid residue at position Cys226 or Pro230 to the carboxy-terminus, although its boundaries may vary.
  • the C-terminal lysine of the Fc region may or may not be present.
  • Fc may also refer to this region in isolation, or in the case of an Fc-containing protein polypeptide, such as an "Fc region-containing binding protein", also referred to as an "Fc fusion protein” (eg, an antibody or an immunoadhesin).
  • the native sequence Fc region of the antibody of the present invention includes human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.
  • the Fc region contains the CH2 and CH3 constant domains of each of the antibody's two heavy chains; the IgM and IgEFc regions contain the three heavy chain constant domains in each polypeptide chain domain (CH domains 2-4). .
  • an “Fv” fragment is the smallest fragment of an antibody that contains the complete target recognition and binding site.
  • This region consists of a dimer of one heavy and one light chain variable domain in tight non-covalent association (VH-VL dimer).
  • VH-VL dimer the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer.
  • six CDRs confer target binding specificity to an antibody.
  • a “single-chain Fv” or “scFv” antibody-binding fragment comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form structures that facilitate target binding.
  • H chain the heavy chain (heavy chain, H chain) is about twice the size of the light chain, contains 450-550 amino acid residues, and has a molecular weight of about 55 or 75kD.
  • Each H chain contains a cyclic peptide composed of 4 to 5 intrachain disulfide bonds. Different H chains have different antigenicities due to the sequence of amino acid composition, the number and position of disulfide bonds, and the types and quantities contained in them.
  • H chains According to the difference in antigenicity of H chains, they can be divided into five categories: ⁇ chains , ⁇ chain, ⁇ chain, ⁇ chain, and ⁇ chain, and different H chains and L chains ( ⁇ or ⁇ chains) to form complete immunoglobulin molecules are called IgM, IgG, IgA, IgD, and IgE, respectively.
  • the ⁇ , ⁇ and ⁇ chains contain 4 peptides, and the ⁇ and ⁇ chains contain 5 cyclic peptides.
  • light chain refers to a polypeptide chain that is smaller in molecular weight relative to the heavy chain in an immunoglobulin monomer molecule.
  • the variable amino acid sequence in the 1/2 region near the amino terminal (N-terminal) of each light chain is the light chain variable region (VL), which is a component of the Ig molecule and the antigen-binding site.
  • the relatively stable amino acid composition and sequence in the remaining 1/2 region is the light chain constant region (CL). Due to certain differences in the amino acid sequence of the light chain constant region, there are two types of light chains, kappa and lambda.
  • Single domain antibody (single domain antibody, sdAb) is a special type of antibody that contains only one antibody heavy chain, also known as nanobody. Similar to traditional diabodies, it can selectively bind to specific antigens. Single-domain antibodies were first discovered in camelids and later in cartilaginous fishes such as nurse sharks. Single Domain Antibody The variable region (VHH) of a single heavy chain antibody is a single functional domain that can completely bind antigen, only 12-15kDa. VHH has a simple structure, and has the advantages of high specificity, high affinity, low immunogenicity, good permeability when binding to an antigen, and the ability to access relatively hidden targets that cannot be accessed by conventional antibodies during tumor treatment.
  • single-domain antibodies have only one chain, there is no mismatch problem when diabody fusion occurs. Based on these advantages, the use of single-domain antibodies as the antigen-binding sequence of bispecific antibodies has great advantages and has gradually become a research hotspot (Serge Muyldermans (2013), Annu.Rev.Biochem.82:775-797).
  • Single variable domain antibody is currently the smallest antibody molecule and was originally discovered in camel blood by Belgian scientist Hamers, R. It is a class of engineered antibody products that has attracted much attention.
  • single variable domain antibodies also have some unique functional characteristics, such as small molecular weight, strong stability, good solubility, easy expression, strong targeting, and simple humanization.
  • VHHs and Nanobodies For a further description of VHHs and Nanobodies, reference is made to the review article by Muyldermans 2001 (Reviews in Molecular Biotechnology 74:277-302), and to the following patent application mentioned as general background art: VrijeUniversiteit Brussel WO 94/04678, WO 95/04079 and WO 96/34103; WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/ 44301, EP 1134231 and WO 02/48193; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of Vlaams Instituutvoor Biotechnologie (VIB); Algonomics N.V.
  • Nanobodies may be characterized in particular by the presence of one or more "signature residues" in one or more framework sequences.
  • a further description of Nanobodies, including humanization and/or camelization of Nanobodies, and other modifications, parts or fragments, derivatives or "Nanobody fusions" can be found, for example, in WO 08/101985 and WO 08/142164. ", multivalent constructs (including some non-limiting examples of linker sequences) and different modifications that increase the half-life of Nanobodies and their preparation.
  • PD-L1 that is, PD-L1 (programmed death ligand 1) full name programmed death receptor ligand 1, also known as surface antigen differentiation cluster 274 (cluster of differentiation 274, CD274) or B7 homolog (B7homolog 1, B7-H1), encoded by the CD274 gene, is the ligand of PD-1 (programmed cell death 1, programmed death receptor 1).
  • PD-L1 is the first type of transmembrane protein with a size of 40kDa. It is expressed on T cells, B cells and other immune cells and tumor cells. Under normal circumstances, the immune system will respond to foreign antigens gathered in lymph nodes or spleen, triggering Antigen-specific cytotoxic T cells (CD8+ Tcell proliferation).
  • the tumor cells When PD-L1 on the tumor cell membrane combines with PD-1 on immune cells such as T cells, the tumor cells send out inhibitory signals, reducing the proliferation of lymph node CD8+ T cells, which in turn causes T cells to fail to recognize tumor cells and to treat tumor cells. Produce a killing effect, and the immune function of the body is suppressed.
  • LAG-3 (Lymphocyte-activation-gene-3) is a gene encoded by LAG-3, mainly expressed in activated T cells, NK cells, and plasmacytoid dendrites, with T cell function Cell surface molecular proteins that regulate function. Discovered in 1990, it was named CD233 at the 7th International Symposium on Human Leukocyte Differentiation Antigen in 2007.
  • the main ligand of LAG-3 is MHCII, which negatively regulates the proliferation and activation of T cells in a similar manner to CTLA-4 and PD-1. Studies have also shown that it plays a role in inhibiting the function of regulatory T cells and maintaining CD8 T cells The effect of being in a state of tolerance, etc. Preclinical studies have shown that inhibiting LAG-3 can allow T cells to restore cytotoxicity, thereby limiting tumor growth and becoming the target of many pharmaceutical companies' tumor immunotherapy
  • bispecific antibodies refers to an antibody structure that binds to different epitopes on the same or different antigens. Thus, bispecific antibodies are able to bridge two different molecules, functioning to recruit effector molecules, effector cells, viruses and drug delivery systems to the target structure. The characteristic of bispecific antibodies that can simultaneously recognize two different molecules (receptors and/or ligands) improves the selectivity and functional affinity of antibodies.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector in which additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in the host cells into which they are introduced (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can integrate into the genome of the host cell after introduction into the host cell and thereby replicate along with the host genome.
  • vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”).
  • expression vectors useful in recombinant DNA techniques are usually in the form of plasmids.
  • viral vectors eg, replication defective retroviruses, adenoviruses and adeno-associated viruses
  • nucleic acid molecule can be single- or double-stranded, and can be cDNA.
  • host cell is a cell in which a vector can be propagated and its DNA can be expressed, which can be prokaryotic or eukaryotic.
  • the term also includes any progeny of the subject host cell. It is understood that not all progeny will be identical to the parental cells as mutations may occur during replication and such progeny are included.
  • immune-related disease refers to an immune-related disease in a mammal that is caused, mediated, or otherwise contributed to by components of the mammalian immune system, and also includes a disease in which stimulating or interfering with the immune response has an ameliorating effect on the development of the disease.
  • the term includes immune-mediated inflammatory diseases, non-immune-mediated inflammatory diseases, infectious diseases, immunodeficiency diseases, tumors, and the like.
  • cancer refers to or describe the physiological condition of a mammal in which a population of cells is characterized by unregulated cell growth.
  • examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, benign or malignant tumors.
  • cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, peritoneal carcinoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma , cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulva cancer, thyroid cancer, brain cancer, hepatic carcinoma and various types of head and neck cancer, neurofibromatosis type I or type II.
  • Other examples of such cancers include those that are therapy resistant, refractory or metastatic.
  • the present invention provides a series of anti-LAG-3/PD-L1 bispecific molecules.
  • humanized anti-LAG-3 antibody for specific information, please refer to the Chinese application with application number: 202210118342.8
  • anti-PD-L1 nanobody B6 for specific information, please refer to the Chinese application with application number: 202210524220.9
  • F2 for specific information, please refer to Application number: Chinese application of 202110433149.9
  • a bispecific antibody with a head-to-tail structure for specific information, please refer to the Chinese application with application number: 202210118342.8
  • anti-PD-L1 nanobody B6 for specific information, please refer to the Chinese application with application number: 202210524220.9
  • F2 for specific information, please refer to Application number: Chinese application of 202110433149.9
  • a bispecific antibody with a head-to-tail structure for specific information, please refer to the Chinese application with application number: 202210524220.9
  • a bispecific antibody with a head-to-tail structure for specific information, please
  • the head-to-tail structure design enables the bispecific binding molecule to effectively bind recombinant and cell surface PD-L1 and LAG-3 at the same time, blocking the binding of PD-1/PD-L1, MHCII/LAG-3 and FSTL-1/LAG A combination of -3.
  • the bispecific molecule can effectively bridge the cells expressing LAG-3 and PD-L1, which is beneficial to gather the de-inhibited T cells around the tumor cells expressing PD-L1, and exert anti-tumor efficacy.
  • both LAG-3 and PD-L1 have been proven to be important targets leading to tumor immune escape.
  • Multiple LAG-3 antibodies are in different clinical stages, and several PD-L1 antibodies have been approved for marketing.
  • the bispecific antibody can maintain the anti-tumor activity of the respective targets, in order to achieve a better anti-tumor therapeutic effect than the combination of the two monoclonal antibodies .
  • the traditional (G4S)2-4 design can be used, or the GGGGSPGGGSPGGGS design can be used, the latter is more conducive to the structural stability of the bispecific binding molecule.
  • LAG-3 molecules are expressed on the surface of T cells
  • PD-L1 molecules are expressed on the surface of tumor cells. Therefore, the bispecific binding molecules of the embodiments of the present invention can be expressed between LAG-3-positive and PD-L1-positive cells Form a bridging effect, thereby recruiting T cells in the tumor environment, and then play a better T cell-specific killing effect.
  • both the bispecific molecules hz7F10-hzF2 and hz7F10-hzB6 can effectively inhibit the growth of tumors, and their efficacy is better than that of anti-PD-L1 monoclonal antibody and LAG-3 monoclonal antibody.
  • the experimental results show that the bispecific binding molecule of the embodiment of the present invention has good application value in anti-tumor therapy.
  • Figure 1 Structural diagram of LAG-3 and PD-L1 bispecific antibody
  • Figure 2 Interaction between LAG-3 binding activity and PD-L1 binding activity of hz7F10-hzB6;
  • Figure 2A hz7F10-hzB6 does not affect the binding to PD-L1 after binding to LAG-3;
  • Figure 3 FACS analysis of the binding of hz7F10-hzB6, hz7F10-hzF2 to cell surface LAG-3;
  • Figure 4 FACS analysis of the binding of hz7F10-hzB6, hz7F10-hzF2 to cell surface PD-L1;
  • Figure 5 The binding specificity of hz7F10-hzB6 and hz7F10-hzF2 to different species of LAG-3/PD-L1.
  • Figure 5B the binding of hz7F10-hzF2 to human LAG-3, cynomolgus LAG-3, and mouse LAG-3
  • Figure 5C the binding of BMS-986016 to human LAG-3, cynomolgus LAG-3, Binding of mouse LAG-3
  • Figure 5D binding of hz7F10-hzB6 to human PD-1, cynomolgus monkey PD-1, mouse PD-L1, and rat PD-L1
  • Figure 5E hz7F10-hzF2 binding to human PD -1. Combination of cynomolgus monkey PD-1, mouse PD-L1, and rat PD-L1;
  • Figure 6 The blocking activity of hz7F10-hzB6 and hz7F10-hzF2 on the combination of MHCII and LAG-3;
  • Figure 7 The blocking activity of hz7F10-hzB6 and hz7F10-hzF2 on the binding of FGL1 and LAG-3;
  • Figure 8 The blocking activity of hz7F10-hzB6 and hz7F10-hzF2 on PD-1/PD-L1;
  • Figure 9 Cross-linking of bispecific antibodies to CHO-PD-L1 cells and HEK293-LAG-3 cells;
  • Figure 10 Inhibitory effect of hz7F10-hzB6 and hz7F10-hzF2 on human hPD-1/hPD-L1/hLAG-3 transgenic mice subcutaneously transplanted with MC38-hPDL1 mouse colon cancer tumor growth, wherein Figure 10A is a schematic diagram of body weight results, and Figure 10B Schematic diagram of tumor growth curve;
  • Figure 11 Inhibitory effect of hz7F10-hzB6 and hz7F10-hzF2 on tumor growth of H1975 human lung adenocarcinoma cells transplanted subcutaneously in human PBMC immune reconstituted mice, wherein Figure 11A is a schematic diagram of body weight results, and Figure 11B is a schematic diagram of tumor growth curves.
  • Example 1 Anti-human LAG-3 humanized antibody hz7F10 and control antibody
  • the murine antibody 7F10 was obtained by immunizing mice with recombinant human LAG-3 protein (NCBI accession number: NP_002277.4) antigen, and then humanized to obtain the humanized antibody hz7F10.
  • the amino acid sequence of the light chain variable region (hz7F10-L) of hz7F10 is shown in SEQ ID NO.1; the amino acid sequence of the light chain constant region is shown in SEQ ID NO.2; the amino acid sequence of the heavy chain variable region (hz7F10-H) is shown in sequence SEQ ID NO.3; the amino acid sequence of the heavy chain constant region is shown in the sequence SEQ ID NO.4, and the CDR is defined by Kabat.
  • SEQ ID NO.1 Amino acid sequence of light chain variable region of humanized antibody hz7F10 (CDRs 1, 2 and 3 are SEQ ID NO.14, 15 and 16, respectively)
  • SEQ ID NO.2 Amino acid sequence of light chain constant region of humanized antibody hz7F10
  • SEQ ID NO.3 Amino acid sequence of heavy chain variable region of humanized antibody hz7F10 (CDRs 1, 2 and 3 are SEQ ID NO.17, 18 and 19, respectively)
  • SEQ ID NO.4 Amino acid sequence of heavy chain constant region of humanized antibody hz7F10
  • the gene encoding the heavy chain variable region of SEQ ID NO: 3 of hz7F10 was cloned into a eukaryotic expression vector (PPT5, purchased from Beijing Huayueyang Co., Ltd. biology, product number VECT6098) to form a heavy chain recombinant expression vector for constructing hz7F10; on the other hand, by cloning the gene encoding the light chain variable region SEQ ID NO: 1 of hz7F10 into a light chain constant region containing SEQ ID NO: 2 On the eukaryotic expression vector of the gene, the light chain recombinant expression vector of hz7F10 was constructed. Then double plasmids were transfected into eukaryotic cells for expression and purification to obtain the humanized monoclonal antibody hz7F10 against LAG-3.
  • PPT5 eukaryotic expression vector
  • control antibody BMS-986016 was fully synthesized, cloned into a eukaryotic transient expression vector using the same strategy, and expressed and purified in eukaryotic cells to obtain the recombinant protein of the control antibody BMS-986016.
  • amino acid sequence of the control antibody BMS-986016 is derived from WHO Drug Information (Vol.32, No.2, 2018), the amino acid sequence of the heavy chain is shown in SEQ ID NO.5, and the amino acid sequence of the light chain is shown in SEQ ID NO.6.
  • SEQ ID NO.5 Amino acid sequence of heavy chain of control antibody BMS-986016
  • SEQ ID NO.6 Control antibody BMS-986016 light chain amino acid sequence
  • Example 2 Humanized anti-PD-L1 single domain antibody Fc fusion proteins hzB6-Fc and hzF2-Fc
  • Human PD-L1 recombinant protein antigen (PD-L1 sequence number: NP_054862.1, 19aa-238aa) was used to immunize camels, and peripheral blood mononuclear cells (PBMC) were isolated and total RNA was extracted for reverse transcription. Template for constructing camel immune library.
  • the constructed camel immune library was screened by solid-phase screening method using PD-L1 recombinant protein to obtain specific phage display single-domain antibodies VHH-B6 and VHH-F2, and then the variable region gene was humanized , obtained the sequences of humanized hzB6 (SEQ ID NO.7) and hzF2 (SEQ ID NO.8), and cloned them respectively into eukaryotic cells containing human Fc (IgG1, hFc, SEQ ID NO.9) coding genes
  • Recombinant antibodies of hzB6-Fc and hzF2-Fc were constructed on the expression vector (PPT5, Beijing Huayueyang Biology Co., Ltd., Cat. No. VECT6098), and the specific sequences are as follows, wherein the CDRs are defined by Kabat.
  • SEQ ID NO.7 Amino acid sequence of variable region of humanized single domain antibody hzB6 (CDRs 1, 2 and 3 are SEQ ID NO.20, 21 and 22, respectively)
  • SEQ ID NO.8 Amino acid sequence of variable region of humanized single domain antibody hzF2 (CDRs 1, 2 and 3 are SEQ ID NO.23, 24 and 25, respectively)
  • SEQ ID NO.9 Amino acid sequence of human IgG1 Fc region
  • a bispecific expression vector was constructed on the heavy chain expression vector of hz7F10 obtained in Example 1 by the method of molecular cloning, and the structure schematic diagram of the bispecific antibody is shown in FIG. 1 .
  • the heavy chain recombinant expression vectors of hz7F10-hzB6 (SEQ ID NO.11) and hz7F10-hzF2 (SEQ ID NO.12) were respectively constructed, wherein the amino acid sequence of the linker is shown in SEQ ID NO.10 (GGGGSPGGGSPGGGS).
  • the hz7F10-hzB6 and hz7F10-hzF2 heavy chain recombinant expression vectors and the hz7F10 light chain (SEQ ID NO.13) recombinant expression vector obtained in Example 1 were co-transfected into HEK293 cells for transient expression, and hz7F10-hzB6 and hz7F10 were obtained after purification - The pure double-antibody recombinant protein of hzF2 was used for subsequent experiments.
  • the affinity of hz7F10-hzB6 and hz7F10-hzF2 was determined by using the 4SPR instrument of Reichert Company, using the Anti-human Fc antibody capture method. Firstly, the anti human Fc antibody was immobilized on the surface of the chip by amino coupling method, followed by affinity measurement: the target antibody was diluted to 40ug/ml with HBS-EP, 25uL/min, and injected for 2min; the antigen was diluted 2 times from 200nM to 8 Concentration, 25uL/min, combined for 2min, dissociated for 3min. Finally, the dextran chip (13206066) was regenerated for 30ses using 3M MgCl 2 to cycle for detection of the next sample.
  • the method of capturing the Fc segment of the antibody with the capture antibody (AHC) bioprobe of the anti-human antibody Fc segment was used to determine the simultaneous binding characteristics of hz7F10-hzB6 to LAG-3/PD-L1.
  • the specific operation is as follows.
  • the hz7F10-hzB6 antibody was diluted to 4 ⁇ g/ml with PBS buffer, and flowed over the surface of the AHC probe (Cat: 18-0015, PALL) for 120 s.
  • Human LAG-3 extracellular domain fusion protein (Shenzhou Yiqiao, Cat: 16498-H08H) 60nM was used as the mobile phase, the binding time was 300s, and then PD-L1 (NCBI serial number: NP_054862.1) 60nM was used as the mobile phase, The binding time is 300s and the dissociation time is 300s.
  • first PD-L1 was used as the first mobile binding phase, and LAG-3 was used as the second mobile binding phase.
  • the experimental results are shown in Figure 2. The results show that after hz7F10-hzB6 binds to LAG-3/PD-L1 on one side, it has no effect on the binding to PD-L1/LAG-3 on the other side.
  • LAG-3 (NCBI accession number: NP_002277.4) full-length plasmid to transiently transfect HEK293 cells, centrifuge to collect cells after 48h, divide cells into 5 ⁇ 10 5 cells/sample/100 ⁇ L, add the antibody to be tested in serial dilution, add The final concentration of the antibody is: the highest concentration 132nM, 3-fold serial dilution of 10 gradients.
  • the tumor cell MBA-MD-231 was taken, 5 ⁇ 10 5 cells/sample/100 ⁇ L, the method was the same as that of HEK293-LAG-3, and the detection results were also shown (Figure 4), hz7F10-hzB6, hz7F10-hzF2 were expressed in
  • the binding activity of PD-L1 on the cell surface is similar to that of the naked antibody hzB6-Fc and hzF2-Fc, and similar to that of the positive control antibody BMS-986016.
  • the EC50 values are shown in Table 2.
  • Human recombinant protein LAG-3 (purchased from Yiqiao Shenzhou, Cat: 16498-H08H), human recombinant protein PD-L1 (NCBI sequence number: NP_054862.1), monkey recombinant protein LAG-3 (Cat: C998, inshore Protein Technology Co., Ltd.), monkey recombinant protein PD-L1 (Cat: CC29, Nearshore Protein Technology Co., Ltd.), mouse recombinant protein LAG-3 (Cat: 53069-M08H, Beijing Sino Biological Science and Technology Co., Ltd.), mouse PD -L1 (Cat: 50010-M08H, Beijing Sino Biological Technology Co., Ltd.
  • hz7F10-hzB6 and hz7F10-hzF2 can significantly block the binding activity of LAG-3 and MHC class II molecules on the surface of A375 cells, and their blocking activity is equivalent to that of the control antibody BMS-986016.
  • the reporter gene system detects the blocking activity of hz7F10-hzB6 and hz7F10-hzF2 on the PD-1/PD-L1 pathway
  • Programmed cell death protein 1 is a receptor expressed on activated T cells, and its binding to ligands PD-L1 and PD-L2 negatively regulates immune responses.
  • TCR on the surface of CHO-K1 cells has a positive regulatory effect and can activate the NFAT pathway in T cells.
  • the PD-1/PD-L1 interaction suppresses T cell activity, allowing cancer cells to escape immune surveillance.
  • antibodies block this pathway the NFAT pathway in T cells mediates substrate luminescence.
  • CHO-PD-L1-CD3L cells were trypsinized, and the reaction was terminated with complete medium.
  • hz7F10-hzB6 and hz7F10-hzF2 can significantly block the PD-1/PD-L1 signaling pathway, and the blocking effect is comparable to that of nanobodies hzB6-Fc and hzF2-Fc.
  • Example 7 Analysis of the cross-linking effect of LAG-3/PD-L1 bispecific antibody on cells expressing LAG-3 and PD-L1
  • Example 8 Anti-tumor efficacy of LAG-3/PD-L1 bispecific antibody on human PD-1/PD-L1/LAG-3 transgenic mice subcutaneously implanted with MC38-hPDL1 mouse colon cancer tumor model
  • Murine colon cancer tumor cells MC38-hPDL1 highly expressing human PD-L1 were inoculated into female hPD-1/hPD-L1/hLAG-3 transgenic mice (C57-derived PD-1/PD-L1/LAG-3 human mice, Biocytogen Jiangsu Gene Biotechnology Co., Ltd., 6-8 weeks) under the skin of the right anterior flank rib, when the tumor grows to about 100mm3 , they are divided into groups, and there are 4 groups, 6 mice in each group, respectively.
  • hz7F10-hzF2 (6mg/kg, ip, Biw ⁇ 3 weeks) group
  • hz7F10-hzB6 (6mg/kg, ip, Biw ⁇ 3 weeks) group
  • hz7F10 (5mg/kg, ip, Biw ⁇ 3 weeks) group
  • NC-hIgG1 (6mg/kg, ip, Biw ⁇ 3 weeks) group.
  • the tumor volume and body weight were measured every week, and the relationship between the body weight and tumor volume of the tumor-bearing mice and the administration time was recorded.
  • Example 9 Anti-tumor efficacy of LAG-3/PD-L1 bispecific antibody on H1975 human lung adenocarcinoma tumor model subcutaneously transplanted into human PBMC immune reconstitution mice
  • Human lung cancer H1975 cells were inoculated into PBMC immune system humanized male NCG mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd., 22-24g) subcutaneously on the right side of the anterior flank, and divided into groups when the tumor grew to about 60mm3 medicine, a total of 5 groups, 6 rats in each group, respectively: hz7F10-hzF2 (20mg/kg, ip, TIW ⁇ 9 times) group, hz7F10-hzB6 (20mg/kg, ip, TIW ⁇ 9 times) group, hz7F10 ( 15mg/kg, ip, TIW ⁇ 9 times) group, hzB6-Fc (12mg/kg, ip, TIW ⁇ 9 times) group, NC-hIgG1 (20mg/kg, ip, TIW ⁇ 9) group.
  • the tumor volume and body weight were measured every week, and the relationship between the body weight and tumor volume of the tumor

Abstract

一种靶向LAG-3和PD-L1的双特异性分子及其应用。在人源化抗人LAG-3抗体的基础上，在抗体重链的C端通过连接子连接抗PD-L1纳米抗体。将纳米抗体通过上述连接子连接于抗体重链C端的方式不仅减少了Fc的效应子功能、避免了活化T细胞的清除，而且使双特异性抗体的结构更加稳定、重组表达的组装效率更高。并且，该双特异性分子能够同时抑制PD-1/PD-L1信号通路和LAG-3/MHCII、LAG3/信号通路，实现LAG-3阳性和PD-L1阳性细胞的桥连，将解除抑制的T细胞聚集在表达PD-L1的肿瘤细胞周围，具有良好的抑瘤效果。

Description

靶向LAG-3和PD-L1的新型双特异抗体及其应用
优先权信息
本申请请求2021年6月23日向中国国家知识产权局提交的、专利申请号为202110695985.4的专利申请的优先权和权益，并且通过参照将其全文并入此处。
技术领域
本发明属于抗体工程领域，具体而言，本发明涉及新型的经人工设计的双特异抗体分子，特别是同时特异性结合LAG-3和PD-L1的新型抗体和抗体片段以及含有所述抗体或抗体片段的组合物，还涉及结合LAG-3和PD-L1的抗体在肿瘤等疾病治疗中的用途。
背景技术
LAG-3(Lymphocyte-activation-gene-3)是重要的免疫检查点蛋白，属于Ⅰ型跨膜蛋白，由LAG-3基因编码，主要表达在活化的T细胞、NK细胞、和浆细胞样树突状等细胞表面。LAG-3通过与其配体作用，调控T细胞功能。到目前为止，已发现的LAG-3配体共有5种：MHC-II，肝窦内皮细胞凝集素(liver sinusoidal endothelial cell lectin，LSECtin)、半乳糖凝集素-3(galectin3)、α-突触核蛋白原纤维(α-synuclein fibrils)，以及纤维蛋白样蛋白1(fibrinogen-likeprotein1，FGL1)，其中最主要的配体是MHC II。正常情况下，LAG-3与其配体介导负性信号，调节T细胞增殖和功能，维持机体T细胞的稳态。在肿瘤微环境中，LAG-3和其配体介导的负性信号减弱CD4+T细胞和CD8+T细胞增殖、分化，并促进Treg细胞的分化，最终实现免疫抑制。抑制或敲除LAG-3可以解除T细胞抑制。LAG-3通路的抑制有助于耗竭性T细胞的功能恢复，提高T细胞抗肿瘤活性。
PD-1及其配体PD-L1是肿瘤免疫的重要靶点。PD-1、PD-L1是一对免疫抑制分子，是免疫系统防止自身免疫过激的重要组成部分，其通路的激活具有抑制肿瘤免疫应答、诱导肿瘤特异性T细胞凋亡的作用,与肿瘤发展关系密切。PD-L1全称是程序性死亡配体-1，可以结合T细胞表面的受体PD-1，发挥免疫抑制作用。PD-L1属于抑制型免疫检查点分子，表达于黑色素瘤、非小细胞肺癌、肾细胞癌、头颈部鳞癌等多种恶性肿瘤细胞表面。PD-L1与T细胞表面的免疫抑制性受体PD-1结合后，可诱导T细胞凋亡、失能、耗竭，进而抑制肿瘤抗原特异性T细胞的激活、增殖和抗肿瘤功能，实现肿瘤免疫逃逸。PD-1/PD-L1阻断型抗体可以解除PD-L1的免疫抑制作用，增强体内免疫细胞T细胞对肿瘤细胞的识别和杀伤，从而达到杀灭肿瘤的作用。
由于双特异抗体具有多种应用，有必要基于双特异抗体研究多种疾病的治疗方法。
发明内容
本发明旨在至少解决现有技术中存在的技术问题之一。为此，本发明的一个目的在于提出一种靶向LAG-3和PD-L1的双特异性抗体及其应用。在人源化抗人LAG-3抗体的基础上，在抗体重链的C端通过连接子连接抗PD-L1纳米抗体。将纳米抗体通过上述连接子连接于抗体重链C端的方式不仅减少了Fc的效应子功能、避免了活化T细胞的清除，而且使双特异性抗体的结构更加稳定、重组表达的组装效率更高。在肿瘤抑制活性方面，本发明制备的靶向LAG-3和PD-L1的双特异性抗体hz7F10-hzF2和hz7F10-hzB6能够同时抑制PD-1/PD-L1信号通路和LAG-3/MHCII、LAG3/信号通路，实现LAG-3阳性和PD-L1阳性细胞的桥连，将解除抑制的T细胞聚集在表达PD-L1的肿瘤细胞周围，从而有效抑制肿瘤的生长。
具体而言：
第一方面，本发明提供了一种双特异性结合分子。根据本发明的实施例，该双特异性结合分子包括：第一抗原结合部，所述第一抗原结合部包括：第一肽段，所述第一肽段包括第一重链可变区和第一轻链可变区，其中，所述第一重链可变区的互补决定区(CDR)包括H1-CDR1、H1-CDR2和H1-CDR3，所述第一轻链可变区的CDR包括L1-CDR1、L1-CDR2和L1-CDR3；第二肽段，所述第二肽段包括第二重链可变区和第二轻链可变区，其中，所述第二重链可变区的CDR包括H2-CDR1、H2-CDR2和H2-CDR3，所述第二轻链可变区的CDR包括L2-CDR1、L2-CDR2和L2-CDR3，且所述第二肽段与所述第一肽段共价连接，具体地，所述第二肽段的N端与所述第一肽段的C端共价连接；第二抗原结合部，所述第二抗原结合部包括:第三肽段，所述第三肽段为纳米抗体，包括第三重链可变区，且与所述第一肽段的C端通过接头相连，所述第三重链可变区的CDR包括H3-CDR1、H3-CDR2和H3-CDR3。
根据本发明的实施例，所述第一抗原结合部含特异性结合程序性细胞死亡-配体1(PD-L1)，所述第二抗原结合部特异性结合淋巴细胞活化基因-3(LAG3)。
根据本发明的实施例，所述双特异性结合分子，优选地，第一抗原结合部包含Fc结构域，所述Fc结构域是IgG，特别是IgG1Fc结构域。
根据本发明的实施例，所述Fc结构域包含L234A和L235A点突变。
根据本发明的实施例，所述H1-CDR1包含SEQ ID NO：17所述的氨基酸序列，所述H1-CDR2包含SEQ ID NO：18所述的氨基酸序列和所述H1-CDR3包含SEQ ID NO：19所述的氨基酸序列，且所述L1-CDR1包含SEQ ID NO：14所述的氨基酸序列、L1-CDR2包含SEQ ID NO：15所述的氨基酸序列和L1-CDR3包含SEQ ID NO：16所述的氨基酸序列。
根据本发明的实施例，所述H2-CDR1、H2-CDR2和H2-CDR3的氨基酸序列分别与所述H1-CDR1、H1-CDR2和H1-CDR3的氨基酸序列相同，且所述L2-CDR1、L2-CDR2和L2-CDR3的氨基酸序列分别与所述L1-CDR1、L1-CDR2和L1-CDR3的氨基酸序列相同。
根据本发明的实施例，所述H3-CDR1、H3-CDR2和H3-CDR3选自下列组之一：(a)所述H3-CDR1包含SEQ ID NO：20所述的氨基酸序列，所述H3-CDR2包含SEQ ID NO：21所述的氨基酸序列和所述H3-CDR3包含SEQ ID NO：22所述的氨基酸序列；(b)所述 H3-CDR1包含SEQ ID NO：23所述的氨基酸序列，所述H3-CDR2包含SEQ ID NO：24所述的氨基酸序列和所述H3-CDR3包含SEQ ID NO：25所述的氨基酸序列。
根据本发明的实施例，所述第一重链可变区包括与SEQ ID NO：3所述的氨基酸序列，所述第一轻链可变区包括SEQ ID NO：1所述的氨基酸序列。
根据本发明的实施例，所述第一肽段进一步包括第一轻链恒定区和第一重链恒定区，且所述第二肽段进一步包括第二轻链恒定区和第二重链恒定区。
根据本发明的实施例，所述第一轻链恒定区与所述第二轻链恒定区均包括SEQ ID NO：2所述的氨基酸序列。
根据本发明的实施例，所述第一重链恒定区与所述第二重链恒定区均包括SEQ ID NO：4所述的氨基酸序列。
根据本发明的实施例，所述第三重链可变区包括SEQ ID NO：7或8所示的氨基酸序列。
根据本发明的实施例，所述多肽复合物进一步包括：第四肽段，所述第四肽段包括第四重链可变区，且与所述第二肽段的C端通过所述接头相连。
根据本发明的实施例，所述第四肽段为纳米抗体，且具有与所述第三肽段相同的CDR序列。
根据本发明的实施例，所述第四重链可变区包括SEQ ID NO：7或8所示的氨基酸序列。
根据本发明的实施例，所述接头的具有(G4S) 2-4或SEQ ID NO:10所示的氨基酸序列。
根据本发明的实施例，所述双特异性结合分子的重链具有SEQ ID NO:11或12所示的氨基酸序列。
根据本发明的实施例，所述双特异性结合分子的轻链具有SEQ ID NO:13所示的氨基酸序列。
第二方面，本发明提供一种双特异性结合分子。根据本发明的实施例，该双特异性结合分子包括两条抗体重链和两条抗体轻链，所述两条抗体重链的可变区和两条抗体轻链的可变区相互作用形成两个第一抗原结合部；所述两条抗体重链中的至少一条在C端通过接头连接第二抗原结合部，所述第二抗原结合部包含纳米抗体。
进一步，根据本发明的实施例，所述抗体重链由N端到C端依次包括：第一抗原结合部重链可变区、CH1区、CH2区、CH3区，以及可选的接头+第二抗原结合部。
进一步，根据本发明的实施例，所述两条抗体重链在Fc段包括L234A、L235A点突变，且所述两条抗体重链相同或不同；
当两条抗体重链不同时，其中一条抗体重链具有第二抗原结合部，另一条抗体重链不具有第二抗原结合部；或者两条抗体重链具有序列不同的第二抗原结合部。
进一步，根据本发明的实施例，所述抗体轻链由N端到C端依次包括：第一抗原结合部轻链可变区、VH。
进一步，根据本发明的实施例，所述接头的选自(G4S)2-4、GGGGSPGGGSPGGGS(SEQ ID NO:10)。
进一步，根据本发明的实施例，所述第一抗原结合部特异性结合LAG-3，第一抗原结合 部包含的轻链可变区具有SEQ ID NO:1所示的氨基酸序列、包含的重链可变区具有SEQ ID NO:3所示的氨基酸序列。
进一步，根据本发明的实施例，所述第二抗原结合部特异性结合PD-L1，第二抗原结合部的纳米抗体包含的氨基酸序列选自SEQ ID NO:7、SEQ ID NO:8组成的组。
进一步，根据本发明的实施例，所述抗体重链具有选自SEQ ID NO:11、SEQ ID NO:12组成的组的氨基酸序列。
进一步，根据本发明的实施例，所述抗体轻链具有SEQ ID NO:13所示的氨基酸序列。
第三方面，本发明提供一种组合物，其包含本发明前述任一项的双特异性结合分子以及可选的药学上可接受的辅料。
进一步，根据本发明的实施例，该组合物还包含生物治疗剂、化学治疗剂、天然活性成分等。
进一步，根据本发明的实施例，所述组合物的剂型为水剂、针剂、粉针剂。
第四方面，本发明提供一种多核苷酸。根据本发明的实施例，该多核苷酸编码前述任一项的双特异性结合分子中的抗体重链和/或抗体轻链。
进一步，本发明还提供多核苷酸组合。根据本发明的实施例，该多核苷酸组合包括编码所述抗体重链的多核苷酸和编码所述抗体轻链的多核苷酸。
第五方面，本发明提供一种核酸构建体。根据本发明的实施例，该核酸构建体包含前述的多核苷酸。
进一步，本发明还提供核酸构建体组合。根据本发明的实施例，该核酸构建体组合包括包含编码所述抗体重链的多核苷酸的核酸构建体和编码所述抗体轻链的多核苷酸的核酸构建体。
第六方面，本发明提供一种宿主细胞。根据本发明的实施例，该宿主细胞包含前述的多核苷酸或多核苷酸组合，或包含前述核酸构建体或核酸构建体组合。
第七方面，本发明提供一种制备前述任一项双特异性结合分子的方法。根据本发明的实施例，该方法包括以下步骤：
(1)在适合表达重组外源蛋白的条件下培养前述宿主细胞；
(2)可选的，从细胞培养物中分离纯化所述双特异性结合分子。
第八方面，本发明提供前述任一项双特异性结合分子、组合物、多核苷酸或多核苷酸组合、核酸构建体、核酸构建体组合、或宿主细胞在结合并抑制LAG3和PD-1功能中的应用，其中所述LAG3和PD-1源自人或食蟹猴。
第九方面，本发明提供了前述任一项双特异性结合分子、组合物、多核苷酸或多核苷酸组合、核酸构建体或核酸构建体组合、或宿主细胞在抑制或阻断LAG-3/MHCII、LAG-3/FGL1、和/或PD-1/PD-L1信号通路中的应用。
第十方面，本发明提供前述任一项双特异性结合分子、组合物、多核苷酸或多核苷酸组合、核酸构建体或核酸构建体组合、或宿主细胞在桥连LAG-3阳性细胞和PD-L1阳性细胞中的应用。
第十一方面，本发明提供了前述任一项双特异性结合分子、组合物、多核苷酸或多核苷酸组合、核酸构建体或核酸构建体组合、或宿主细胞在制备治疗LAG-3和/或PD-L1信号通路异常相关疾病药物中的用途。
进一步，根据本发明的实施例，所述LAG-3和/或PD-L1信号通路异常相关疾病包括异常增生性疾病或免疫相关疾病。
进一步，根据本发明的实施例，异常增殖性疾病包括肿瘤、囊肿、增生等；所述免疫相关疾病包括炎症、免疫缺陷、免疫耐受和过敏等。
根据本发明的实施例，所述肿瘤为结肠癌或肺癌。
第十二方面，本发明提供了一种治疗患有异常增生性疾病或免疫相关疾病个体的方法。根据本发明的实施例，该方法包括向所述个体施用前述双特异性结合分子或前述组合物。
第十三方面，本发明提供了一种抑制个体中肿瘤细胞生长的方法。根据本发明的实施例，该方法包括向所述个体施用有效剂量的前述双特异性结合分子或前述组合物以抑制所述肿瘤细胞的生长。
为更好理解本发明，首先定义一些术语。其他定义则贯穿具体实施方式部分而列出。
术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。术语“免疫结合”是指发生在抗体分子和抗原(对于该抗原而言抗体为特异性的)之间的特异性结合反应。免疫结合相互作用的强度或亲和力可以相互作用的平衡解离常数(KD)表示，其中KD值越小，表示亲和力越高。两分子间的的免疫结合性质可使用本领域中公知的方法定量。一种方法涉及测量抗原结合位点/抗原复合物形成和解离的速度。指特定抗体-抗原相互作用的“结合速率常数”(Ka或Kon)和“解离速率常数”(Kd或Koff)两者都可通过浓度及缔合和解离的实际速率而计算得出，参见Malmqvist M,1993,Nature,361:186-187。Kd/Ka的比率等于解离常数KD，参见Davies DR等,1990,Annual Rev Biochem.,59:439-473。可用任何有效的方法测量KD、Ka和Kd值。在优选的实施方案中，用生物发光干涉测量法来测量解离常数。在其它优选的实施方案中，可用表面等离子共振技术(例如Biacore)或KinExa来测量解离常数。
本文中的术语“抗体”意在包括全长抗体及其任何抗原结合片段(即，抗原结合部分)或单链。全长抗体是包含至少两条重(H)链和两条轻(L)链的糖蛋白，重链和轻链由二硫键连接。各重链由重链可变区(简称VH)和重链恒定区构成。重链恒定区由三个结构域构成，即CH1、CH2和CH3。各轻链由轻链可变区(简称VL)和轻链恒定区构成。轻链恒定区由一个结构域CL构成。VH和VL区还可以划分为称作互补决定区(CDR)的高变区，其由较为保守的框架区(FR)区分隔开。各VH和VL由三个CDR以及四个FR构成，从氨基端到羧基端以FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4的顺序排布。重链和轻链的可变区包含与抗原相互作用的结合域。抗体的恒定区可以介导免疫球蛋白与宿主组织或因子的结合，包括多种免疫系统细胞(例如，效应细胞)和传统补体系统的第一组分(C1q)。
术语“单克隆抗体”或“单抗”或“单克隆抗体组成”是指单一分子组成的抗体分子制品。单克隆抗体组成呈现出对于特定表位的单一结合特异性和亲和力。
本文中的术语，抗体的“抗原结合片段”(或简称为抗体部分)，是指抗体的保持有特异结合抗原能力的一个或多个片段。已证实，抗体的抗原结合功能可以通过全长抗体的片段来实施。包含在抗体的“抗原结合部分”中的结合片段的例子包括(i)Fab片段，由VL、VH、CL和CH1构成的单价片段；(ii)F(ab′)2片段，包含铰链区二硫桥连接的两个Fab片段的二价片段；(iii)由VH和CH1构成的Fd片段；(iv)由抗体单臂VL和VH构成的Fv片段；(v)由VH构成的dAb片段(Ward et al.，(1989)Nature 341：544-546)；(vi)分离的互补决定区(CDR)；以及(vii)纳米抗体，一种包含单可变结构域和两个恒定结构域的重链可变区。此外，尽管Fv片段的两个结构域VL和VH由不同的基因编码，它们可以通过重组法经由使两者成为单蛋白链的合成接头而连接，其中VL和VH区配对形成单价分子(称为单链Fc(scFv)；参见例如Bird et al.，(1988)Science 242：423-426；and Huston et al.，(1988)Proc.Natl.Acad.Sci.USA 85：5879-5883)。这些单链抗体也意在包括在术语涵义中。这些抗体片段可以通过本领域技术人员已知的常用技术而得到，且片段可以通过与完整抗体相同的方式进行功能筛选。本发明的抗原结合片段的实例包括例如但不限于Fab、Fab'、F(ab') 2、Fv片段、单链Fv(scFv)片段和单结构域片段。
Fab片段含有轻链的恒定结构域和重链的第一恒定结构域(CH1)。Fab'片段与Fab片段的不同之处在于在重链CH1结构域的羧基末端处的少数残基的添加，包括来自抗体铰链区的一个或多个半胱氨酸。通过切割在F(ab')2胃蛋白酶消化产物的铰链半胱氨酸处的二硫键产生Fab'片段。抗体片段的另外化学偶联是本领域普通技术人员已知的。Fab和F(ab')2片段缺乏完整抗体的片段可结晶(Fc)区，从动物的循环中更快速地清除，并且可能具有比完整抗体更少的非特异性组织结合(参见例如，Wahl等人，1983，J.Nucl.Med.24:316)。
术语“Fc区”或“Fc”是指免疫球蛋白重链的C端区，其含有铰链区的至少一部分、CH2结构域和CH3结构域，其介导免疫球蛋白与宿主组织或因子的结合，包括与位于免疫系统的各种细胞(例如，效应细胞)上的Fc受体结合或与经典补体系统的第一组分(例如C1q)结合，包括天然序列Fc区和变异Fc区。通常，人IgG重链Fc区为自其Cys226或Pro230位置的氨基酸残基至羧基末端的区段，但其边界可能有变化。Fc区的C-末端赖氨酸(残基447，依照EU编号系统)可以存在或可以不存在。Fc还可以指隔离的这一区域，或在包含Fc的蛋白多肽的情况下，例如“包含Fc区的结合蛋白”，还称为“Fc融合蛋白”(例如，抗体或免疫粘合素)。本发明的抗体中天然序列Fc区包括人IgG1，IgG2(IgG2A，IgG2B)，IgG3和IgG4。在IgG、IgA和IgD抗体同种型中，Fc区包含抗体的两条重链的每一条的CH2和CH3恒定结构域；IgM和IgEFc区包含在每条多肽链中的三个重链恒定结构域(CH结构域2-4)。。
“Fv”片段是含有完整靶识别和结合位点的抗体的最小片段。该区域由以紧密的非共价结合的一个重链和一个轻链可变结构域的二聚体(VH-VL二聚体)组成。在该构型中，每个可变结构域的三个CDR相互作用，以限定在VH-VL二聚体的表面上的靶结合位点。通常，六个CDR对抗体赋予靶结合特异性。然而，在一些情况下，甚至单个可变结构域(或仅包含对于靶特异性的三个CDR的Fv的一半)可以具有识别且结合靶的能力，尽管其亲和力低 于整个结合位点。
“单链Fv”或“scFv”抗体结合片段包含抗体的VH和VL结构域，其中这些结构域存在于单条多肽链中。一般地，Fv多肽进一步包含在VH和VL结构域之间的多肽接头，其致使scFv能够形成有利于靶结合的结构。
术语“重链”，重链(heavy chain，H链)大小约为轻链的2倍，含450～550个氨基酸残基，分子量约为55或75kD。每条H链含有4～5个链内二硫键所组成的环肽。不同的H链由于氨基酸组成的排列顺序、二硫键的数目和位置、含的种类和数量不同，其抗原性也不相同，根据H链抗原性的差异可将其分为5类：μ链、γ链、α链、δ链和ε链，不同H链与L链(κ或λ链)组成完整免疫球蛋白的分子分别称之为IgM、IgG、IgA、IgD和IgE。γ、α和δ链上含有4个肽，μ和ε链含有5个环肽。
术语“轻链”，轻链(light chain，L)指在免疫球蛋白单体分子中相对于重链而论，在分子量上较小的多肽链。每条轻链近氨基末端(N端)1/2区域内的氨基酸组成序列多变处为轻链可变区(VL)，是Ig分子与抗原结合部位的一个组成部分。其余1/2区域内的氨基酸组成及排列顺序相对稳定处为轻链恒定区(CL)。由于轻链恒定区内氨基酸序列存在某些差异，故轻链有k和λ两型。
单域抗体(single domain antibody，sdAb)是一类特殊的，只包含一条抗体重链的抗体，也称为纳米抗体。和传统双链抗体类似，它可以选择性的与特定抗原结合。单域抗体最早在骆驼科动物中被发现，之后在护士鲨等软骨鱼纲动物中也被发现。单域抗体单个重链抗体可变区(VHH)是能完整结合抗原的单个功能域，只有12-15kDa。VHH结构简单，在与抗原结合时具有高特异性、高亲和力、免疫原性低、渗透性好以及在进行肿瘤治疗时具有接触到不能被常规抗体接触的较为隐蔽靶点的能力等优点。此外，因为单域抗体只有一条链，所以不会产生双链抗体融合时的错配问题。基于这些优点，利用单域抗体作为双特异性抗体的抗原结合序列具有很大的优势，逐渐成为研发热点(Serge Muyldermans(2013)，Annu.Rev.Biochem.82:775-797)。单可变域抗体是目前最小的抗体分子最初由比利时科学家Hamers，R在骆驼血液中发现，它是工程化抗体产品中备受关注的一类。单可变域抗体除具备单克隆抗体的抗原反应性外，还拥有一些独特的功能特性，如分子质量小，稳定性强、可溶性好、易表达、靶向性强、人源化简单等，尤其是适合进行双/多特异治疗性抗体的开发和Car-T/M/NK等疗法的开发。目前单可变域抗体和/或基于单可变域抗体的双/多特异性抗体开发已成为研发热点。
对于VHH和纳米抗体的进一步描述，参考Muyldermans 2001(分子生物技术中的综述(Reviews in Molecular Biotechnology)74:277-302)的综述文章，以及参考作为一般背景技术提及的以下专利申请：VrijeUniversiteit Brussel的WO 94/04678、WO 95/04079和WO 96/34103；Unilever的WO 94/25591、WO 99/37681、WO 00/40968、WO 00/43507、WO00/65057、WO 01/40310、WO 01/44301、EP 1134231和WO 02/48193；Vlaams Instituutvoor Biotechnologie(VIB)的WO 97/49805、WO 01/21817、WO 03/035694、WO 03/054016和WO 03/055527；Algonomics N.V.和埃博灵克斯股份有限公司的WO 03/050531；加拿大国立研究委员会(National Research  Council of Canada)的WO 01/90190；抗体研究所(Institute of Antibodies)的WO 03/025020；以及埃博灵克斯股份有限公司的WO 04/041867、WO 04/041862、WO 04/041865、WO 04/041863、WO 04/062551、WO 05/044858、WO06/40153、WO 06/079372、WO 06/122786、WO 06/122787和WO 06/122825和埃博灵克斯股份有限公司的另外的公布的专利申请。还参考在这些申请中提及的另外的现有技术，并且尤其参考在国际申请WO 06/040153的第41-43页提及的参考文献清单，该清单和参考文献通过引用结合在本文中。如在这些参考文献中描述的，纳米抗体(尤其是VHH序列和部分人源化的纳米抗体)的特征可以尤其在于在一个或多个构架序列中存在一个或多个“标志残基”。可以例如在WO 08/101985和WO 08/142164中找到纳米抗体的进一步描述，包括纳米抗体的人源化和/或骆驼源化，以及其他修饰、部分或片段、衍生物或“纳米抗体融合体”、多价构建体(包括接头序列的一些非限制性实例)和增加纳米抗体半衰期的不同修饰和它们的制备。
术语“PD-L1”，即PD-L1(programmed death ligand 1)全称程序性死亡受体配体1，也称为表面抗原分化簇274(cluster of differentiation 274，CD274)或B7同源体(B7homolog 1，B7-H1)，由CD274基因编码，是PD-1(programmed cell death 1,程序性死亡受体1)的配体。PD-L1是大小为40kDa的第一型跨膜蛋白，表达在T细胞、B细胞等免疫细胞以及肿瘤细胞上，正常情形下免疫系统会对聚集在淋巴结或脾脏的外来抗原产生反应，促发具抗原特异性的细胞毒杀性T细胞(CD8+Tcell增生)。当肿瘤细胞膜上的PD-L1与T细胞等免疫细胞上的PD-1结合后，肿瘤细胞发出抑制性信号，减低淋巴结CD8+T细胞的增殖，进而导致T细胞不能识别肿瘤细胞和对肿瘤细胞产生杀伤作用，机体的免疫功能受到抑制。
术语“LAG-3”，(Lymphocyte-activation-gene-3)是一种由LAG-3基因编码的，主要表达在活化的T细胞、NK细胞、和浆细胞样树突状，具有T细胞功能调节功能的细胞表面分子蛋白。于1990年被发现，2007年第7届国际人类白细胞分化抗原专题会议命名为CD233。LAG-3的主要配体是MHCII，它以CTLA-4和PD-1相似的方式负性调节T细胞的增殖与活化，也有研究表明它在抑制调节性T细胞功能中发挥作用，维持CD8T细胞处于耐受性状态的作用等。临床前研究显示，抑制LAG-3能够让T细胞重新恢复细胞毒性作用，从而限制肿瘤的生长，成为许多制药公司肿瘤免疫疗法的靶点
术语“双特异性抗体”(bispecific antibodies)，一种可与相同或不同抗原上的不同表位结合的抗体结构。因此，双特异性抗体能够桥连两种不同的分子，起到将效应分子、效应细胞、病毒和药物载体系统招募至靶标结构的作用。双特异性抗体这种可同时识别两种不同分子(受体和/或配体)的特点，提高了抗体的选择性和功能性亲和力。
术语“载体”、“核酸构建体”是指能够运输与其连接的另一种核酸的核酸分子。一种类型的载体是“质粒”，其是指其中可以连接另外的DNA区段的环状双链DNA环。另一种类型的载体是病毒载体，其中额外的DNA区段可以连接到病毒基因组中。某些载体能够在它们被导入的宿主细胞中自主复制(例如，具有细菌复制起点和游离型哺乳动物载体的细菌载体)。其他载体(例如非附加型哺乳动物载体)可以在导入宿主细胞后整合到宿主细胞的基因组中，并由此与宿主基因组一起复制。此外，某些载体能够指导它们有效连接的基因的表达。 这种载体在本文中被称为“重组表达载体”(或简称为“表达载体”)。通常，在重组DNA技术中有用的表达载体通常以质粒的形式存在。然而，也包括其他形式的表达载体，如病毒载体(例如，复制缺陷型逆转录病毒，腺病毒和腺伴随病毒)，其起到等同的功能。
术语“多核苷酸”旨在包括DNA分子和RNA分子。核酸分子可以是单链或双链的，并且可以是cDNA。
术语“宿主细胞”在其中载体可以增殖并且其DNA可以表达的细胞，所述细胞可以是原核细胞或者真核细胞。该术语还包括受试宿主细胞的任何后代。应理解，并不是所有的后代都与亲本细胞相同，因为在复制过程中可能会发生突变，这类后代被包括在内。
术语“免疫相关疾病”指哺乳动物中由哺乳动物免疫系统成分引起、介导、或以其他方式促成发病的免疫相关疾病，还包括刺激或干预免疫应答对该疾病发展具有改善作用的疾病。此术语包括免疫介导的炎性疾病、非免疫介导的炎性疾病、感染性疾病、免疫缺陷病、肿瘤等。
术语“癌症”和“肿瘤”是指或描述哺乳动物的其中细胞群体的特征在于细胞生长不受调控的生理病状。癌症的实例包括但不限于癌瘤、淋巴瘤、胚细胞瘤、肉瘤、白血病、良性或恶性肿瘤。所述癌症的更特定实例包括鳞状细胞癌、小细胞肺癌、非小细胞肺癌、肺腺癌、肺鳞状癌、腹膜癌、肝细胞癌、胃肠癌、胰腺癌、成胶质细胞瘤、子宫颈癌、卵巢癌、肝癌(liver cancer)、膀胱癌、肝细胞瘤、乳癌、结肠癌、结肠直肠癌、子宫内膜癌或子宫癌、唾液腺癌、肾癌、肝癌、前列腺癌、阴门癌、甲状腺癌、脑癌、肝癌(hepatic carcinoma)和各种类型的头颈部癌、I型或II型神经纤维瘤病。所述癌症的其它实例包括具有疗法抗性、难治性或转移性的那些。
本发明取得了以下有益的技术效果：
第一、本发明提供一系列抗LAG-3/PD-L1双特异分子。具体地，以人源化抗LAG-3抗体(具体信息参见申请号:202210118342.8的中国申请)与抗PD-L1纳米抗体B6(具体信息参见申请号：202210524220.9的中国申请)或F2(具体信息参见申请号：202110433149.9的中国申请)组成首尾结构的双特异抗体。该首尾结构设计使双特异性结合分子能够同时有效结合重组及细胞表面的PD-L1和LAG-3，阻断PD-1/PD-L1的结合、MHCII/LAG-3以及FSTL-1/LAG-3的结合。同时，双特异分子可以有效实现对表达LAG-3和PD-L1的细胞的桥连作用，有利于将解除抑制的T细胞聚集在表达PD-L1的肿瘤细胞周围，发挥抗肿瘤药效。
第二、LAG-3与PD-L1均被证实为导致肿瘤免疫逃逸的重要靶点，LAG-3多个抗体处于不同临床阶段，且PD-L1抗体已有多个获批上市品种。本发明实施例通过构建LAG-3与PD-L1双特异性结合分子，使得双特异抗体在保持各自靶点的抗肿瘤活性同时，以期达到比两株单抗联用更优的抗肿瘤治疗效果。在PD-L1纳米抗体与Fc连接的linker设计上，可以采用传统的(G4S)2-4设计，也可以采用GGGGSPGGGSPGGGS的设计，后者更利于双特异性结合分子的结构稳定性。
第三、LAG-3分子表达于T细胞表面，PD-L1分子表达于肿瘤细胞表面，从而，本发 明实施例的双特异性结合分子可以在LAG-3阳性和PD-L1阳性的细胞之间形成桥接的作用，从而在肿瘤为环境内募集T细胞，进而起到更好的T细胞特异杀伤作用。根据本发明实施例的数据，双特异分子hz7F10-hzF2和hz7F10-hzB6均能有效抑制肿瘤的生长，其疗效优于抗PD-L1单抗和LAG-3单抗。实验结果表明，本发明实施例的双特异性结合分子在抗肿瘤治疗上具有良好的应用价值。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1：LAG-3和PD-L1双特异抗体结构模式图；
图2：hz7F10-hzB6的LAG-3结合活性和PD-L1结合活性的相互影响；
图2A：hz7F10-hzB6在结合LAG-3后不影响对PD-L1的结合；
图2B：hz7F10-hzB6在结合PD-L1后不影响对LAG-3的结合；
图3：FACS分析hz7F10-hzB6、hz7F10-hzF2与细胞表面LAG-3的结合；
图4：FACS分析hz7F10-hzB6、hz7F10-hzF2与细胞表面PD-L1的结合；
图5：hz7F10-hzB6、hz7F10-hzF2对不同种属LAG-3/PD-L1结合特异性，其中，图5A：hz7F10-hzB6对人LAG-3、食蟹猴LAG-3、小鼠LAG-3的结合；图5B：hz7F10-hzF2对人LAG-3、食蟹猴LAG-3、小鼠LAG-3的结合；图5C：BMS-986016对人LAG-3、食蟹猴LAG-3、小鼠LAG-3的结合；图5D：hz7F10-hzB6对人PD-1、食蟹猴PD-1、小鼠PD-L1、大鼠PD-L1的结合；图5E：hz7F10-hzF2对人PD-1、食蟹猴PD-1、小鼠PD-L1、大鼠PD-L1的结合；
图6：hz7F10-hzB6、hz7F10-hzF2对MHCII与LAG-3结合的阻断活性；
图7：hz7F10-hzB6、hz7F10-hzF2对FGL1与LAG-3结合的阻断活性；
图8：hz7F10-hzB6、hz7F10-hzF2对PD-1/PD-L1的阻断活性；
图9：双特异性抗体对CHO-PD-L1细胞和HEK293-LAG-3细胞的交联；
图10：hz7F10-hzB6、hz7F10-hzF2对人hPD-1/hPD-L1/hLAG-3转基因小鼠皮下移植MC38-hPDL1鼠结肠癌肿瘤生长抑制作用，其中，图10A为体重结果示意图，图10B为肿瘤生长曲线示意图；
图11：hz7F10-hzB6、hz7F10-hzF2对人PBMC免疫重建小鼠皮下移植H1975人肺腺癌细胞肿瘤生长的抑制作用，其中，图11A为体重结果示意图，图11B为肿瘤生长曲线示意图。
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的 实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
下面参考具体实施例，对本发明进行说明，需要说明的是，这些实施例仅仅是说明性的，而不能理解为对本发明的限制。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品，例如可以采购自Sigma公司。
实施例1：抗人LAG-3人源化抗体hz7F10及对照抗体
通过用重组人LAG-3蛋白(NCBI登录号：NP_002277.4)抗原免疫小鼠获得鼠源抗体7F10，然后对其进行人源化改造，获得人源化抗体hz7F10。hz7F10的轻链可变区(hz7F10-L)氨基酸序列见序列SEQ ID NO.1；轻链恒定区氨基酸序列见序列SEQ ID NO.2；重链可变区(hz7F10-H)氨基酸序列见序列SEQ ID NO.3；重链恒定区氨基酸序列见序列SEQ ID NO.4，CDR以Kabat定义。
SEQ ID NO.1：人源化抗体hz7F10轻链可变区氨基酸序列(CDRs 1、2和3分别为SEQ ID NO.14、15和16)
Figure PCTCN2022100816-appb-000001
SEQ ID NO.2：人源化抗体hz7F10轻链恒定区氨基酸序列
Figure PCTCN2022100816-appb-000002
SEQ ID NO.3：人源化抗体hz7F10重链可变区氨基酸序列(CDRs 1、2和3分别为SEQ ID NO.17、18和19)
Figure PCTCN2022100816-appb-000003
SEQ ID NO.4：人源化抗体hz7F10重链恒定区氨基酸序列
Figure PCTCN2022100816-appb-000004
在本实施例中，将hz7F10的重链可变区SEQ ID NO:3的编码基因克隆至含有重链恒定区SEQ ID NO:4编码基因的真核表达载体(PPT5，购自北京华越洋生物，货号VECT6098) 上，形成构建hz7F10的重链重组表达载体；另一方面通过将hz7F10的轻链可变区SEQ ID NO:1的编码基因克隆至含有轻链恒定区SEQ ID NO:2编码基因的真核表达载体上，构建hz7F10的轻链重组表达载体。然后双质粒转染真核细胞进行表达和纯化，获得针对LAG-3人源化单克隆抗体hz7F10。
类似地，将对照抗体BMS-986016轻重链序列编码基因进行全合成，采用相同策略克隆至真核瞬时表达载体中，并在真核细胞中进行表达纯化，获得对照抗体BMS-986016重组蛋白。其中，对照抗体BMS-986016氨基酸序列来源于WHO Drug Information(Vol.32,No.2,2018)，重链氨基酸序列见SEQ ID NO.5，轻链氨基酸序列见SEQ ID NO.6。
SEQ ID NO.5：对照抗体BMS-986016重链氨基酸序
Figure PCTCN2022100816-appb-000005
SEQ ID NO.6：对照抗体BMS-986016轻链氨基酸序列
Figure PCTCN2022100816-appb-000006
实施例2：人源化抗PD-L1单域抗体Fc融合蛋白hzB6-Fc和hzF2-Fc
利用人PD-L1重组蛋白抗原(PD-L1序列号：NP_054862.1，19aa-238aa)免疫骆驼，分离外周血单核细胞(PBMC)并提取总RNA进行反转录，以反转录产物为模板构建骆驼免疫库。随后利用PD-L1重组蛋白通过固相筛选的方法对所构建的骆驼免疫库进行筛选，获得特异性噬菌体展示单域抗体VHH-B6和VHH-F2，然后将可变区基因进行人源化改造，获得人源化的hzB6(SEQ ID NO.7)和hzF2(SEQ ID NO.8)的序列，将其分别克隆入含有人Fc(IgG1，hFc，SEQ ID NO.9)编码基因的真核表达载体(PPT5，北京华越洋生物，货号VECT6098)上，构建hzB6-Fc和hzF2-Fc的重组抗体，具体序列如下，其中，CDR以Kabat定义。
SEQ ID NO.7：人源化单域抗体hzB6可变区氨基酸序列(CDRs 1、2和3分别为SEQ ID NO.20、21和22)
Figure PCTCN2022100816-appb-000007
Figure PCTCN2022100816-appb-000008
SEQ ID NO.8：人源化单域抗体hzF2可变区氨基酸序列(CDRs 1、2和3分别为SEQ ID NO.23、24和25)
Figure PCTCN2022100816-appb-000009
SEQ ID NO.9：人IgG1Fc区氨基酸序列
Figure PCTCN2022100816-appb-000010
实施例3：抗人LAG-3与PD-L1双特异抗体的制备
利用分子克隆的方法在实施例1得到的hz7F10的重链表达载体上进行双特异表达载体的构建，所述双特异抗体结构示意图如图1所示。分别构建hz7F10-hzB6(SEQ ID NO.11)和hz7F10-hzF2(SEQ ID NO.12)的重链重组表达载体，其中linker的氨基酸序列如SEQ ID NO.10(GGGGSPGGGSPGGGS)所示。
SEQ ID NO.10：linker氨基酸序列
Figure PCTCN2022100816-appb-000011
SEQ ID NO.11：hz7F10-hzB6重链氨基酸序列
Figure PCTCN2022100816-appb-000012
SEQ ID NO.12：hz7F10-hzF2重链氨基酸序列
Figure PCTCN2022100816-appb-000013
Figure PCTCN2022100816-appb-000014
将hz7F10-hzB6和hz7F10-hzF2的重链重组表达载体与实施例1得到hz7F10的轻链(SEQ ID NO.13)重组表达载体共转染HEK293细胞进行瞬时表达，纯化后获得hz7F10-hzB6和hz7F10-hzF2的双抗重组蛋白纯品用于后续实验。
SEQ ID NO.13：hz7F10轻链氨基酸序列
Figure PCTCN2022100816-appb-000015
实施例4：抗人LAG-3/PD-L1双特异抗体结合活性分析
4.1 SRP测定与重组蛋白的结合活性
使用Reichert公司的4SPR型号仪器，利用Anti-human Fc antibody捕获法对hz7F10-hzB6、hz7F10-hzF2进行亲和力测定。首先通过氨基偶联方法将anti human Fc antibody固定在芯片表面，随后进行亲和力测定：用HBS-EP将目标抗体稀释至40ug/ml，25uL/min，注射2min；将抗原从200nM 2倍梯度稀释8个浓度，25uL/min，结合2min，解离3min。最后，使用3M MgCl 2对葡聚糖芯片(13206066)再生30ses，以循环检测下一样品。结果显示(表1)，当前实验条件下，hz7F10-hzB6、hz7F10-hzF2与LAG-3结合的亲和力与裸抗体hz7F10相近，与阳性对照抗体BMS-986016也相近；hz7F10-hzB6、hz7F10-hzF2与PD-L1结合的亲和力与裸抗体hzB6-Fc和hzF2-Fc，与阳性对照抗体BMS-986016也相近。
表1.hz7F10-hzB6、hz7F10-hzF2与人LAG-3/PD-L1重组蛋白亲和力测定
Figure PCTCN2022100816-appb-000016
Figure PCTCN2022100816-appb-000017
4.2 BLI测定LAG-3/PD-L1双特异抗体同时结合LAG-3/PD-L1的情况
利用Fortebio公司的OctetQKesystem仪器，采用抗人抗体Fc段的捕获抗体(AHC)生物探针捕获抗体Fc段的方法测定hz7F10-hzB6同时与LAG-3/PD-L1结合的特性。具体操作如下。
将hz7F10-hzB6抗体用PBS缓冲液稀释至4μg/ml，流经AHC探针(Cat:18-0015，PALL)表面，时间为120s。使用人LAG-3胞外区融合蛋白(义翘神州，Cat:16498-H08H)60nM作为流动相，结合时间为300s，然后以PD-L1(NCBI序列号：NP_054862.1)60nM作为流动相，结合时间300s，解离时间300s。类似的进行首先PD-L1作为第一流动结合相，LAG-3作为第二流动结合相。实验结果如图2，结果表明hz7F10-hzB6在结合了一侧的LAG-3/PD-L1后，对与另一侧PD-L1/LAG-3的结合不产生影响。
4.3 FACS分析LAG-3/PD-L1双特异抗体与细胞表面抗原的结合活性
使用LAG-3(NCBI登录号：NP_002277.4)全长质粒瞬转HEK293细胞，48h后离心收集细胞，将细胞分为5×10 5cells/样品/100μL，加入梯度稀释的待测抗体，加入的抗体终浓度为：最高浓度132nM，3倍连续稀释10个梯度。冰上孵育2h，冰冷PBS洗涤细胞2遍；加入FITC标记的抗人Fc二抗(Cat.:F9512，Sigma)，冰上孵育1h，冰冷PBS(含0.05％吐温)洗涤细胞2遍，重悬于200μL流式缓冲液中，流式细胞仪(型号B49007AD，SNAW31211，BECKMANCOULTER)检测细胞的平均荧光强度(MFI)。检测结果显示(图3)，hz7F10-hzB6、hz7F10-hzF2与表达在细胞表面的LAG-3结合活性与裸抗体hz7F10相当，EC50值见表2。
与之类似，取肿瘤细胞MBA-MD-231，5×10 5cells/样品/100μL，方法同HEK293-LAG-3，检测结果同样显示(图4)，hz7F10-hzB6、hz7F10-hzF2与表达在细胞表面的PD-L1的结合活性与裸抗体hzB6-Fc和hzF2-Fc相近，与阳性对照抗体BMS-986016也相近，EC50值见表2。
表2.hz7F10-hzB6、hz7F10-hzF2与细胞表面抗原结合活性(EC50：nM)
Figure PCTCN2022100816-appb-000018
实施例5：LAG-3/PD-L1双特异抗体种属交叉分析
将人重组蛋白LAG-3(购自义翘神州，Cat:16498-H08H)、人重组蛋白PD-L1(NCBI序列号：NP_054862.1)，猴重组蛋白LAG-3(Cat:C998，近岸蛋白质科技有限公司)、猴重组蛋白PD-L1(Cat:CC29，近岸蛋白质科技有限公司)，鼠重组蛋白LAG-3(Cat:53069-M08H，北京义翘神州科技股份有限公司)、鼠PD-L1(Cat:50010-M08H，北京义翘神州科技股份有限公司50010-M08H-B)、大鼠PD-L1(Cat:80450-R08H，北京义翘神州科技股份有限公司)，用PBS稀释至1μg/mL，100μL/孔包被酶联板，4℃包被过夜；5％BSA封闭液37℃恒温培养箱封闭60min，PBST洗板3次；加入稀释至1μg/mL的hz7F10-hzB6、hz7F10-hzF2、hz7F10、BMS-986016，37℃反应60min，PBST洗板4次；加入1:5000稀释的HRP-羊抗人IgG(Cat:109-035-098，Jackson Immuno Research)反应45min，PBST洗板4次；最后加入TMB底物显色，37℃恒温培养箱反应15min，2MHCl终止反应，读取并记录波长450nm下孔板的吸光度。结果如图5、表3所示，hz7F10-hzB6、hz7F10-hzF2与人猴LAG-3/PD-L1均特异性结合，而与鼠LAG-3/PD-L1不结合；抗LAG-3对照抗体BMS-986016与人、猴LAG-3特异性结合，与小鼠LAG-3不结合。
表3.hz7F10-hzB6、hz7F10-hzF2与人、猴、鼠LAG-3/PD-L1重组蛋白的结合活性(EC50，nM)
Figure PCTCN2022100816-appb-000019
实施例6：LAG-3/PD-L1双特异抗体阻断活性分析
6.1 FACS鉴定hz7F10-hzB6对LAG-3/MHCII类分子的阻断活性
消化并离心收集A375细胞，计数为3×10 5cells/样品/100μL；稀释hz7F10-hzB6、hz7F10-hzF2及对照抗体BMS-986016，以40nM起始，1.3倍倍比稀释10个梯度；加入重悬好的A375细胞，冰上孵育1h，冰冷PBS洗涤细胞2遍；稀释LAG-3-mFc至30nM,加入已结合抗体的A375细胞，冰上孵育1h，冰冷PBS洗涤细胞2遍；加入FITC标记的抗鼠Fc二抗(Cat：F9006，Sigma)，冰上孵育1h，冰冷PBS洗涤细胞2遍，重悬于200μL流式缓冲液中，流式细胞仪检测。检测结果如图6、表4所示，hz7F10-hzB6、hz7F10-hzF2可明显 阻断LAG-3与A375细胞表面MHCII类分子的结合活性，其阻断活性与对照抗体BMS-986016相当。
表4.hz7F10-hzB6、hz7F10-hzF2对A375细胞表面MHCII类分子与LAG-3结合的阻断活性
Figure PCTCN2022100816-appb-000020
6.2 ELISA鉴定hz7F10-hzB6、hz7F10-hzF2对LAG-3/FGL1的阻断活性
将人重组蛋白FGL1(Cat:13484-H08B，北京义翘神州科技股份有限公司)，用PBS稀释至1μg/mL，100μL/孔包被酶联板，4℃包被过夜；5％BSA封闭液37℃恒温培养箱封闭60min，PBST洗板3次；梯度稀释hz7F10-hzB6、hz7F10-hzF2、hz7F10、BMS-986016，10nM起始3倍稀释12个梯度，然后加入终浓度0.25ug/ml的LAG-3-mFc,混合后加入酶联板，37℃反应60min，PBST洗板4次；加入1:5000稀释的HRP抗小鼠IgG(Cat：115-035-071，Jackson Immuno Research)反应45min，PBST洗板4次；最后加入TMB底物显色，37℃恒温培养箱反应15min，2MHCl终止反应，读取并记录波长450nm下孔板的吸光度。结果如图7、表5所示，hz7F10-hzB6、hz7F10-hzF2均可特异性阻断LAG-3与FGL1的结合，其阻断活性与对照抗体BMS-986016相当。
表5.hz7F10-hzB6、hz7F10-hzF2对FGL1与LAG-3结合的阻断活性
Figure PCTCN2022100816-appb-000021
6.3报告基因系统检测hz7F10-hzB6、hz7F10-hzF2对PD-1/PD-L1通路的阻断活性
程序性细胞死亡蛋白1(PD-1)是一种在活化T细胞上表达的受体，它与配体PD-L1和PD-L2的结合对免疫应答具有负向调节作用。CHO-K1细胞表面的TCR具有正向调节作用，可激活T细胞内的NFAT通路。当两种细胞共同培养时，PD-1/PD-L1相互作用抑制T细胞活性，使癌细胞逃避免疫监视。当有抗体阻断该通路时，T细胞内的NFAT通路则可介导底物发光。
CHO-PD-L1-CD3L细胞，胰酶消化，用完全培养基终止反应。细胞计数，用完全培养基调整细胞密度为4x10 5cell/ml,100μl每孔铺到板子上。(4×10 4cell/孔)，37℃，5％CO 2培养，过夜；用工作培养基稀释抗体至2×起始浓度,再进行1.5倍稀释，共12个点，抗体初始浓度最大稀释倍数为10倍；Jurkat-PD1-NFAT细胞，细胞计数,1000rpm离心5min，用工作培养基重悬，将细胞密度调整至1.2×10 6cell/ml；弃CHO-PD-L1-CD3L细胞上清，将已梯度稀释的样品加入至96孔白色细胞板，50μl/孔，每个浓度点设置复孔。室温放置1h；将制备好的Jurkat-PD1-NFAT细胞悬液50μl/孔加入至96孔板相应位置(6×104cell/孔)，完成 Jurkat-PD1-NFAT细胞的铺板后，将白色96孔板置于37℃、5％CO 2恒温培养箱中孵育6h；提前1～2h取出Bio-Lite Luciferase Assay System底物并融化，室温避光放置至室温。从培养箱取出细胞板，平衡至室温后(约10～15min)，向细胞板50μl/孔加入Bio-Lite底物，避光孵育5min。用酶标仪为Luminescence模式，Intergration选择100，读取RLU。检测结果显示(图8，表6)，hz7F10-hzB6、hz7F10-hzF2可明显阻断PD-1/PD-L1信号通路，且阻断效果与纳米抗体hzB6-Fc、hzF2-Fc相当。
表6.hz7F10-hzB6对PD-1/PD-L1信号系统的的阻断活性(单位nM)
Figure PCTCN2022100816-appb-000022
实施例7：LAG-3/PD-L1双特异性抗体对表达LAG-3和PD-L1的细胞交联作用分析
瞬时转染带有OFP红光标签的LAG-3-OFP(NCBI登录号：NP_002277.4)质粒至HEK293细胞，待2天后收集细胞；消化高表达人PD-L1的CHO细胞并计数，调细胞密度为3×10 7cells/ml，将CHO-PD-L1-CD3L细胞按照1:500加入CFSE(CFSE Cell Division Tracker Kit,Cat:423801)染料，于37°放置30min，在离心机以400g离心5min，去除上清液，用PBS洗一次细胞；稀释不同抗体(hz7F10\hzB6-Fc\hzF2-Fc\hz7F10-hzB6\hz7F10-hzB6\NC-IgG1)，100nM起始，3倍稀释8个梯度，分别加入U型底96孔板中，加入PBS洗涤后的CHO-PD-L1-CD3L细胞混合，细胞终密度：1.5×10 5cells/well，4℃放置30min，于400g离心5min，用PBS洗4次细胞，并用PBS重悬细胞；向U型底96孔板的上述CHO-PD-L1-CD3L的细胞悬液中加入计数好的HEK-293-LAG3-OFP细胞，该细胞终密度：1×10 5cells/well，室温放置1h后进行流式细胞仪检测；FITC通道和PC 5.5通道的双阳性细胞的比例可反映出双特异性抗体引起的细胞交联情况。结果显示(图9)，hz7F10-hzB6可剂量依赖地引起CHO-PD-L1细胞与HEK293-LAG-3细胞发生交联。
实施例8：LAG-3/PD-L1双特异抗体对人PD-1/PD-L1/LAG-3转基因小鼠皮下移植MC38-hPDL1鼠结肠癌肿瘤模型的抗肿瘤药效
将高表达人PD-L1的鼠结肠癌肿瘤细胞MC38-hPDL1接种于雌性hPD-1/hPD-L1/hLAG-3转基因小鼠(C57来源的PD-1/PD-L1/LAG-3人源化小鼠，百奥赛图江苏基因生物技术有限公司，6-8周)右侧前胁肋部皮下，在肿瘤生长至100mm 3左右时分组给药，共4组，每组6只，分别为：hz7F10-hzF2(6mg/kg,ip,Biw×3周)组、hz7F10-hzB6(6mg/kg,ip,Biw×3周)组、hz7F10(5mg/kg,ip,Biw×3周)组、NC-hIgG1(6mg/kg,ip,Biw×3周)组。每周测量肿瘤体积及体重，记录荷瘤鼠体重和肿瘤体积的变化与给药时间的关系。
结果显示(图10)，受试药物hz7F10(TGI:13％)无抑制肿瘤生长的活性，而hz7F10-hzF2 (TGI:58％)及hz7F10-hzB6(TGI:69％)均有效地抑制了肿瘤的生长，同时各组间小鼠体重生长无显著性差异，表明hz7F10、hz7F10-hzF2和hz7F10-hzB6的毒副作用很小甚至没有。
实施例9：LAG-3/PD-L1双特异抗体对人PBMC免疫重建小鼠皮下移植H1975人肺腺癌细胞肿瘤模型的抗肿瘤药效
将人源肺癌H1975细胞接种于PBMC免疫系统人源化雄性NCG小鼠(江苏集萃药康生物科技有限公司，22-24g)右侧前胁肋部皮下，在肿瘤生长至约60mm3左右时分组给药，共5组，每组6只，分别为：hz7F10-hzF2(20mg/kg,ip,TIW×9次)组、hz7F10-hzB6(20mg/kg,ip,TIW×9次)组、hz7F10(15mg/kg,ip,TIW×9次)组、hzB6-Fc(12mg/kg,ip,TIW×9次)组、NC-hIgG1(20mg/kg,ip,TIW×9)组。每周测量肿瘤体积及体重，记录荷瘤鼠体重和肿瘤体积的变化与给药时间的关系。
结果显示(图11)，本条件下，抗LAG-3抗体hz7F10(TGI:-1％)与抗PD-L1抗体hzB6-Fc(TGI:14％)均无抑制肿瘤生长的活性，而双特异性抗体hz7F10-hzF2(TGI:52％)及hz7F10-hzB6(TGI:60％)可显著抑制肿瘤生长。同时各组间小鼠体重生长无显著性差异，表明hz7F10、hz7F10-hzF2和hz7F10-hzB6的毒副作用很小甚至没有。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
尽管已经示出和描述了本发明的实施例，本领域的普通技术人员可以理解：在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由权利要求及其等同物限定。

Claims (34)

  1. 一种双特异性结合分子，其特征在于，包括：
    第一抗原结合部，所述第一抗原结合部包括：
    第一肽段，所述第一肽段包括第一重链可变区和第一轻链可变区，其中，所述第一重链可变区的互补决定区(CDR)包括H1-CDR1、H1-CDR2和H1-CDR3，所述第一轻链可变区的CDR包括L1-CDR1、L1-CDR2和L1-CDR3；
    第二肽段，所述第二肽段包括第二重链可变区和第二轻链可变区，其中，所述第二重链可变区的CDR包括H2-CDR1、H2-CDR2和H2-CDR3，所述第二轻链可变区的CDR包括L2-CDR1、L2-CDR2和L2-CDR3，且所述第二肽段与所述第一肽段共价连接；
    第二抗原结合部，所述第二抗原结合部包括:
    第三肽段，所述第三肽段为纳米抗体，包括第三重链可变区，且与所述第一肽段的C端通过接头相连，所述第三重链可变区的CDR包括H3-CDR1、H3-CDR2和H3-CDR3。
  2. 根据权利要求1所述的双特异性结合分子，其特征在于，所述第一抗原结合部含特异性结合程序性细胞死亡-配体1(PD-L1)，所述第二抗原结合部特异性结合淋巴细胞活化基因-3(LAG3)。
  3. 根据权利要求2所述的双特异性结合分子，其特征在于，所述双特异性结合分子包含Fc结构域，所述Fc结构域是IgG，特别是IgG1 Fc结构域。
  4. 根据权利要求3所述的双特异性结合分子，其特征在于，所述Fc结构域包含L234A和L235A点突变。
  5. 根据权利要求2所述的双特异性结合分子，其特征在于，所述H1-CDR1包含SEQ ID NO：17所述的氨基酸序列，所述H1-CDR2包含SEQ ID NO：18所述的氨基酸序列和所述H1-CDR3包含SEQ ID NO：19所述的氨基酸序列，且所述L1-CDR1包含SEQ ID NO：14所述的氨基酸序列、L1-CDR2包含SEQ ID NO：15所述的氨基酸序列和L1-CDR3包含SEQ ID NO：16所述的氨基酸序列。
  6. 根据权利要求5所述的双特异性结合分子，其特征在于，所述H2-CDR1、H2-CDR2和H2-CDR3的氨基酸序列分别与所述H1-CDR1、H1-CDR2和H1-CDR3的氨基酸序列相同，且所述L2-CDR1、L2-CDR2和L2-CDR3的氨基酸序列分别与所述L1-CDR1、L1-CDR2和L1-CDR3的氨基酸序列相同。
  7. 根据权利要求2所述的双特异性结合分子，其特征在于，所述H3-CDR1、H3-CDR2和H3-CDR3选自下列组之一：
    (a)所述H3-CDR1包含SEQ ID NO：20所述的氨基酸序列，所述H3-CDR2包含SEQ ID NO：21所述的氨基酸序列和所述H3-CDR3包含SEQ ID NO：22所述的氨基酸序列；
    (b)所述H3-CDR1包含SEQ ID NO：23所述的氨基酸序列，所述H3-CDR2包含SEQ ID NO：24所述的氨基酸序列和所述H3-CDR3包含SEQ ID NO：25所述的氨基酸序列。
  8. 根据权利要求2所述的双特异性结合分子，其特征在于，所述第一重链可变区包括SEQ ID NO：3所述的氨基酸序列，所述第一轻链可变区包括SEQ ID NO：1所述的氨基酸序列。
  9. 根据权利要求2所述的双特异性结合分子，其特征在于，所述第一肽段进一步包括第一轻链恒定区和第一重链恒定区，且所述第二肽段进一步包括第二轻链恒定区和第二重链恒定区。
  10. 根据权利要求9所述的双特异性结合分子，其特征在于，所述第一轻链恒定区与所述第二轻链恒定区均包括SEQ ID NO：2所述的氨基酸序列。
  11. 根据权利要求10所述的双特异性结合分子，其特征在于，所述第一重链恒定区与所述第二重链恒定区均包括SEQ ID NO：4所述的氨基酸序列。
  12. 根据权利要求2所述的双特异性结合分子，其特征在于，所述第三重链可变区包括SEQ ID NO：7或8所示的氨基酸序列。
  13. 根据权利要求2所述的多肽复合物，其特征在于，所述多肽复合物进一步包括：
    第四肽段，所述第四肽段包括第四重链可变区，且与所述第二肽段的C端通过所述接头相连。
  14. 根据权利要求13所述的双特异性结合分子，其特征在于，所述第四肽段为纳米抗体，且具有与所述第三肽段相同的CDR序列。
  15. 根据权利要求14所述的双特异性结合分子，其特征在于，所述第四重链可变区包括SEQ ID NO：7或8所示的氨基酸序列。
  16. 根据权利要求1所述的双特异性结合分子，其特征在于，所述接头具有(G4S) 2-4或SEQ ID NO:10所示的氨基酸序列。
  17. 根据权利要求2所述的双特异性结合分子，其特征在于，所述双特异性结合分子的重链具有SEQ ID NO:11或12所示的氨基酸序列。
  18. 根据权利要求2所述的双特异性结合分子，其特征在于，所述双特异性结合分子的轻链具有SEQ ID NO:13所示的氨基酸序列。
  19. 一种组合物，其特征在于，包含：
    权利要求1-18中任一项所述的双特异性结合分子；以及
    可选的药学上可接受的辅料。
  20. 根据权利要求19所述组合物，其特征在于，还包含生物治疗剂、化学治疗剂和天然活性成分中的至少一种。
  21. 根据权利要求20所述组合物，其特征在于，所述组合物的剂型为水剂、针剂或粉针剂。
  22. 一种多核苷酸，其特征在于，编码权利要求1-18中任一项所述双特异性结合分子中的抗体重链和/或抗体轻链。
  23. 一种核酸构建体，其特征在于，包含权利要求22所述的多核苷酸。
  24. 一种宿主细胞，其特征在于，包含权利要求22所述的多核苷酸或权利要求23所述 的核酸构建体。
  25. 一种制备权利要求1-18中任一项所述双特异性结合分子的方法，其特征在于，包括以下步骤：
    (1)在适合表达重组外源蛋白的条件下培养权利要求24所述宿主细胞；
    (2)可选的，从细胞培养物中分离纯化所述双特异性结合分子。
  26. 权利要求1-18中任一项所述双特异性结合分子、权利要求19-21中任一项所述组合物、权利要求22所述多核苷酸、权利要求23所述核酸构建体、或权利要求24所述宿主细胞在结合并抑制LAG3和PD-1功能中的应用，其中，所述LAG3和PD-1源自人或食蟹猴。
  27. 权利要求1-18中任一项所述双特异性结合分子、权利要求19-21中任一项所述组合物、权利要求22所述多核苷酸、权利要求23所述核酸构建体、或权利要求24所述宿主细胞在抑制或阻断LAG-3/MHCII、LAG-3/FGL1、和/或PD-1/PD-L1信号通路中的应用。
  28. 权利要求1-18中任一项所述双特异性结合分子、权利要求19-21中任一项所述组合物、权利要求22所述多核苷酸、权利要求23所述核酸构建体、或权利要求24所述宿主细胞在桥连LAG-3阳性细胞和PD-L1阳性细胞中的应用。
  29. 权利要求1-18中任一项所述双特异性结合分子、权利要求19-21中任一项所述组合物、权利要求22所述多核苷酸、权利要求23所述核酸构建体、或权利要求24所述宿主细胞在制备治疗LAG-3和/或PD-L1信号通路异常相关疾病药物中的用途。
  30. 如权利要求29所述的用途，其特征在于，所述LAG-3和/或PD-L1信号通路异常相关疾病包括异常增生性疾病或免疫相关疾病。
  31. 根据权利要求30所述用途，其特征在于，所述异常增殖性疾病包括肿瘤、囊肿和增生；所述免疫相关疾病包括炎症、免疫缺陷、免疫耐受和过敏。
  32. 根据权利要求30所述用途，其特征在于，所述肿瘤为结肠癌或肺癌。
  33. 一种治疗患有异常增生性疾病或免疫相关疾病个体的方法，其特征在于，包括向所述个体施用权利要求1-18中任一项所述双特异性结合分子或权利要求19-21中任一项所述组合物。
  34. 一种抑制个体中肿瘤细胞生长的方法，其特征在于，包括向所述个体施用有效剂量的权利要求1-18中任一项所述双特异性结合分子或权利要求19-21中任一项所述组合物以抑制所述肿瘤细胞的生长。
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