WO2023088337A1 - 抗tigit-抗pd-l1双特异性抗体、其药物组合物及用途 - Google Patents

抗tigit-抗pd-l1双特异性抗体、其药物组合物及用途 Download PDF

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WO2023088337A1
WO2023088337A1 PCT/CN2022/132422 CN2022132422W WO2023088337A1 WO 2023088337 A1 WO2023088337 A1 WO 2023088337A1 CN 2022132422 W CN2022132422 W CN 2022132422W WO 2023088337 A1 WO2023088337 A1 WO 2023088337A1
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tigit
antibody
bispecific antibody
seq
amino acid
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French (fr)
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戴爽
翟天航
黄威峰
彭绍岗
孙左宇
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Biotheus Inc
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Biotheus Inc
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Priority to US18/711,482 priority Critical patent/US20250019436A1/en
Priority to AU2022389530A priority patent/AU2022389530A1/en
Priority to JP2024529415A priority patent/JP2024542192A/ja
Priority to CA3238571A priority patent/CA3238571A1/en
Priority to CN202280075442.5A priority patent/CN118251419A/zh
Priority to EP22894869.1A priority patent/EP4435010A4/en
Priority to KR1020247019886A priority patent/KR20240103034A/ko
Publication of WO2023088337A1 publication Critical patent/WO2023088337A1/zh
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Definitions

  • the invention belongs to the field of medicine, and relates to an anti-TIGIT-anti-PD-L1 bispecific antibody, its pharmaceutical composition and application.
  • T cell immunoglobulin and ITIM domains T cell immunoreceptor with Ig and ITIM domains, TIGIT, also known as WUCAM, Vstm3, VSIG9
  • TIGIT T cell immunoreceptor with Ig and ITIM domains
  • WUCAM WUCAM
  • Vstm3 VSIG9
  • TIGIT participates in a complex regulatory network in tumor immunity involving multiple immunosuppressive receptors (e.g., CD96/TACTILE, CD112R/PVRIG), a competitive costimulatory receptor (DNAM-1/CD226) and multiple ligands.
  • CD96/TACTILE multiple immunosuppressive receptors
  • CD112R/PVRIG a competitive costimulatory receptor
  • DNAM-1/CD226 multiple ligands.
  • CD155 PVR/NECL-5
  • CD112 Nectin-2/PVRL2
  • DNAM-1, TIGIT, and CD96 are expressed on T cells and NK cells and share CD155 as a ligand.
  • TIGIT TIGIT-mediated effector T cells and NK cell inhibition: (1) TIGIT on the surface of T/NK cells Bind to CD155, phosphorylate the immunoreceptor tyrosine inhibitory motif (ITIM) in TIGIT cells, and directly transduce inhibitory signals; (2) TIGIT promotes the formation of immune tolerance DCs by binding to CD155 on DCs , reducing the production of interleukin (IL)-12 and increasing IL-10, indirectly suppressing T cell responses; (3) TIGIT with inhibitory effect competes for binding to CD155 with a higher affinity than costimulatory receptor CD226, thereby limiting CD226-mediated activation.
  • ITIM immunoreceptor tyrosine inhibitory motif
  • TIGIT also directly binds CD226 in cis on cells, destroying its ability to bind to CD155 homodimers; (4) Tregs expressing TIGIT are highly inhibitory, and TIGIT+Tregs are activated to produce IL-10 and fibroblasts Proteoprotein-like protein 2 (Fgl2), which mediates T-cell suppression; (5) Fap2 protein from Fusobacterium nucleatum, an anaerobic Gram commensal bacterium associated with colorectal cancer, which directly binds to TIGIT , but does not bind to CD226, thereby inhibiting tumor immunity mediated by NK cells and T cells and regulating innate immune responses.
  • Fgl2 Proteoprotein-like protein 2
  • Programmed death 1 ligand 1 (programmed death 1 ligand 1, PD-L1), also known as CD274, is a member of the B7 family and a ligand for PD-1.
  • PD-L1 is a type I transmembrane protein with a total of 290 amino acids, including an IgV-like region, an IgC-like region, a transmembrane hydrophobic region and an intracellular region consisting of 30 amino acids.
  • PD-L1 negatively regulates the immune response. Studies have found that PD-L1 is mainly expressed in activated T cells, B cells, macrophages, and dendritic cells.
  • PD-L1 In addition to lymphocytes, PD-L1 is also expressed in other tissues such as thymus, heart, placenta, etc. Endothelial cells, and various non-lymphoid systems such as melanoma, liver cancer, gastric cancer, renal cell carcinoma, ovarian cancer, colon cancer, breast cancer, esophageal cancer, head and neck cancer, etc. (Akintunde Akinleye&Zoaib Rasool, Journal of Hematology&Oncology volume 12, Article number: 92 (2019)). PD-L1 has a certain generality in regulating autoreactive T and B cells and immune tolerance, and plays a role in peripheral tissue T and B cell responses. High expression of PD-L1 on tumor cells is associated with poor prognosis of cancer patients.
  • bispecific antibodies are a direction in the development of antibody drugs, they face many challenges, such as preclinical evaluation models, low expression levels, poor stability, complex processes, and large differences in quality control. Therefore, the development of bispecific antibodies has always been Development is difficult.
  • the inventors After in-depth research and creative work, the inventors obtained an anti-TIGIT antibody, and constructed an anti-TIGIT-anti-PD-L1 bispecific antibody based on the antibody.
  • the present inventors have surprisingly found that the anti-TIGIT antibody of the present invention (also referred to as the antibody or the antibody of the present invention) and the anti-TIGIT-anti-PD-L1 bispecific antibody (also referred to as the bispecific antibody of the present invention or the bispecific antibody of the present invention) Antibody) has high binding affinity to PD-L1 and TIGIT (even better than the positive control antibody, the bispecific antibody in one or more aspects), and can block the binding of PD-L1 to its ligand PD-1 and TIGIT respectively. Binding to its ligand CD155/CD112 reduces or eliminates the inhibitory signal transmitted to cells, and the administration of the antibody of the present invention in animal models can significantly inhibit tumor growth.
  • the following inventions are thus provided:
  • bispecific antibody comprising:
  • a second protein domain targeting a target other than TIGIT e.g. PD-L1;
  • the first protein functional region is an anti-TIGIT immunoglobulin or an antigen-binding fragment thereof;
  • the heavy chain variable region of the anti-TIGIT immunoglobulin comprises HCDR1 with an amino acid sequence as shown in SEQ ID NO: 10, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 11, and an amino acid sequence as shown in SEQ ID NO: 12 HCDR3; and
  • the light chain variable region of the anti-TIGIT immunoglobulin comprises an amino acid sequence such as LCDR1 shown in SEQ ID NO: 13, an amino acid sequence such as LCDR2 shown in SEQ ID NO: 14, and an amino acid sequence as shown in SEQ ID NO: 15 LCDR3.
  • variable regions of the light chain and the heavy chain determine the binding of antigens; the variable region of each chain contains three hypervariable regions, called complementarity determining regions (CDR), and the CDR of the heavy chain (H) includes HCDR1, HCDR2, HCDR3, the CDR of the light chain (L) includes LCDR1, LCDR2, LCDR3.
  • CDRs contained in the antibodies of the present invention or antigen-binding fragments thereof can be identified according to various numbering systems known in the art.
  • an antibody or antigen-binding fragment thereof of the invention contains CDRs preferably identified by the Kabat, Chothia, AbM HVR or IMGT numbering system.
  • the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention are preferably numbered according to the Kabat numbering system (kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institute of Health, Bethesda , Md.(1991)) determined the numbering position.
  • the bispecific antibody wherein the first protein functional domain and the second protein functional domain are directly connected or connected through a linker (linker, also called a linker peptide);
  • the connecting segment is (GGGGS)m, where m is a positive integer, such as 1, 2, 3, 4, 5 or 6;
  • the connecting segment is (GGGGS)nG, n is a positive integer, such as 1, 2, 3, 4, 5 or 6;
  • amino acid sequence of the connecting fragment is shown in SEQ ID NO:2.
  • GGGGS in brackets is represented as SEQ ID NO:22.
  • the first protein functional domain and the second protein functional domain are independently 1, 2 or more than 2.
  • the bispecific antibody in the bispecific antibody, there is one first protein functional domain and two second protein functional domains.
  • the bispecific antibody in the bispecific antibody, there are two first protein functional domains, and one second protein functional domain.
  • the bispecific antibody wherein,
  • the first protein functional region is an anti-TIGIT immunoglobulin or an antigen-binding fragment thereof, and the second protein functional region is a single domain antibody or a single chain antibody whose target is different from TIGIT;
  • the single domain antibody is an anti-PD-L1 single domain antibody
  • the single-chain antibody is an anti-PD-L1 single-chain antibody.
  • the bispecific antibody wherein,
  • the first protein functional region is an anti-TIGIT immunoglobulin or an antigen-binding fragment thereof
  • the second protein functional region is an anti-PD-L1 single domain antibody or an anti-PD-L1 single-chain antibody
  • the bispecific antibody wherein,
  • the first protein functional region is an anti-TIGIT single-chain antibody
  • the second protein functional region is an immunoglobulin whose target is different from TIGIT or an antigen-binding fragment thereof;
  • the immunoglobulin with a target other than TIGIT is anti-PD-L1 immunoglobulin.
  • the bispecific antibody wherein,
  • the first protein functional domain is an anti-TIGIT single-chain antibody
  • the second protein functional domain is an anti-PD-L1 immunoglobulin or an antigen-binding fragment thereof.
  • the bispecific antibody wherein,
  • the anti-PD-L1 single domain antibody comprises a heavy chain variable region, the heavy chain variable region comprises HCDR1 with an amino acid sequence as shown in SEQ ID NO: 16, and HCDR2 with an amino acid sequence as shown in SEQ ID NO: 17 And the HCDR3 whose amino acid sequence is shown in SEQ ID NO:18;
  • amino acid sequence of the anti-PD-L1 single domain antibody is shown in SEQ ID NO:3.
  • the bispecific antibody wherein,
  • amino acid sequence of the heavy chain variable region of the anti-TIGIT immunoglobulin is as shown in SEQ ID NO: 19, and the amino acid sequence of its light chain variable region is as shown in SEQ ID NO: 20;
  • amino acid sequence of the heavy chain of the anti-TIGIT immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of its light chain is shown in SEQ ID NO: 5.
  • the bispecific antibody wherein the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb and complementary determining region fragments.
  • the bispecific antibody wherein,
  • the constant region of the anti-TIGIT immunoglobulin or the immunoglobulin whose target is different from TIGIT is from a human antibody
  • the constant region is selected from the constant region of human IgG1, IgG2, IgG3 or IgG4.
  • the bispecific antibody wherein,
  • the heavy chain constant region of the anti-TIGIT immunoglobulin or the immunoglobulin whose target point is different from TIGIT is human Ig gamma-1 chain C region (such as NCBI ACCESSION: P01857) or human Ig gamma-4 chain C region (such as NCBI ACCESSION :P01861.1), and its light chain constant region is human Ig kappa chain C region (such as NCBI ACCESSION: P01834); preferably, the heavy chain constant region of the anti-TIGIT immunoglobulin also comprises the L234A mutation according to the EU numbering system and L235A mutations.
  • the bispecific antibody wherein the single domain antibody or single chain antibody is linked to the C-terminal or N-terminal of anti-TIGIT immunoglobulin, for example, the single domain antibody or There are two single-chain antibodies, and one end of each single-domain antibody or single-chain antibody is respectively connected to the C-terminal or N-terminal of the two heavy chains of anti-TIGIT immunoglobulin.
  • the bispecific antibody wherein the anti-PD-L1 single domain antibody or anti-PD-L1 single-chain antibody is linked to the C-terminal or N-terminal of the anti-TIGIT immunoglobulin,
  • the anti-PD-L1 single domain antibody or anti-PD-L1 single-chain antibody is linked to the C-terminal or N-terminal of the anti-TIGIT immunoglobulin.
  • there are two anti-PD-L1 single-domain antibodies or anti-PD-L1 single-chain antibodies and one end of each anti-PD-L1 single-domain antibody or anti-PD-L1 single-chain antibody is respectively connected to an anti-TIGIT immunoglobulin C-terminal or N-terminal of the two heavy chains.
  • the bispecific antibody wherein the single domain antibody is an anti-PD-L1 single domain antibody, the amino acid sequence of the peptide chain obtained by linking the single domain antibody to the anti-TIGIT immunoglobulin As shown in SEQ ID NO:4.
  • the bispecific antibody is a tetramer formed by two identical first peptide chains and two identical second peptide chains, wherein,
  • amino acid sequence of the first peptide chain is shown in SEQ ID NO:4; and the amino acid sequence of the second peptide chain is shown in SEQ ID NO:5.
  • the bispecific antibody includes:
  • the first protein functional region is an anti-TIGIT immunoglobulin
  • the second protein functional region is an anti-PD-L1 single domain antibody
  • amino acid sequence of the heavy chain of the anti-TIGIT immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of its light chain is shown in SEQ ID NO: 5;
  • amino acid sequence of the anti-PD-L1 single domain antibody is shown in SEQ ID NO: 3;
  • Two anti-PD-L1 single domain antibodies are connected to the C-terminus of each heavy chain of anti-TIGIT immunoglobulin through the same or different connecting fragments;
  • the connecting segment is (GGGGS)m, where m is a positive integer, such as 1, 2, 3, 4, 5 or 6;
  • the connecting segment is (GGGGS)nG, n is a positive integer, such as 1, 2, 3, 4, 5 or 6;
  • amino acid sequence of the connecting fragment is shown in SEQ ID NO:2.
  • GGGGS in brackets is represented as SEQ ID NO:22.
  • the bispecific antibody is in IgG-VHH format.
  • the bispecific antibody is in the form of IgG-scFv, ie Morrison pattern.
  • the bispecific antibody wherein the single domain antibody or single chain antibody is linked to the C-terminus of the heavy chain of immunoglobulin.
  • immunoglobulins consist of two heavy chains
  • one immunoglobulin molecule is linked with two single domain antibody molecules or two single chain antibody molecules.
  • the two single domain antibody molecules are identical.
  • the two single chain antibody molecules are identical.
  • the single domain antibody or single chain antibody forms an amide bond connection with the C-terminus of the heavy chain of the immunoglobulin through the aforementioned connecting fragment.
  • the monovalent affinity of the bispecific antibody binding to human PD-L1, cynomolgus monkey PD-L1, human TIGIT and/or cynomolgus monkey TIGIT antigen respectively
  • the monovalent affinity of its single-end antibody molecule binding to human PD-L1, cynomolgus monkey PD-L1, human TIGIT and/or cynomolgus TIGIT anti-antigen is the same or better.
  • Another aspect of the invention relates to an isolated nucleic acid molecule encoding a bispecific antibody according to any one of the invention.
  • a further aspect of the invention relates to a vector comprising an isolated nucleic acid molecule of the invention.
  • a further aspect of the invention relates to a host cell comprising an isolated nucleic acid molecule of the invention, or a vector of the invention.
  • Another aspect of the present invention relates to a method for preparing the bispecific antibody described in any one of the present invention, which comprises culturing the host cell of the present invention under suitable conditions, and recovering the bispecific antibody from the cell culture. Antibody steps.
  • Another aspect of the present invention relates to a conjugate, which includes a bispecific antibody and a coupling part, wherein the bispecific antibody is the bispecific antibody described in any one of the present invention, and the conjugate
  • the coupling part is a detectable label; preferably, the coupling part is a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme.
  • kits which comprises the bispecific antibody according to any one of the present invention, or comprises the conjugate of the present invention
  • the kit further comprises a second antibody capable of specifically binding to the bispecific antibody; optionally, the second antibody further comprises a detectable label, such as a radioactive isotope, a fluorescent substance, a luminescent substance , colored substances or enzymes.
  • a detectable label such as a radioactive isotope, a fluorescent substance, a luminescent substance , colored substances or enzymes.
  • Another aspect of the present invention relates to a pharmaceutical composition, which comprises the bispecific antibody according to any one of the present invention or the conjugate of the present invention; optionally, it also includes pharmaceutically acceptable excipients .
  • Another aspect of the present invention relates to the use of any one of the bispecific antibodies of the present invention or the conjugates of the present invention in the preparation of drugs for the prevention and/or treatment of malignant tumors; preferably, the malignant tumors are selected from From melanoma, liver cancer, stomach cancer, renal cell cancer, ovarian cancer, colon cancer, breast cancer, esophageal cancer and head and neck cancer.
  • Another aspect of the present invention relates to a method for treating and/or preventing malignant tumors, comprising administering to a subject in need an effective amount of the bispecific antibody of any one of the present invention or the conjugated antibody of the present invention
  • the malignant tumor is selected from the group consisting of melanoma, liver cancer, gastric cancer, renal cell carcinoma, ovarian cancer, colon cancer, breast cancer, esophagus cancer and head and neck cancer.
  • the step of administering an effective amount of the bispecific antibody described in any one of the present invention to a subject in need is: Before or after surgery, and/or before or after radiation therapy.
  • the method for treating and/or preventing malignant tumors wherein,
  • the single administration dose of the bispecific antibody of the present invention is 0.1-100 mg per kilogram body weight, preferably 4.8-24 mg or 1-10 mg; or, the single administration dose of the bispecific antibody of the present invention is Or 10-1000mg, preferably 50-500mg, 100-400mg, 150-300mg, 150-250mg or 200mg;
  • it is administered once every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks or 3 weeks;
  • the administration method is intravenous drip or intravenous injection.
  • the bispecific antibody according to any one of the present invention or the conjugate of the present invention is used for treating and/or preventing malignant tumors; preferably, the malignant tumors are selected from melanoma, liver cancer, gastric cancer, kidney cancer Cell carcinoma, ovarian cancer, colon cancer, breast cancer, esophagus cancer and head and neck cancer.
  • EC 50 refers to the half-maximal effect concentration (concentration for 50% of maximal effect), which refers to the concentration that can cause 50% of the maximal effect.
  • antibody refers to an immunoglobulin molecule generally composed of two pairs of polypeptide chains, each pair having a "light” (L) chain and a “heavy” (H) chain.
  • Antibody light chains can be classified as kappa and lambda light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also comprising a "D" region of about 3 or more amino acids.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3).
  • Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • the constant regions of the antibodies 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.
  • the VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, from amino-terminus to carboxy-terminus.
  • the variable regions (VH and VL) of each heavy chain/light chain pair form the antibody binding site, respectively. Assignment of amino acids to regions or domains follows Bethesda M.d., Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987and 1991)), or Chothia & Lesk J. Mol. Biol. 1987; 196:901-917; Chothia et al.
  • antibody is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
  • IgG eg, IgGl, IgG2, IgG3, or IgG4 subtype
  • IgAl IgA2, IgD, IgE, or IgM antibodies.
  • mAb and “monoclonal antibody” refer to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, ie, a population of identical antibody molecules except for natural mutations that may occur spontaneously. mAbs are highly specific for a single epitope on an antigen. Compared with monoclonal antibodies, polyclonal antibodies usually contain at least two or more different antibodies, and these different antibodies usually recognize different epitopes on antigens. Monoclonal antibodies can usually be obtained using hybridoma technology first reported by Kohler et al. ( G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. Nature, 1975; 256(5517): 495), but it can also be obtained by recombinant DNA technology (see US Patent 4,816,567).
  • single chain fragment variable refers to a molecule comprising an antibody heavy chain variable region (V H ) and an antibody light chain variable region (V L ) connected by a linker. where the VL and VH domains pair to form a monovalent molecule via a linker that enables production as a single polypeptide chain (see, e.g., Bird et al, Science 1988; 242:423-426 and Huston et al, Proc. Natl. Acad. Sci. USA 1988; 85:5879-5883).
  • Such scFv molecules may have the general structure: NH2- VL -Linker- VH -COOH or NH2- VH -Linker- VL -COOH.
  • Suitable prior art linkers consist of the repeated GGGGS amino acid sequence or variants thereof.
  • a linker having the amino acid sequence (GGGGS)4 SEQ ID NO: 23
  • variants thereof can also be used (Holliger et al, Proc. Natl. Acad. Sci. USA 1993; 90:6444-6448) .
  • Other linkers that can be used in the present invention are described by Alfthan et al, Protein Eng. 1995; 8:725-731, Choi et al, Eur.J.
  • isolated means obtained from the natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be that its natural environment has been altered, the substance has been isolated from its natural environment, or both. For example, an unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of.
  • isolated or “isolated” do not exclude the admixture of artificial or synthetic substances, nor the presence of other impurities which do not affect the activity of the substance.
  • vector refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector is called an expression vector.
  • a vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phage such as lambda phage or M13 phage and animal viruses.
  • artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC)
  • Phage such as lambda phage or M13 phage and animal viruses.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, papillomaviruses, Polyoma vacuolar virus (eg SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • papillomaviruses papillomaviruses
  • papillomaviruses papillomaviruses
  • Polyoma vacuolar virus eg
  • the term "host cell” refers to cells that can be used to introduce vectors, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • prokaryotic cells such as Escherichia coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • Insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • an antibody that specifically binds to an antigen refers to an antibody that is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, Binds the antigen with an affinity (K D ) of 10 ⁇ 8 M, 10 ⁇ 9 M or 10 ⁇ 10 M or less.
  • KD refers to the dissociation equilibrium constant for a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen.
  • the antibody has a dissociation equilibrium constant (K D ) of less than about 10 ⁇ 5 M, such as less than about 10 ⁇ 6 M, 10 ⁇ 7 M, 10 ⁇ 8 M, 10 ⁇ 9 M, or 10 ⁇ 10 M or less.
  • K D dissociation equilibrium constant
  • Binding to antigen eg, TIGIT protein or PD-L1 protein.
  • KD can be determined using methods known to those skilled in the art, for example using a Fortebio Molecular Interaction Instrument.
  • amino acids are generally represented by single-letter and three-letter abbreviations known in the art.
  • alanine can be represented by A or Ala.
  • the term "pharmaceutically acceptable excipient” refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, which are well known in the art (See eg Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19 th ed. Pennsylvania: Mack Publishing Company, 1995), and include but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers.
  • pH regulators include but not limited to phosphate buffer; surfactants include but not limited to cationic, anionic or nonionic surfactants such as Tween-80; ionic strength enhancers include but not limited to sodium chloride.
  • an effective amount refers to an amount sufficient to achieve, or at least partially achieve, the desired effect.
  • an effective amount for preventing a disease refers to an amount sufficient to prevent, arrest, or delay the occurrence of a disease (such as a tumor);
  • an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent the occurrence of a disease in a patient Amount of disease and its complications. Determining such an effective amount is well within the capability of those skilled in the art.
  • amounts effective for therapeutic use will depend on the severity of the disease being treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered concomitantly etc.
  • the amino acid sequence of the PD-L1 protein when referring to the amino acid sequence of the PD-L1 protein (Programmed death-ligand 1, NCBI GenBank ID: NP_054862.1), it includes the full length of the human PD-L1 protein, or the human PD-L1
  • the extracellular fragment PD-L 1 ECD (for example, the amino acid sequence is shown in SEQ ID NO: 6) or a fragment containing PD-L 1 ECD; also includes fusion proteins of PD-L1 ECD, such as Fc with mouse or human IgG Fragments fused to protein fragments (mFc or hFc).
  • the term "PD-L1 protein” shall include all such sequences, including the indicated sequences and their natural or artificial variants. And, when describing the sequence fragments of PD-L1 protein, it not only includes the sequence fragments of PD-L1 protein, but also includes the corresponding sequence fragments in its natural or artificial variants.
  • the amino acid sequence of the TIGIT protein when referring to the amino acid sequence of the TIGIT protein (T cell immunoreceptor with Ig and ITIM domains, NCBI GenBank ID: NP_776160.2), it includes the amino acid sequence of the human TIGIT protein Full length, or the extracellular fragment TIGIT ECD of human TIGIT (for example, the amino acid sequence is shown in SEQ ID NO: 8) or a fragment comprising TIGIT ECD; also include a full-length fusion protein of TIGIT protein or a fusion protein of TIGIT ECD, for example Fragments fused to mouse or human IgG Fc protein fragments (mFc or hFc).
  • the term "additional protein” shall include all such sequences, including their natural or artificial variants. And, when describing the sequence fragments of TIGIT protein, it also includes the corresponding sequence fragments in its natural or artificial variants.
  • the amino acid sequence of the PD-1 protein when referring to the amino acid sequence of the PD-1 protein (NCBI GenBank: NP_005009.2), it includes the full length of the human PD-1 protein, or the extracellular fragment PD-1ECD of human PD-1 Or a fragment containing PD-1 ECD; also includes a full-length fusion protein of PD-1 protein or a fusion protein of PD-1 ECD, such as a fragment fused with a mouse or human IgG Fc protein fragment (mFc or hFc) .
  • mutations or variations including but not limited to substitutions, deletions and/or additions
  • the term "additional protein” shall include all such sequences, including their natural or artificial variants.
  • the sequence fragment of PD-1 protein when describing the sequence fragment of PD-1 protein, it also includes the corresponding sequence fragment in its natural or artificial variant.
  • single domain antibody VHH
  • nanobody cloning the variable region of the heavy chain of an antibody to construct a nanobody (VHH) consisting of only one heavy chain variable region , which is the smallest fully functional antigen-binding fragment.
  • VHH single domain antibody
  • CH1 light chain and heavy chain constant region 1
  • first such as the first protein functional region, the first peptide chain
  • second such as the second protein functional region, the second peptide chain
  • the single-end antibody molecule refers to an antibody molecule that is the same as or similar to the first protein functional domain molecule or the second protein functional domain molecule in the bispecific antibody, unless otherwise specified.
  • an anti-TIGIT monoclonal antibody, an anti-PD-L1 monoclonal antibody, or an anti-PD-L1 single domain antibody that is identical or similar to the first protein functional domain molecule or the second protein functional domain molecule in the bispecific antibody.
  • the anti-TIGIT antibody of the present invention has superior affinity and specificity
  • the bispecific antibody of the present invention can specifically bind to TIGIT well
  • the bispecific antibody of the present invention can specifically bind to PD-L1 well.
  • the bispecific antibody of the present invention can simultaneously bind human TIGIT and human PD-L1 proteins;
  • the anti-TIGIT-anti-PD-L1 bispecific antibody of the present invention can respectively release the CD155/CD112-mediated TIGIT downstream inhibitory signaling pathway and PD-L1-mediated PD-1 downstream inhibitory signaling pathway, and the activity is better than that of the two single The combination of single-terminal antibody molecules; and/or the anti-tumor activity in vivo is better than the combination of two single-terminal antibody molecules.
  • the bispecific antibody of the present invention has good stability and long half-life.
  • FIG. 1 Schematic diagram of the structure of the bispecific antibody of the present invention.
  • Figure 2A The binding curve of the bispecific antibody of the present invention to human PD-L1 overexpressed on CHO cells.
  • Fig. 2B The binding curve of the bispecific antibody of the present invention to cynomolgus monkey PD-L1 overexpressed on CHO cells.
  • Fig. 2C a graph showing that the bispecific antibody of the present invention blocks the binding of human PD-L1 to overexpressed human PD-1 on CHO cells.
  • Fig. 3A Binding curve of the bispecific antibody of the present invention to human TIGIT overexpressed on CHO cells.
  • Fig. 3B The binding curve of the bispecific antibody of the present invention to cynomolgus TIGIT overexpressed on CHO cells.
  • Fig. 3C The binding curve of the bispecific antibody of the present invention to mouse TIGIT overexpressed on CHO cells.
  • Fig. 3D a graph showing that the bispecific antibody of the present invention blocks the binding of human CD155 to overexpressed human TIGIT on CHO cells.
  • Fig. 3E a graph showing that the bispecific antibody of the present invention blocks the binding of mouse CD155 to mouse TIGIT overexpressed on CHO cells.
  • Fig. 4 A graph showing that the bispecific antibody of the present invention simultaneously binds to human PD-L1 and human TIGIT proteins.
  • Fig. 5 A graph showing that the bispecific antibody of the present invention blocks PD-1/PD-L1 and TIGIT/CD155/CD112 signaling pathways.
  • Fig. 6A and Fig. 6B Statistical graphs of the cytokine release amount of the bispecific antibody of the present invention in the mixed lymphocyte test.
  • the PBMC cell samples used in Figure 6A and Figure 6B were from different donors.
  • Fig. 7 The pharmacodynamic curve of the bispecific antibody of the present invention in the B-NDG mouse model inoculated with A375 and human PBMC.
  • Fig. 8 The dose-dependent pharmacodynamic curve of the bispecific antibody of the present invention in the B-NDG mouse model inoculated with A375 and human PBMC.
  • Figure 9 The pharmacodynamic curve of the bispecific antibody of the present invention in the human PD-L1/PD-1/TIGIT transgenic mouse CT26 tumor model.
  • Figure 10 The half-life curve of the bispecific antibody of the present invention in mice.
  • Fig. 11 The binding curve of the anti-TIGIT antibody of the present invention to human TIGIT overexpressed on CHO cells.
  • Figure 12 The binding curve of the anti-TIGIT antibody of the present invention to cynomolgus monkey TIGIT overexpressed on CHO cells.
  • Figure 13 The binding curve of the anti-TIGIT antibody of the present invention to mouse TIGIT overexpressed on CHO cells.
  • Fig. 14 is a graph showing that the anti-TIGIT antibody of the present invention blocks the binding of human CD155 to human TIGIT overexpressed on CHO cells.
  • Fig. 15 is a graph showing that the anti-TIGIT antibody of the present invention blocks the binding of mouse CD155 to mouse TIGIT overexpressed on CHO cells.
  • Figure 16 The binding curve of the anti-TIGIT antibody of the present invention to TIGIT on activated human primary T cells.
  • Control antibody Atezolizumab anti-PD-L1 monoclonal antibody, trade name Tecentriq, Roche.
  • Control antibody Tiragolumab anti-TIGIT monoclonal antibody, Roche.
  • Example 1 Expression and purification of anti-TIGIT-anti-PD-L1 bispecific antibody
  • the anti-TIGIT antibody heavy chain variable region (VH) sequence (SEQ ID NO: 19) was synthesized by whole gene synthesis, and recombined into the wild-type hIgG1 antibody heavy chain to form a complete "VH-CH1-CH2 -CH3" antibody heavy chain (SEQ ID NO: 1), and connect anti-PD-L1 VHH (SEQ ID NO: 3) through 2 connecting fragments G 4 S (SEQ ID NO: 2) at the C-terminus of its heavy chain to obtain The full-length amino acid sequence of the peptide chain #1 is shown in SEQ ID NO:4.
  • VL anti-TIGIT light chain variable region
  • the heavy chain and light chain sequences were respectively constructed into the pcDNA3.1 expression frame by molecular cloning technology, and were routinely expressed through the Expi-293 expression system.
  • the transfection method was operated according to the manufacturer’s instructions, and the supernatant was collected after the cells were cultured for 5 days, and the target protein was purified by protein A magnetic beads (purchased from GenScript) sorting method. Resuspend the magnetic beads with an appropriate volume of binding buffer (PBS+0.1% Tween 20, pH 7.4) (1-4 times the volume of magnetic beads), add them to the sample to be purified, and incubate at room temperature for 1 hour with gentle shaking.
  • PBS+0.1% Tween 20, pH 7.4 an appropriate volume of binding buffer
  • the sample was placed on a magnetic stand (purchased from Beaver), the supernatant was discarded, and the magnetic beads were washed 3 times with binding buffer.
  • Add elution buffer (0.1M sodium citrate, pH 3.2) according to 3-5 times the volume of the magnetic beads, shake at room temperature for 5-10min, put it back on the magnetic stand, collect the elution buffer, and transfer to the added neutralization buffer (1M Tris, pH 8.54) collection tube and mix well.
  • anti-TIGIT-anti-PD-L1 bispecific antibody also referred to as anti-TIGIT/PD-L1 double antibody in the present invention
  • Figure 1 The anti-TIGIT-anti-PD-L1 bispecific antibody (also referred to as anti-TIGIT/PD-L1 double antibody in the present invention) was obtained, and its schematic structure is shown in Figure 1.
  • the binding dissociation constant (K D ) of the bispecific antibody obtained in Example 1 and its corresponding single-end antibody molecule binding to human and cynomolgus monkey PD-L1 and TIGIT was determined by biofilm layer optical interference technology (ForteBio). Fortebio affinity determination was carried out according to the existing method (Este, P et al. High throughput solution-based measurement of antibody-antigen affinity and epitope binning.
  • the sensor is equilibrated offline for 30 minutes in the analysis buffer, and then the baseline is established by online detection for 60 s, and the purified intact antibody is loaded onto the AHQ sensor to a thickness of 1 nm for affinity detection.
  • Antibody-loaded sensors were plateaued in 100 nM human or cynomolgus PD-L1, TIGIT-his antigen, and then transferred to assay buffer for at least 2 minutes for off-rate measurement.
  • Kinetic analysis was performed using a 1:1 binding model.
  • Table 1 K D values of anti-TIGIT-anti-PD-L1 double antibody and corresponding single-end antibody
  • Example 3 Anti-TIGIT-anti-PD-L1 bispecific antibody and overexpression of human/cynomolgus PD-L1 CHO cells Binding activity and blocking activity
  • CHO-S cells overexpressing human PD-L1 (CHO-huPD- L1 cells) and CHO-S cells overexpressing cynomolgus monkey PD-L1 (CHO-cynoPD-L1 cells). Adjust the overexpressed cells in the expanded culture to a suitable cell density and add them to a 96-well flow plate. After centrifugation, add serially diluted samples to be tested and incubate at 4°C for 30 minutes. Wash twice with PBS, add fluorescent secondary antibody correspondingly diluted to an appropriate concentration, and incubate at 4°C for 30 minutes. Wash twice with PBS, add PBS to resuspend cells, detect on CytoFlex flow cytometer and calculate corresponding MFI. The EC 50 value was obtained by graph analysis using Graphpad software.
  • Table 2 Binding blocking activity of anti-TIGIT-anti-PD-L1 bisantibody to overexpressed PD-L1/PD-1 cells
  • the results show that the binding activity of the anti-TIGIT-anti-PD-L1 bispecific antibody of the present invention to human/cynomolgus monkey PD-L1 overexpressed on CHO cells and its PD-L1 end monoclonal antibody molecule (anti-PD-L1 VHH )resemblance.
  • CHO-S cells (CHO-huPD-1 cells) overexpressing human PD-1 were generated by transfecting pCHO1.0 vector of human PD-1 cloned into MCS (purchased from Invitrogen) and pressurized selection.
  • the purified antibody to be tested was diluted with PBS, and 60 ⁇ L/well of the diluted sample was added to a 96-well flow plate. Then 60 ⁇ L/well of biotinylated human PD-L1 protein was added to the final concentration of 0.5 ⁇ g/mL, mixed and incubated at 4°C for 30 minutes.
  • the expanded cultured CHO-huPD-1 cells were adjusted to a cell density of 2 ⁇ 10 6 cells/mL, 100 ⁇ L/well was added to a 96-well flow plate, and the supernatant was discarded after centrifugation. Add 100 ⁇ L/well of the above co-incubated antibody-antigen mixture and incubate at 4°C for 30 minutes. Wash twice with PBS, add 100 ⁇ L/well of streptavidin-R-phycoerythrin conjugate (SAPE) diluted 100 times with PBS, and incubate at 4°C for 30 minutes. Wash twice with PBS, add 100 ⁇ L/well of PBS to resuspend cells, detect on CytoFlex flow cytometer and calculate corresponding MFI. Graphpad software was used to analyze and obtain IC50 values.
  • SAPE streptavidin-R-phycoerythrin conjugate
  • the results are shown in Table 2 and Figure 2C.
  • the results show that the anti-TIGIT-anti-PD-L1 bispecific antibody of the present invention has the blocking activity for the binding of human PD-L1 to human PD-1 overexpressed on CHO cells and its PD-L1 end monoclonal antibody molecule (anti- PD-L1 VHH) similar or even better.
  • Example 4 Anti-TIGIT-anti-PD-L1 bispecific antibody and overexpression of human/cynomolgus monkey/mouse TIGIT CHO Cell binding activity and blocking activity
  • the pCHO1.0 vector purchased from Invitrogen
  • human TIGIT human TIGIT
  • cynomolgus TIGIT mouse TIGIT cDNA cloned into MCS was transfected to produce CHO-S cells (CHO-huTIGIT cells) overexpressing human TIGIT.
  • CHO-S cells overexpressing cynomolgus TIGIT
  • CHO-muTIGIT cells CHO-muTIGIT cells
  • Table 3 Binding blocking activity of anti-TIGIT-anti-PD-L1 double antibody and overexpressed TIGIT cells
  • the expanded cultured CHO-huTIGIT cells were adjusted to a cell density of 2 ⁇ 10 6 cells/mL, 100 ⁇ L/well was added to a 96-well flow plate, and centrifuged for later use. Dilute the purified monoclonal antibody with PBS, starting at 400nM and diluting 3 times for a total of 12 points. Add 60 ⁇ L/well of the diluted sample into the above-mentioned 96-well flow plate with cells, and incubate at 4°C for 30 minutes. Then 60 ⁇ L/well of human CD155 protein with Mouse IgG2a Fc Tag was added to the final concentration of 2 ⁇ g/mL, and incubated at 4°C for 30 minutes.
  • Example 5 Co-binding activity of anti-TIGIT-anti-PD-L1 bispecific antibody with human TIGIT and human PD-L1
  • the co-binding activity of the anti-TIGIT-anti-PD-L1 bispecific antibody of the present invention with human TIGIT and human PD-L1 proteins was detected based on an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • human TIGIT protein was dissolved according to the instructions, diluted to 1 ⁇ g/mL with 1 ⁇ ELISA coating solution, 100 ⁇ L/well was coated in a 96-well ELISA plate, and covered overnight at 4°C. Discard the coating solution, wash 3 times with 1 ⁇ PBST, add 200 ⁇ L/well of 5% BSA/PBS to block at room temperature for 2 hours. Discard the blocking solution, add 100 ⁇ L/well of the antibody to be tested in a gradient dilution of 1% BSA/PBS, and incubate at room temperature for 2 hours.
  • the anti-TIGIT-anti-PD-L1 bispecific antibody of the present invention can simultaneously bind to human TIGIT and human PD-L1 proteins.
  • Example 6 Anti-TIGIT-anti-PD-L1 bispecific antibody in luciferase reporter gene system for PD-1/PD- Blocking activity of L1 and TIGIT/CD155/CD112 signaling pathway
  • a CHO-K1 cell line (CHO-K1-CD155-CD112-PD-L1) overexpressing human CD155, human CD112, human PD-L1 and OKT-3 scFv was constructed by using lentivirus transfection cells.
  • a Jurkat cell line (Jurkat-TIGIT-PD-1-luc) overexpressing human TIGIT, human PD-1 and NF-AT luciferase reporter genes (purchased from Promega) was obtained, and this reporter gene system was used to carry out related experiments.
  • CHO-K1-CD155-CD112-PD-L1 functional cells were obtained by digestion, the cell density was adjusted, 100 ⁇ L/well was added to a 96-well white bottom plate, and adherent culture was carried out overnight. The next day, a Jurkat-TIGIT-PD-1-luc effector cell suspension was prepared, and the sample to be tested was serially diluted with the reaction medium.
  • the result is shown in Figure 5.
  • the results show that the anti-TIGIT-anti-PD-L1 bispecific antibody of the present invention can release the CD155/CD112-mediated TIGIT downstream inhibitory signaling pathway and PD-L1-mediated PD-1 downstream inhibitory signaling pathway respectively, and up-regulate the reporter gene Luciferase expression, and the activity is better than the combination of two single-end antibody molecules.
  • Example 7 Effect of anti-TIGIT-anti-PD-L1 bispecific antibody on T cell activation in mixed lymphocyte assay active
  • the activation of T cells by the anti-TIGIT-anti-PD-L1 bispecific antibody was detected by mixed lymphocyte reaction assay (MLR).
  • MLR mixed lymphocyte reaction assay
  • PBMC cells purchased from SAILYBIO, XFB-HP100B
  • centrifuge resuspend PBMCs with 10mL X-VIVO-15 medium (purchased from LONZA), culture in a cell incubator at 37°C for 2 hours, and suck off the non-adhered cell.
  • Add 10mL DC medium add 10ng/mL GM-CSF (purchased from R&D) and 20ng/mL IL-4 (purchased from R&D) to X-VIVO-15 medium, culture for 3 days, add 5mL DC medium, and continue to cultivate On day 6, DC maturation medium was added: X-VIVO-15 medium was added with 1000 U/mL TNF- ⁇ (purchased from R&D), 10 ng/mL IL-6 (purchased from R&D), 5 ng/mL IL-1 ⁇ ( Purchased from R&D), 1 ⁇ M PGE2 (purchased from Tocris), cultured for 2 days, collected mature DC cells, and adjusted the cell density to 2 ⁇ 10 5 cells/mL with X-VIVO-15 medium.
  • TNF- ⁇ purchasedd from R&D
  • 10 ng/mL IL-6 purchasedd from R&D
  • 5 ng/mL IL-1 ⁇ Purchased from R&D
  • 1 ⁇ M PGE2 purchasedd from Tocris
  • PBMC cells from another donor (purchased from SAILY BIO, XFB-HP100B), centrifuge, and resuspend PBMC with 10 mL X-VIVO-15 medium.
  • Enrich T cells with a T cell sorting kit (purchased from Stemcell), resuspend T cells in X-VIVO-15, and adjust the cell density to 2 ⁇ 10 6 cells/mL.
  • Example 8 Anti-TIGIT-anti-PD-L1 bispecific antibody mixed with A375 and human inoculation in B-NDG mice In vivo pharmacodynamic study of PBMC
  • B-NDG mice were inoculated with A375 (purchased from Addexbio, C0020004, a kind of human malignant melanoma cells), human PBMC cells (Shanghai Miaoshun, A10S033014/PB100C) model (A375huPBMC model) to measure the Antitumor effect of anti-TIGIT-anti-PD-L1 bispecific antibody.
  • A375 purchased from Addexbio, C0020004, a kind of human malignant melanoma cells
  • human PBMC cells Sthanghai Miaoshun, A10S033014/PB100C model
  • A375huPBMC model an antitumor effect of anti-TIGIT-anti-PD-L1 bispecific antibody.
  • PBMC human immune cells
  • A375 cells and human PBMCs were mixed at 1:1 equal volume to form 0.1 mL of cell suspension, and A375huPBMC models were established by subcutaneous injection in the right abdominal groove of mice, and grouped when the average tumor volume grew to about 200 mm 3 .
  • Different doses and the same volume of PBS or antibody treatment were administered intraperitoneally, with 6 mice in each group.
  • the changes in the tumor volume and body weight of the mice in each group were monitored, and the monitoring frequency was 2-3 days/time, and the monitoring was continuous for 2 to 3 weeks.
  • the dosage and method of administration are shown in Table 4.
  • the molecular weights of Anti-PD-L1 VHH, Anti-TIGIT mAb, and anti-TIGIT/PD-L1 double antibody are about 75KD, 150KD, and 175KD, respectively, and the dosage is given according to the equimolar concentration of each component.
  • Example 9 Anti-TIGIT-anti-PD-L1 bispecific antibody mixed with A375 and human inoculation in B-NDG mice Dose-dependent pharmacodynamic study of PBMC in vivo
  • the A375 huPBMC model was established by subcutaneous mixed inoculation (the modeling steps were the same as in Example 8).
  • the average tumor volume grew to about 300 mm 3 , they were divided into groups, and different doses of PBS with the same volume of administration were administered intraperitoneally.
  • Specific antibody treatment 6 mice per group.
  • the changes in tumor volume and body weight of the mice in each group were monitored.
  • the monitoring frequency was 2-3 days/time, and the monitoring was continuous for 2 to 3 weeks.
  • the dosage and method of administration are shown in Table 5.
  • the anti-TIGIT-anti-PD-L1 bispecific antibody of the present invention can significantly inhibit the growth of mouse tumors in a dose-dependent manner.
  • CT-26-huPD-L1 tumor cells human PD-L1 knock-in CT26 Mouse colon cancer cells (purchased from Jiangsu Jicui Yaokang) were used to measure the anti-tumor effect of the anti-TIGIT-anti-PD-L1 bispecific antibody of the present invention.
  • CT-26-huPD-L1 cell suspension firstly prepare CT-26-huPD-L1 cell suspension, and subcutaneously inject 0.1 mL of about 5 ⁇ 10 5 cells into the right inguinal groove of the mouse to establish the CT-26-huPD-L1 tumor-bearing mouse model.
  • the length reached 80-120mm 3 , they were divided into groups, and treated with different doses of PBS or antibody in the same administration volume by intraperitoneal injection, with 6 mice in each group.
  • the changes in tumor volume and body weight of the mice in each group were monitored, and the monitoring frequency was 2-3 days/time, and the monitoring was continuous for 2 to 3 weeks.
  • the dosage and method of administration are shown in Table 6.
  • the anti-TIGIT-anti-PD-L1 bispecific antibody of the present invention has similar efficacy to the clinical drug combination (atezolizumab+tiragolumab), and the anti-tumor activity is better than the two monoclonal antibody drug groups .
  • mice The half-life of the anti-TIGIT-anti-PD-L1 bispecific antibody of the present invention in mice was detected by a single tail vein injection method.
  • the experiment uses Balb/c mice, 3 males and 3 males, 12/12 hours light/dark adjustment, temperature 24°C ⁇ 2°C, humidity 40%-70%, free access to water and diet.
  • Balb/c mice were given a single tail vein injection of monoclonal antibody molecules at a dose of 10 mg/kg.
  • Blood collection time point 5 minutes, 0.5 hours, 2 hours, 6 hours, 24 hours, 48 hours, 96 hours, 168 hours, 336 hours, 504 hours after administration, blood was collected from the mouse orbit.
  • the heavy chain variable region of the anti-TIGIT monoclonal antibody (the amino acid sequence is shown in SEQ ID NO: 19) was recombined into the human IgG1 heavy chain constant region and the human IgG1 heavy chain L234A, L235A modified constant region respectively.
  • the light chain variable region (amino acid sequence shown in SEQ ID NO: 20) was recombined into the human kappa light chain constant region, and the anti-TIGIT monoclonal antibodies were named 55796-G1 and 55796-G1LALA, respectively.
  • the specific operation is as follows: the pcDNA3.1 vector carrying the antibody heavy chain and light chain was transferred into HEK293 cells by chemical transfection method, and cultured for 7 days at 37° C. and 8% CO 2 . The cell fluid was collected and centrifuged at 13000rpm for 20 minutes. Take the supernatant, Protein A purification supernatant, SEC to detect the purity of the antibody, while controlling the endotoxin content.
  • the prepared anti-TIGIT monoclonal antibodies 55796-G1 and 55796-G1LALA were used in the following test examples 1-3.
  • Test Example 1 Affinity Detection of Anti-TIGIT Monoclonal Antibody
  • the binding dissociation constant (K D ) of the anti-TIGIT monoclonal antibody prepared in Preparation Example 1 binding to human, cynomolgus monkey, and mouse TIGIT was determined by biofilm layer optical interference technology (ForteBio). Fortebio affinity was determined according to the existing method (Este, P et al. High throughput solution-based measurement of antibody-antigen affinity and epitope binning. Mabs, 2013.5 (2): p.270-8).
  • the amino acid sequence of the extracellular segment of human TIGIT, the amino acid sequence of the extracellular segment of TIGIT of cynomolgus monkey, and the amino acid sequence of the extracellular segment of mouse TIGIT are shown in SEQ ID NOs: 8-9 and 21, respectively.
  • Test Example 2 The binding activity of anti-TIGIT monoclonal antibody to overexpressed human/cynomolgus monkey/mouse TIGIT CHO cells and blocking activity
  • the pCHO1.0 vector purchased from Invitrogen
  • human TIGIT human TIGIT
  • cynomolgus TIGIT mouse TIGIT cDNA cloned into MCS was transfected to produce CHO-S cells (CHO-huTIGIT cells) overexpressing human TIGIT.
  • CHO-S cells overexpressing cynomolgus TIGIT
  • CHO-muTIGIT cells CHO-muTIGIT cells
  • the expanded cultured CHO-huTIGIT cells were adjusted to a cell density of 2 ⁇ 10 6 cells/mL, 100 ⁇ L/well was added to a 96-well flow plate, and centrifuged for later use. Dilute the purified monoclonal antibody with PBS, starting at 400nM and diluting 3 times for a total of 12 points. Add 60 ⁇ L/well of the diluted sample into the above-mentioned 96-well flow plate with cells, and incubate at 4°C for 30 minutes.
  • Table 8 Summary table of anti-TIGIT monoclonal antibody overexpression cell binding blocking activity
  • the anti-TIGIT monoclonal antibody of the present invention (1) has a better binding activity with the human TIGIT protein overexpressed on the surface of CHO cells than the control molecule Tiragolumab; (2) binds with the overexpressed human TIGIT protein on the surface of CHO cells.
  • the binding activity of cynomolgus monkey TIGIT protein is better than that of the control molecule Tiragolumab; (3) It has obvious binding to mouse TIGIT protein overexpressed on the surface of CHO cells.
  • the anti-TIGIT monoclonal antibody of the present invention (1) is superior to the control molecule Tiragolumab in blocking the binding of human CD155 to the human TIGIT protein overexpressed on the surface of CHO cells;
  • the anti-TIGIT antibody molecules bound to the overexpressed mouse TIGIT protein on the cell surface can significantly block the binding of mouse CD155 to the overexpressed mouse TIGIT protein on the surface of CHO cells.
  • the binding activity of the invented anti-TIGIT antibody to TIGIT on the surface of activated T cells was detected based on the flow cytometry detection method.
  • human PBMCs were sorted according to the protocol provided by STEMCELL (stemcell, catalog number: #17951C) to obtain human total T cells.
  • Use X-VIVO15 medium (purchased from lonza, product number: 04-418Q) to adjust the concentration of T cells to 1.0 ⁇ 10 6 cells/mL, add 1 ⁇ L IL-2 stock solution (1 million IU), and simultaneously 1:1 (bead-to- -cell) was added with CD3/CD28 Dynabeads (purchased from gibco, product number: 11132D), and cultured in a 5% CO 2 incubator at 37° C. for 48 hours.
  • the activated T cells were adjusted to a suitable cell density and added to a 96-well flow plate, after centrifugation, a gradient dilution of the test sample was added, and incubated at 4°C for 30 minutes. Wash twice with PBS, add corresponding fluorescent secondary antibody diluted to an appropriate concentration, incubate at 4°C for 30 minutes, wash twice with PBS. Add PBS to resuspend cells, detect on CytoFlex flow cytometer and calculate corresponding MFI.

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Abstract

本发明属于生物医药领域，涉及一种抗TIGIT-抗PD-L1双特异性抗体、其药物组合物及用途。本发明的双特异性抗体包括：靶向TIGIT的第一蛋白功能区，和靶向PD-L1的第二蛋白功能区；其中：所述第一蛋白功能区为抗TIGIT抗体或其抗原结合片段；所述抗TIGIT抗体的重链可变区包含氨基酸序列如SEQ ID NO:10所示的HCDR1、氨基酸序列如SEQ ID NO:11所示的HCDR2以及氨基酸序列如SEQ ID NO:12所示的HCDR3；并且所述抗TIGIT抗体的轻链可变区包含氨基酸序列如SEQ ID NO:13所示的LCDR1、氨基酸序列如SEQ ID NO:14所示的LCDR2以及氨基酸序列如SEQ ID NO:15所示的LCDR3。本发明的双特异性抗体具有针对TIGIT和PD-L1双靶点的高亲和力，具有良好的应用前景。

Description

抗TIGIT-抗PD-L1双特异性抗体、其药物组合物及用途 技术领域
本发明属于医药领域，涉及一种抗TIGIT-抗PD-L1双特异性抗体、其药物组合物及用途。
背景技术
T细胞免疫球蛋白和ITIM结构域(T cell immunoreceptor with Ig and ITIM domains,TIGIT,又称WUCAM、Vstm3、VSIG9)是一种新型免疫抑制性受体，由活化的CD8 +T和CD4 +T细胞、自然杀伤(NK)细胞、调节性T细胞(Tregs)和滤泡辅助性T细胞表达。
TIGIT在肿瘤免疫中参与了一个复杂的调控网络，涉及多个免疫抑制受体(例如，CD96/TACTILE，CD112R/PVRIG)、一个竞争性共刺激受体(DNAM-1/CD226)和多个配体(例如，CD155(PVR/NECL-5)，CD112(Nectin-2/PVRL2))。DNAM-1、TIGIT和CD96在T细胞和NK细胞上表达，并以配体形式共享CD155。
TIGIT在肿瘤免疫抑制中的作用和PD-1/PD-L1类似，目前的研究已经提出了几种TIGIT介导的效应T细胞和NK细胞抑制作用的机制:(1)T/NK细胞表面TIGIT与CD155结合，使TIGIT胞内的免疫受体酪氨酸抑制基序(ITIM)磷酸化，直接转导抑制性信号；(2)TIGIT通过与DCs上的CD155结合，促进形成免疫耐受性DCs，降低白细胞介素(IL)-12的产生和IL-10的增加，间接抑制T细胞应答；(3)具有抑制作用的TIGIT以比共刺激受体CD226更高的亲和力竞争结合CD155，从而限制CD226介导的激活。另外，TIGIT还直接在细胞上顺式结合CD226，破坏其与CD155的同源二聚体的结合能力；(4)表达TIGIT的Tregs具有高度抑制性，TIGIT+Tregs激活后产生IL-10和纤维蛋白原样蛋白2(Fgl2)，介导T细胞抑制；(5)来自核梭杆菌(Fusobacterium nucleatum,一种与结直肠癌相关的厌氧革兰氏共生菌)的Fap2蛋白，可直接与TIGIT结合，但不与CD226结合，从而抑制NK细胞和T细胞介导的肿瘤免疫，调节先天免疫反应。
程序性死亡因子1配体1(programmed death 1 ligand 1，PD-L1)又称CD274，为B7家族成员，是PD-1的配体。PD-L1属于I型跨膜蛋白，共290个氨基酸，包含1个IgV样区、1个IgC样区、1个跨膜疏水区和1个由30个氨基酸组成的胞内区。与其 他B7家族分子不同的是，PD-L1具有负向调节免疫应答的作用。研究发现，PD-L1主要表达于活化的T细胞、B细胞、巨噬细胞和树突状细胞等，除淋巴细胞外，PD-L1也表达于其它多种组织如胸腺、心脏、胎盘等的内皮细胞，以及各类非淋巴系如黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、结肠癌、乳腺癌、食道癌、头颈癌等(Akintunde Akinleye&Zoaib Rasool，Journal of Hematology&Oncology volume 12,Article number:92(2019))。PD-L1在调节自身反应性T、B细胞和免疫耐受方面具有一定广泛性，并且在外周组织T和B细胞应答起作用。PD-L1在肿瘤细胞上的高表达与癌症患者的不良预后相关。
双特异性抗体虽然是抗体药物研发的一个方向，但面临诸多挑战，比如临床前评价模型、表达量低、稳定性差、工艺复杂、质控差异性大等问题，因此一直以来双特异性抗体的研发困难重重。
因此，需要开发一种特异性佳、疗效好且易于制备的针对TIGIT和PD-L1两个靶点的双特异性抗体。
发明内容
本发明人经过深入的研究和创造性的劳动，得到了一种抗TIGIT抗体，并基于该抗体构建了抗TIGIT-抗PD-L1的双特异性抗体。本发明人惊奇地发现，本发明的抗TIGIT抗体(也简称为抗体或本发明的抗体)和抗TIGIT-抗PD-L1双特异性抗体(也简称为双特异性抗体或本发明的双特异性抗体)与PD-L1和TIGIT的结合亲和力高(甚至在一个或多个方面优于阳性对照抗体该双特异性抗体)，可以分别阻断PD-L1与其配体PD-1的结合以及TIGIT与其配体CD155/CD112的结合，减少或消除传递至细胞的抑制信号，在动物模型中施用本发明的抗体可以显著抑制肿瘤生长。由此提供了下述发明：
本发明的一个方面设计一种双特异性抗体，其包括：
靶向TIGIT的第一蛋白功能区，和
靶向不同于TIGIT的靶点(例如PD-L1)的第二蛋白功能区；
其中：
所述第一蛋白功能区为抗TIGIT免疫球蛋白或其抗原结合片段；
所述抗TIGIT免疫球蛋白的重链可变区包含氨基酸序列如SEQ ID NO:10所示的HCDR1、氨基酸序列如SEQ ID NO:11所示的HCDR2以及氨基酸序列如SEQ ID NO: 12所示的HCDR3；并且
所述抗TIGIT免疫球蛋白的轻链可变区包含氨基酸序列如SEQ ID NO:13所示的LCDR1、氨基酸序列如SEQ ID NO:14所示的LCDR2以及氨基酸序列如SEQ ID NO:15所示的LCDR3。
轻链和重链的可变区决定抗原的结合；每条链的可变区均含有三个高变区，称互补决定区(CDR)，其中重链(H)的CDR包含HCDR1、HCDR2、HCDR3，轻链(L)的CDR包含LCDR1、LCDR2、LCDR3。本发明的抗体或其抗原结合片段含有的CDR可根据本领域已知的各种编号系统确定。在某些实施方案中，本发明的抗体或其抗原结合片段含有的CDR优选地通过Kabat、Chothia，AbM HVR或IMGT编号系统确定。除非另有说明，否则本发明中抗体或其抗原结合片段含有的CDR优选地通过Kabat编号系统(kabat等人,Sequences of Proteins of Immunological Interest,5 th Ed.Public Health Service,National Institute of Health,Bethesda,Md.(1991))确定的编号位置。
在本发明的一些实施方式中，所述的双特异性抗体，其中，所述第一蛋白功能区和第二蛋白功能区直接连接或者通过连接片段(linker，也称为连接肽)连接；
优选地，所述连接片段为(GGGGS)m，m为正整数，例如1、2、3、4、5或6；
优选地，所述连接片段为(GGGGS)nG，n为正整数，例如1、2、3、4、5或6；
优选地，所述连接片段的氨基酸序列如SEQ ID NO:2所示。
其中，括号内的GGGGS表示为SEQ ID NO:22。
在本发明的一些实施方式中，所述的双特异性抗体，其中，所述第一蛋白功能区和第二蛋白功能区独立地为1个、2个或者2个以上。
在本发明的一些实施方式中，所述的双特异性抗体，其中，所述第一蛋白功能区为1个，并且所述第二蛋白功能区为2个。
在本发明的一些实施方式中，所述的双特异性抗体，其中，所述第一蛋白功能区为2个，并且所述第二蛋白功能区为1个。
在本发明的一些实施方式中，所述的双特异性抗体，其中，
所述第一蛋白功能区为抗TIGIT免疫球蛋白或其抗原结合片段，并且所述第二蛋白功能区为靶点不同于TIGIT的单域抗体或单链抗体；
优选地，所述单域抗体为抗PD-L1单域抗体；
优选地，所述单链抗体为抗PD-L1单链抗体。
在本发明的一些实施方式中，所述的双特异性抗体，其中，
所述第一蛋白功能区为抗TIGIT免疫球蛋白或其抗原结合片段，并且所述第二蛋白功能区为抗PD-L1单域抗体或抗PD-L1单链抗体
在本发明的一些实施方式中，所述的双特异性抗体，其中，
所述第一蛋白功能区为抗TIGIT单链抗体，并且所述第二蛋白功能区为靶点不同于TIGIT的免疫球蛋白或其抗原结合片段；
优选地，靶点不同于TIGIT的免疫球蛋白为抗PD-L1免疫球蛋白。
在本发明的一些实施方式中，所述的双特异性抗体，其中，
所述第一蛋白功能区为抗TIGIT单链抗体，并且所述第二蛋白功能区为抗PD-L1免疫球蛋白或其抗原结合片段。
在本发明的一些实施方式中，所述的双特异性抗体，其中，
所述抗PD-L1单域抗体包含一个重链可变区，所述重链可变区包含氨基酸序列如SEQ ID NO:16所示的HCDR1、氨基酸序列如SEQ ID NO:17所示的HCDR2以及氨基酸序列如SEQ ID NO:18所示的HCDR3；
优选地，所述抗PD-L1单域抗体的氨基酸序列如SEQ ID NO:3所示。
在本发明的一些实施方式中，所述的双特异性抗体，其中，
所述抗TIGIT免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:19所示，并且其轻链可变区的氨基酸序列如SEQ ID NO:20所示；
优选地，
所述抗TIGIT免疫球蛋白的重链的氨基酸序列如SEQ ID NO:1所示，其轻链的氨基酸序列如SEQ ID NO:5所示。
在本发明的一些实施方式中，所述的双特异性抗体，其中，所述抗原结合片段选自Fab、Fab'、F(ab')2、Fd、Fv、dAb和互补决定区片段。
在本发明的一些实施方式中，所述的双特异性抗体，其中，
所述抗TIGIT免疫球蛋白或者靶点不同于TIGIT的免疫球蛋白的恒定区来自人抗体；
优选地，所述恒定区选自人IgG1、IgG2、IgG3或IgG4的恒定区。
在本发明的一些实施方式中，所述的双特异性抗体，其中，
所述抗TIGIT免疫球蛋白或者靶点不同于TIGIT的免疫球蛋白的重链恒定区为人Ig gamma-1 chain C region(例如NCBI ACCESSION:P01857)或人Ig gamma-4 chain C  region(例如NCBI ACCESSION:P01861.1)，并且其轻链恒定区为人Ig kappa chain C region(例如NCBI ACCESSION:P01834)；优选地，所述抗TIGIT免疫球蛋白的重链恒定区还包含按照EU编号系统的L234A突变和L235A突变。
在本发明的一些实施方式中，所述的双特异性抗体，其中，所述单域抗体或单链抗体连接在抗TIGIT免疫球蛋白的C末端或N末端，例如，所述单域抗体或单链抗体为两个，每个单域抗体或单链抗体的一端分别连接在抗TIGIT免疫球蛋白的两条重链的C末端或N末端。
在本发明的一些实施方式中，所述的双特异性抗体，其中，所述抗PD-L1单域抗体或抗PD-L1单链抗体连接在抗TIGIT免疫球蛋白的C末端或N末端，例如，所述抗PD-L1单域抗体或抗PD-L1单链抗体为两个，每个抗PD-L1单域抗体或抗PD-L1单链抗体的一端分别连接在抗TIGIT免疫球蛋白的两条重链的C末端或N末端。
在本发明的一些实施方式中，所述的双特异性抗体，其中，所述单域抗体为抗PD-L1单域抗体，单域抗体连接在抗TIGIT免疫球蛋白得到的肽链的氨基酸序列如SEQ ID NO:4所示。
在本发明的一些实施方式中，所述的双特异性抗体，其为由两条相同的第一肽链和两条相同的第二肽链形成的四聚体，其中，
所述第一肽链的氨基酸序列如SEQ ID NO:4所示；并且所述第二肽链的氨基酸序列如SEQ ID NO:5所示。
在本发明的一些实施方式中，所述的双特异性抗体，包括：
靶向TIGIT的第一蛋白功能区，和
靶向PD-L1的第二蛋白功能区；
所述第一蛋白功能区为1个，所述第二蛋白功能区为2个；
其中，所述第一蛋白功能区为抗TIGIT免疫球蛋白，所述第二蛋白功能区为抗PD-L1单域抗体；
所述抗TIGIT免疫球蛋白的重链的氨基酸序列如SEQ ID NO:1所示，并且其轻链的氨基酸序列如SEQ ID NO:5所示；
所述抗PD-L1单域抗体的氨基酸序列如SEQ ID NO:3所示；
两个抗PD-L1单域抗体分别通过相同或不同的连接片段连接在抗TIGIT免疫球蛋白的每条重链的C末端；
优选地，所述连接片段为(GGGGS)m，m为正整数，例如1、2、3、4、5或6；
优选地，所述连接片段为(GGGGS)nG，n为正整数，例如1、2、3、4、5或6；
优选地，所述连接片段的氨基酸序列如SEQ ID NO:2所示。
其中，括号内的GGGGS表示为SEQ ID NO:22。
在本发明的一个或多个实施方式中，所述的双特异性抗体，其为IgG-VHH形式。
在本发明的一个或多个实施方式中，所述的双特异性抗体，其为IgG-scFv形式即Morrison模式。
在本发明的一个或多个实施方式中，所述的双特异性抗体，其中，所述单域抗体或单链抗体连接在免疫球蛋白的重链的C末端。由于免疫球蛋白由两条重链，因此，一个免疫球蛋白分子连接有两个单域抗体分子或两个单链抗体分子。优选地，两个单域抗体分子相同。优选地，两个单链抗体分子相同。优选地，所述单域抗体或单链抗体通过前述连接片段与免疫球蛋白的重链的C末端形成酰胺键连接。
在本发明的一个或多个实施方式中，所述的双特异性抗体，其与人PD-L1、食蟹猴PD-L1、人TIGIT和/或食蟹猴TIGIT抗原结合的单价亲和力，分别与其单端抗体分子与人PD-L1、食蟹猴PD-L1、人TIGIT和/或食蟹猴TIGIT抗抗原结合的单价亲和力相同或更优。
本发明的另一方面涉及分离的核酸分子，其编码权本发明中任一项所述的双特异性抗体。
本发明的再一方面涉及一种载体，其包含本发明的分离的核酸分子。
本发明的再一方面涉及一种宿主细胞，其包含本发明的分离的核酸分子，或者本发明的载体。
本发明的再一方面涉及制备本发明中任一项所述的双特异性抗体的方法，其包括在合适的条件下培养本发明的宿主细胞，以及从细胞培养物中回收所述双特异性抗体的步骤。
本发明的再一方面涉及一种偶联物，其包括双特异性抗体以及偶联部分，其中，所述双特异性抗体为本发明中任一项所述的双特异性抗体，所述偶联部分为可检测的标记；优选地，所述偶联部分为放射性同位素、荧光物质、发光物质、有色物质或酶。
本发明的再一方面涉及一种试剂盒，其包含本发明中任一项所述的双特异性抗体， 或者包含本发明的偶联物；
优选地，所述试剂盒还包含第二抗体，其能够特异性结合所述双特异性抗体；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、发光物质、有色物质或酶。
本发明的再一方面涉及一种药物组合物，其包含本发明中任一项所述的双特异性抗体或者包含本发明的偶联物；可选地，其还包括药学上可接受的辅料。
本发明的再一方面涉及本发明中任一项所述的双特异性抗体或者本发明的偶联物在制备预防和/或治疗恶性肿瘤的药物中的用途；优选地，所述恶性肿瘤选自黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、结肠癌、乳腺癌、食道癌和头颈癌。
本发明的再一方面涉及一种治疗和/或预防恶性肿瘤的方法，包括给予有需求的受试者以有效量的本发明中任一项所述的双特异性抗体或者本发明的偶联物的步骤；优选地，所述恶性肿瘤选自黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、结肠癌、乳腺癌、食道癌和头颈癌。
在本发明的一些实施方式中，所述治疗和/或预防恶性肿瘤的方法，其中，给予有需求的受试者以有效量的本发明中任一项所述的双特异性抗体的步骤为在手术治疗之前或之后，和/或在放射治疗之前或之后。
在本发明的一些实施方式中，所述治疗和/或预防恶性肿瘤的方法，其中，
本发明的双特异性抗体的单次给药剂量为每千克体重0.1-100mg，优选4.8-24mg或1-10mg；或者，本发明的双特异性抗体的单次给药剂量为每位受试者10-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg或200mg；
优选地，每3天、4天、5天、6天、10天、1周、2周或3周给药一次；
优选地，给药方式为静脉滴注或静脉注射。
根据本发明中任一项所述的双特异性抗体或者本发明的偶联物，其用于治疗和/或预防恶性肿瘤；优选地，所述恶性肿瘤选自黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、结肠癌、乳腺癌、食道癌和头颈癌。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明， 下面提供相关术语的定义和解释。
术语“EC 50”是指半最大效应浓度(concentration for 50％of maximal effect)，是指能引起50％最大效应的浓度。
术语“抗体”是指通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循Bethesda M.d.,Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health,(1987and 1991))，或Chothia&Lesk J.Mol.Biol.1987；196:901-917；Chothia等人Nature 1989；342:878-883，或者IMGT编号系统定义，见Ehrenmann F,Kaas Q,Lefranc M P.IMGT/3Dstructure-DB and IMGT/DomainGapAlign:a database and a tool for immunoglobulins or antibodies,T cell receptors,MHC,IgSF and MhcSF[J].Nucleic acids research,2009；38(suppl_1):D301-D307的定义。
术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
术语“单抗”和“单克隆抗体”是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片段，也即除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。单克隆抗体通常可采用Kohler等首次报道的杂交瘤技术获得(
Figure PCTCN2022132422-appb-000001
G,Milstein C.Continuous cultures of fused cells secreting antibody of predefined specificity[J].nature,1975；256(5517): 495)，但也可采用重组DNA技术获得(如参见U.S.Patent 4,816,567)。
术语“单链抗体(single chain fragment variable，ScFv)”是指，包含通过连接体连接的抗体重链可变区(V H)和抗体轻链可变区(V L)的分子。其中V L和V H结构域通过使其能够产生为单个多肽链的连接体配对形成单价分子(参见，例如,Bird et al,Science 1988；242:423-426和Huston et al,Proc.Natl.Acad.Sci.USA 1988；85:5879-5883)。此类scFv分子可具有一般结构：NH2-V L-连接片段-V H-COOH或NH2-V H-连接片段-V L-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4(SEQ ID NO:23)的接头，但也可使用其变体(Holliger et al，Proc.Natl.Acad.Sci.USA 1993；90:6444-6448)。可用于本发明的其他接头由Alfthan et al，Protein Eng.1995；8:725-731，Choi et al，Eur.J.Immunol.2001；31:94-106，Hu et al，Cancer Res.1996；56:3055-3061，Kipriyanov et al，J.Mol.Biol.1999；293:41-56和Roovers et al,Cancer Immunology,Immunotherapy,2001,50(1):51-59.描述。
如本文中所使用的，术语“分离的”或“被分离的”指的是，从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”或“被分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草杆菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果 蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，GS细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中使用的，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。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的亲和力(K D)结合该抗原。
如本文中所使用的，术语“K D”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的解离平衡常数(K D)结合抗原(例如，TIGIT蛋白或PD-L1蛋白)。可以使用本领域技术人员知悉的方法测定K D，例如使用Fortebio分子相互作用仪测定。
如本文中所使用的，术语“单克隆抗体”和“单抗”具有相同的含义且可互换使用；术语“多克隆抗体”和“多抗”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“药学上可接受的辅料”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19 th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。例如，pH调节剂包括但不限于磷酸盐缓冲液；表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80；离子强度增强剂包括但不限于氯化钠。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如肿瘤)有效量是指，足以预防，阻止，或延迟疾病(例如肿瘤)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
如本文中所使用的，当提及PD-L1蛋白(Programmed death-ligand 1,NCBI GenBank ID:NP_054862.1)的氨基酸序列时，其包括人PD-L1蛋白的全长，或者人PD- L1的胞外片段PD-L 1 ECD(例如氨基酸序列如SEQ ID NO:6所示)或者包含PD-L 1 ECD的片段；还包括PD-L1ECD的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)进行融合的片段。然而，本领域技术人员理解，在PD-L1蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“PD-L1蛋白”应包括所有此类序列，包括所示的序列以及其天然或人工的变体。并且，当描述PD-L1蛋白的序列片段时，其不仅包括的序列片段，还包括其天然或人工变体中的相应序列片段。
如本文中所使用的，当提及TIGIT蛋白(T细胞免疫球蛋白和ITIM结构域T cell immunoreceptor with Ig and ITIM domains，NCBI GenBank ID:NP_776160.2)的氨基酸序列时，其包括人TIGIT蛋白的全长，或者人TIGIT的胞外片段TIGIT ECD(例如氨基酸序列如SEQ ID NO:8所示)或者包含TIGIT ECD的片段；还包括TIGIT蛋白的全长的融合蛋白或TIGIT ECD的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)进行融合的片段。然而，本领域技术人员理解，在TIGIT蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“添加)，蛋白”应包括所有此类序列，包括其天然或人工的变体。并且，当描述TIGIT蛋白的序列片段时，其还包括其天然或人工变体中的相应序列片段。
如本文中所使用的，当提及PD-1蛋白(NCBI GenBank:NP_005009.2)的氨基酸序列时，其包括人PD-1蛋白的全长，或者人PD-1的胞外片段PD-1ECD或者包含PD-1 ECD的片段；还包括PD-1蛋白的全长的融合蛋白或PD-1 ECD的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)进行融合的片段。然而，本领域技术人员理解，在PD-1蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“添加)，蛋白”应包括所有此类序列，包括其天然或人工的变体。并且，当描述PD-1蛋白的序列片段时，其还包括其天然或人工变体中的相应序列片段。
本发明中，术语“单域抗体”、“VHH”、“纳米抗体”具有相同的含义，指克隆抗体重链的可变区，构建仅由一个重链可变区组成的纳米抗体(VHH)，它是具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的纳米抗体(VHH)。
在本发明中，如果没有特别说明，所述“第一”(例如第一蛋白功能区、第一肽链) 和“第二”(例如第二蛋白功能区、第二肽链)是为了指代上的区分或表述上的清楚，并不具有典型的次序上的含义。
本发明中，所述单端抗体分子，如果没有特别说明，是指与双特异性抗体中的第一蛋白功能区分子或第二蛋白功能区分子相同或类似的抗体分子。例如与双特异性抗体中的第一蛋白功能区分子或第二蛋白功能区分子相同或类似的抗TIGIT单抗、抗PD-L1单抗、或抗PD-L1单域抗体。
发明的有益效果
本发明取得了如下效果中的一项或多项：
(1)本发明的抗TIGIT抗体具有优越的亲和力和特异性；
(2)本发明的双特异性抗体能够很好地特异性与TIGIT结合；
(3)本发明的双特异性抗体能够很好地特异性与PD-L1结合。
(4)本发明的双特异性抗体能够同时结合人TIGIT和人PD-L1蛋白；
(5)本发明的双特异性抗体中的第一蛋白功能区和第二蛋白功能区之间具有协同作用。例如：本发明的抗TIGIT-抗PD-L1双特异性抗体可以分别解除CD155/CD112介导的TIGIT下游抑制信号通路以及PD-L1介导的PD-1下游抑制信号通路，活性优于两单端抗体分子的联合；和/或体内抗肿瘤活性优于两单端抗体分子联用。
(6)本发明的双特异性抗体的生产简便。
(7)本发明的双特异性抗体的稳定性好，半衰期长。
附图说明
图1：本发明的双特异性抗体的结构示意图。
图2A：本发明的双特异性抗体对于CHO细胞上过表达的人PD-L1的结合曲线图。
图2B：本发明的双特异性抗体对于CHO细胞上过表达的食蟹猴PD-L1的结合曲线图。
图2C：本发明的双特异性抗体阻断人PD-L1与CHO细胞上过表达的人PD-1结合的曲线图。
图3A：本发明的双特异性抗体对于CHO细胞上过表达的人TIGIT的结合曲线图。
图3B：本发明的双特异性抗体对于CHO细胞上过表达的食蟹猴TIGIT的结合曲线图。
图3C：本发明的双特异性抗体对于CHO细胞上过表达的小鼠TIGIT的结合曲线图。
图3D：本发明的双特异性抗体阻断人CD155与CHO细胞上过表达的人TIGIT结合的曲线图。
图3E：本发明的双特异性抗体阻断小鼠CD155与CHO细胞上过表达的小鼠TIGIT结合的曲线图。
图4：本发明的双特异性抗体同时结合人PD-L1和人TIGIT蛋白的曲线图。
图5：本发明的双特异性抗体阻断PD-1/PD-L1与TIGIT/CD155/CD112信号通路的曲线图。
图6A和图6B：本发明的双特异性抗体在混合淋巴细胞试验中细胞因子释放量的统计图。其中图6A和图6B中使用的PBMC细胞样品来自不同的捐献者。
图7：本发明的双特异性抗体在混合接种A375和人PBMC的B-NDG小鼠模型的药效曲线图。
图8：本发明的双特异性抗体在混合接种A375和人PBMC的B-NDG小鼠模型的剂量依赖药效曲线图。
图9：本发明的双特异性抗体在人PD-L1/PD-1/TIGIT转基因小鼠CT26肿瘤模型中的药效曲线图。
图10：本发明的双特异性抗体在小鼠体内半衰期曲线。
图11：本发明抗TIGIT抗体对于CHO细胞上过表达的人TIGIT的结合曲线图。
图12：本发明抗TIGIT抗体对于CHO细胞上过表达的食蟹猴TIGIT的结合曲线图。
图13：本发明抗TIGIT抗体对于CHO细胞上过表达的小鼠TIGIT的结合曲线图。
图14：本发明抗TIGIT抗体阻断人CD155与CHO细胞上过表达的人TIGIT结合的曲线图。
图15：本发明抗TIGIT抗体阻断小鼠CD155与CHO细胞上过表达的小鼠TIGIT结合的曲线图。
图16：本发明抗TIGIT抗体与激活的人原代T细胞上TIGIT的结合曲线图。
本发明涉及的序列如下面的表A所示。其中各CDR按照Kabat编号系统确定。
表A
Figure PCTCN2022132422-appb-000002
Figure PCTCN2022132422-appb-000003
Figure PCTCN2022132422-appb-000004
Figure PCTCN2022132422-appb-000005
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
对照抗体Atezolizumab：抗PD-L1单抗，商品名Tecentriq，罗氏Roche。
对照抗体Tiragolumab：抗TIGIT单抗，罗氏Roche。
实施例1：抗TIGIT-抗PD-L1双特异性抗体的表达与纯化
在本实施例中，采用全基因合成方式合成抗TIGIT抗体重链可变区(VH)序列(SEQ ID NO:19)，将其重组至野生型hIgG1抗体重链构成完整"VH-CH1-CH2-CH3"抗体重链(SEQ ID NO:1)，并在其重链C末端通过2个连接片段G 4S(SEQ ID NO:2)连接抗PD-L1VHH(SEQ ID NO:3)，得到的肽链#1的全长氨基酸序列如SEQ ID NO:4所示。此外，全基因合成抗TIGIT轻链可变区(VL)序列(SEQ ID NO:20)，将其重组 至kappa轻链构成完整“VL-CL”抗体轻链结构，得到的肽链#2的全长氨基酸序列如SEQ ID NO:5所示。
利用分子克隆技术将重链和轻链序列分别构建到pcDNA3.1表达框内，通过Expi-293表达系统进行常规表达。转染方法按照商品说明书操作，细胞培养5天后收集上清，采用蛋白A磁珠(购自金斯瑞)分选法纯化目的蛋白。将磁珠用适当体积的结合缓冲液(PBS+0.1％吐温20，pH 7.4)重悬(1-4倍磁珠体积)后加入至待纯化样品中，室温孵育1小时，期间温柔振荡。样品置于磁力架上(购自海狸)，弃去上清，磁珠用结合缓冲液清洗3遍。按照磁珠体积的3-5倍体积加入洗脱缓冲液(0.1M sodium citrate,pH 3.2)室温振荡5-10min，置回磁力架上，收集洗脱缓冲液，转移至已加入中和缓冲液(1M Tris，pH 8.54)的收集管中混匀。
获得抗TIGIT-抗PD-L1双特异性抗体(本发明中也称为抗TIGIT/PD-L1双抗)，其示意结构如图1所示。
实施例2：抗体亲和力检测
采用生物膜层光学干涉技术(ForteBio)测定实施例1获得的双特异性抗体及其对应单端抗体分子结合人和食蟹猴PD-L1、TIGIT的结合解离常数(K D)。Fortebio亲和力测定按照现有方法(Este,P等人High throughput solution-based measurement of antibody-antigen affinity and epitope binning.Mabs,2013.5(2):p.270-8)进行，其中人PD-L1的胞外段氨基酸序列、食蟹猴PD-L1的胞外段氨基酸序列、人TIGIT的胞外段氨基酸序列、食蟹猴TIGIT的胞外段氨基酸序列分别如SEQ ID NOs:6-9所示。
具体操作如下：传感器在分析缓冲液中线下平衡30分钟，然后线上检测60s建立基线，加载经纯化获得的完整抗体至AHQ传感器至厚度1nm进行亲和力检测。将已加载抗体的传感器于100nM人或食蟹猴PD-L1、TIGIT-his抗原中作用至平台期，然后将传感器转移到分析缓冲液中至少2分钟用于解离速率测量。使用1:1结合模型进行动力学分析。
双特异性抗体及其对应单端抗体分子结合人、食蟹猴PD-L1及TIGIT的K D值如下面的表1所示。
表1：抗TIGIT-抗PD-L1双抗及对应单端抗体的K D值
Figure PCTCN2022132422-appb-000006
Figure PCTCN2022132422-appb-000007
注："N.B.":no binding。
结果显示，抗TIGIT/PD-L1双抗与人PD-L1、食蟹猴PD-L1、人TIGIT、食蟹猴TIGIT抗原结合的单价亲和力，分别与单端抗体分子与人PD-L1、食蟹猴PD-L1、人TIGIT、食蟹猴TIGIT抗原结合的单价亲和力相似甚至更优。
实施例3：抗TIGIT-抗PD-L1双特异性抗体与过表达人/食蟹猴PD-L1 CHO细胞 的结合活性及阻断活性
3.1基于流式细胞术检测法检测抗TIGIT-抗PD-L1双特异性抗体与过表达在CHO细胞上人/食蟹猴PD-L1的结合活性。
具体地，通过转染克隆到MCS的人PD-L1和食蟹猴PD-L1的pCHO1.0载体(购自Invitrogen)加压筛选产生过表达人PD-L1的CHO-S细胞(CHO-huPD-L1细胞)和过表达食蟹猴PD-L1的CHO-S细胞(CHO-cynoPD-L1细胞)。将扩大培养的过表达细胞调整至合适细胞密度加入96孔流式板，离心后加入梯度稀释的待测样品，4℃孵育30分钟。PBS清洗两次，加入对应稀释至合适浓度的荧光二抗，4℃孵育30分钟。PBS清洗两次，加入PBS重悬细胞，在CytoFlex流式细胞仪上进行检测并计算对应的MFI。采用Graphpad软件作图分析获得EC 50数值。
结果如表2、图2A和图2B所示。
表2：抗TIGIT-抗PD-L1双抗与过表达PD-L1/PD-1细胞结合阻断活性
Figure PCTCN2022132422-appb-000008
结果显示，本发明的抗TIGIT-抗PD-L1双特异性抗体与过表达在CHO细胞上人/食蟹猴PD-L1的结合活性与其PD-L1端单抗分子(anti-PD-L1 VHH)相似。
3.2基于流式细胞术检测法检测抗TIGIT-抗PD-L1双特异性抗体对于人PD-L1与过表达在CHO细胞上人PD-1的结合的阻断活性。
具体地，通过转染克隆到MCS的人PD-1的pCHO1.0载体(购自Invitrogen)加压筛选产生过表达人PD-1的CHO-S细胞(CHO-huPD-1细胞)。将纯化的待测抗体用PBS稀释，将稀释好的样品60μL/孔加入96孔流式板中。然后60μL/孔加入生物素化标记的人PD-L1蛋白，终浓度为0.5μg/mL，混匀后4℃孵育30分钟。将扩大培养的CHO-huPD-1细胞调整细胞密度至2×10 6细胞/mL，100μL/孔加入96孔流式板，离心后弃上清。100μL/孔加入上述共孵育完成的抗体抗原混合液，4℃孵育30分钟。PBS清洗两次，100μL/孔加入用PBS稀释100倍的链霉素亲和素-R-藻红蛋白偶联物(SAPE)，4℃孵育30分钟。PBS清洗两次，100μL/孔加入PBS重悬细胞，在CytoFlex流式细胞仪上进行检测并计算对应的MFI。采用Graphpad软件作图分析获得IC 50数值。
结果如表2和图2C所示。结果显示，本发明的抗TIGIT-抗PD-L1双特异性抗体对于人PD-L1与过表达在CHO细胞上人PD-1的结合的阻断活性与其PD-L1端单抗分子(anti-PD-L1 VHH)相似甚至更优。
实施例4：抗TIGIT-抗PD-L1双特异性抗体与过表达人/食蟹猴/小鼠TIGIT CHO 细胞的结合活性及阻断活性
4.1基于流式细胞术检测法检测抗TIGIT-抗PD-L1双特异性抗体与过表达在CHO细胞上人/食蟹猴/小鼠TIGIT的结合活性。
具体地，通过转染克隆到MCS的人TIGIT、食蟹猴TIGIT、小鼠TIGIT cDNA的pCHO1.0载体(购自Invitrogen)加压筛选产生过表达人TIGIT的CHO-S细胞(CHO-huTIGIT细胞)，过表达食蟹猴TIGIT的CHO-S细胞(CHO-cynoTIGIT细胞)以及过表达小鼠TIGIT的CHO-S细胞(CHO-muTIGIT细胞)。将扩大培养的过表达细胞调整至合适细胞密度加入96孔流式板，离心后加入梯度稀释的待测样品，4℃孵育30分钟。PBS清洗两次，加入对应稀释至合适浓度的荧光二抗，4℃孵育30分钟。PBS清洗两次，加入PBS重悬细胞，在CytoFlex流式细胞仪上进行检测并计算对应的MFI。采用Graphpad软件作图分析获得EC 50数值。
结果如表3、图3A至图3C所示。
表3：抗TIGIT-抗PD-L1双抗与过表达TIGIT细胞结合阻断活性
Figure PCTCN2022132422-appb-000009
Figure PCTCN2022132422-appb-000010
结果显示，本发明的抗TIGIT-抗PD-L1双特异性抗体与过表达在CHO细胞上人/食蟹猴/小鼠TIGIT的结合活性与其TIGIT端单抗分子(anti-TIGIT mAb)相似或者更优。
4.2基于流式细胞术检测法检测抗TIGIT-抗PD-L1双特异性抗体对于人CD155与过表达在CHO细胞上人TIGIT的结合，小鼠CD155与过表达在CHO细胞上小鼠TIGIT的结合的阻断活性。
具体地，将扩大培养的CHO-huTIGIT细胞调整细胞密度至2×10 6细胞/mL，100μL/孔加入96孔流式板，离心备用。将纯化的单克隆抗体用PBS稀释，400nM开始3倍稀释共12个点。将稀释好的样品60μL/孔加入上述带有细胞的96孔流式板中，4℃共孵育30分钟。然后60μL/孔加入带有Mouse IgG2a Fc Tag的人CD155蛋白，终浓度为2μg/mL，4℃共孵育30分钟。PBS清洗两次，100μL/孔加入用PBS稀释100倍的APC羊抗鼠IgG抗体，4℃孵育30分钟。PBS清洗两次，100μL/孔加入PBS重悬细胞，在CytoFlex流式细胞仪上进行检测并计算对应的MFI。
将扩大培养的CHO-muTIGIT细胞调整细胞密度至2×10 6细胞/mL，100μL/孔加入96孔流式板，离心备用。将纯化的单克隆抗体用PBS稀释，400nM开始3倍稀释共12个点。将稀释好的样品60μL/孔加入上述带有细胞的96孔流式板中，4℃共孵育30分钟。然后60μL/孔加入带有Mouse IgG2a Fc Tag的小鼠CD155蛋白，终浓度为2μg/mL，4℃共孵育30分钟。PBS清洗两次，100μL/孔加入用PBS稀释100倍的APC羊抗鼠IgG抗体，4℃孵育30分钟。PBS清洗两次，100μL/孔加入PBS重悬细胞，在CytoFlex流式细胞仪上进行检测并计算对应的MFI。采用Graphpad软件作图分析获得IC 50数值。
结果如表3、图3D至图3E所示。结果显示，本发明的抗TIGIT-抗PD-L1双特异性抗体对于人CD155与过表达在CHO细胞上人TIGIT的结合，小鼠CD155与过表达在CHO细胞上小鼠TIGIT的结合的阻断活性与其TIGIT端单抗分子(anti-TIGIT mAb)相似。
实施例5：抗TIGIT-抗PD-L1双特异性抗体与人TIGIT和人PD-L1共结合活性
基于酶联免疫吸附剂检测法(ELISA)检测本发明抗TIGIT-抗PD-L1双特异性抗体与人TIGIT和人PD-L1蛋白共结合活性。
具体地，人TIGIT蛋白按照说明书溶解，用1×ELISA包被液稀释至1μg/mL，100μL/孔包被96孔ELISA板中，4℃覆膜过夜。弃去包被液，1×PBST清洗3次，200μL/孔加入5％BSA/PBS室温封闭2小时。弃去封闭液，100μL/孔加入1％BSA/PBS梯度稀释的待测抗体，室温孵育2小时。弃去抗体稀释液，1×PBST清洗3次，100μL/孔加入1％BSA/PBS稀释的生物素标记的PD-L1蛋白，终浓度为1μg/mL，室温孵育1小时。弃去抗原稀释液，1×PBST清洗3次，100μL/孔加入1％BSA/PBS稀释的SA-HRP,室温孵育1小时。弃去SA-HRP稀释液，1×PBST清洗3次，100μL/孔加入ELISA显色液室温反应1-3分钟，50μL/孔加入ELISA终止液，读取450nm处吸光度数值。采用Graphpad软件作图获得浓度-吸光度值的结合曲线图。
结果如图4所示，本发明的抗TIGIT-抗PD-L1双特异性抗体可同时结合人TIGIT和人PD-L1蛋白。
实施例6：抗TIGIT-抗PD-L1双特异性抗体在荧光素酶报告基因系统对PD-1/PD- L1与TIGIT/CD155/CD112信号通路的阻断活性
为进一步在细胞水平检测抗TIGIT-抗PD-L1双特异性抗体同时阻断PD-1/PD-L1与TIGIT/CD155/CD112信号通路的活性，本实施例构建了如下荧光素酶报告基因系统。简言之，利用慢病毒转染细胞，构建了过表达人CD155、人CD112、人PD-L1和OKT-3scFv的CHO-K1细胞株(CHO-K1-CD155-CD112-PD-L1)，构建了过表达人TIGIT、人PD-1和NF-AT luciferase报告基因(购自promega)的Jurkat细胞株(Jurkat-TIGIT-PD-1-luc)，后续利用这一报告基因系统开展相关试验。
具体地，消化获取CHO-K1-CD155-CD112-PD-L1功能细胞，调整细胞密度，100μL/孔加入96孔白底板中，贴壁培养过夜。第二天，制备Jurkat-TIGIT-PD-1-luc效应细胞悬液，将待测样品用反应培养基梯度稀释。取出白底板，吸去培养上清，将上述稀释好的样品40μL/孔加入白底板，同时40μL/孔加入Jurkat-TIGIT-PD-1-luc效应细胞悬液，置于37℃，5％CO 2培养箱培养6h，期间将Bio-Glo TM reagent恢复至室温。培养完成，取出细胞，室温平衡5分钟，80μL/孔加入Bio-Glo TM reagent，使用多功能酶标仪读取荧光信号值。
结果如图5所示。结果显示，本发明的抗TIGIT-抗PD-L1双特异性抗体可以分别解除CD155/CD112介导的TIGIT下游抑制信号通路，以及PD-L1介导的PD-1下游抑制信号通路，上调报告基因荧光素酶表达，且活性优于两单端抗体分子的联合。
实施例7：抗TIGIT-抗PD-L1双特异性抗体在混合淋巴细胞试验中对T细胞激活的 活性
本实施例通过混合淋巴细胞反应实验(MLR)检测抗TIGIT-抗PD-L1双特异性抗体激活T细胞的活性。具体实验方法如下：
复苏PBMC细胞(购自SAILYBIO,XFB-HP100B)，离心，用10mL X-VIVO-15培养基(购自LONZA)重悬PBMC，于细胞培养箱内37℃贴壁培养2h，吸去未贴壁细胞。加入10mL DC培养基：X-VIVO-15培养基加入10ng/mLGM-CSF(购自R&D)，20ng/mL IL-4(购自R&D)，培养3天，补加5mL DC培养基，继续培养至第6天，加入DC成熟培养基：X-VIVO-15培养基加入1000U/mL TNF-α(购自R&D)，10ng/mL IL-6(购自R&D)，5ng/mL IL-1β(购自R&D)，1μM PGE2(购自Tocris)，培养2天，收集成熟的DC细胞，用X-VIVO-15培养基调整细胞密度为2×10 5细胞/mL。
复苏另一位捐献者的PBMC细胞(购自SAILY BIO,XFB-HP100B)，离心，用10mL X-VIVO-15培养基重悬PBMC。用T细胞分选试剂盒(购自Stemcell)富集T细胞，X-VIVO-15重悬T细胞，调整细胞密度为2×10 6细胞/mL.将T细胞悬液与上述收集的成熟DC细胞悬液按1:1体积比混合，100μL/孔加入96孔U底板。
用X-VIVO-15培养基稀释待测抗体样品，200nM开始10倍稀释共5个点，100μL/孔加入上述混合细胞孔，培养5天，收集上清，ELISA(购自eBioscience)方法检测IFN-γ表达量。
结果如图6A和图6B所示。结果显示，抗TIGIT-抗PD-L1双特异性抗体在MLR实验中显示较好的生物学活性，且T细胞激活水平与两单端抗体分子联合的活性相似。
实施例8：抗TIGIT-抗PD-L1双特异性抗体在B-NDG小鼠混合接种A375和人 PBMC体内药效学研究
本实验在B-NDG小鼠混合接种A375(购自Addexbio,C0020004，一种人恶性黑色素瘤细胞)，人PBMC细胞(上海妙顺,A10S033014/PB100C)的模型(A375huPBMC模型)中测定本发明的抗TIGIT-抗PD-L1双特异性抗体的抗肿瘤作用。其中，通过将 人的免疫细胞(PBMC)接种至免疫缺陷小鼠中，产生局部重组人类免疫系统的人源化肿瘤小鼠模型。
具体地，首先A375细胞与人PBMC 1：1等体积混合成0.1mL细胞悬液，在小鼠右腹沟以皮下注射的方式建立A375huPBMC模型，待平均肿瘤体积长至200mm 3左右时进行分组，腹腔注射给予不同剂量、相同给药体积的PBS或抗体治疗，每组6只小鼠。监测各组小鼠瘤体积和体重变化，监测频率均为2-3天/次，连续监测2到3周，给药剂量和方式如表4。
表4：抗TIGIT-抗PD-L1双特异性抗体A375 huPBMC模型给药方案
Figure PCTCN2022132422-appb-000011
备注：Anti-PD-L1 VHH、Anti-TIGIT mAb、抗TIGIT/PD-L1双抗分子量分别约为75KD、150KD、175KD，给药剂量按各组分等摩尔浓度剂量给药。
结果如图7所示。结果表明，本发明的抗TIGIT-抗PD-L1双特异性抗体具有显著的抗肿瘤作用，且效果优于两单端抗体分子联用。
实施例9：抗TIGIT-抗PD-L1双特异性抗体在B-NDG小鼠混合接种A375和人 PBMC体内剂量依赖药效学研究
本实验采用皮下混合接种的方式建立A375 huPBMC模型(建模步骤与实施例8相同)，待平均肿瘤体积长至300mm 3左右时进行分组，腹腔注射给予不同剂量、相同给药体积的PBS或双特异性抗体治疗，每组6只小鼠。监测各组小鼠瘤体积和体重变化，监测频率均为2-3天/次，连续监测2到3周，给药剂量和方式如表5。
表5：抗TIGIT-抗PD-L1双抗剂量依赖性肿瘤抑制活性试验方案
组别 给药剂量 给药频率/次数
PBS N/A Q3-4d×3
抗TIGIT/PD-L1双抗 1.2mg/kg Q3-4d×3
抗TIGIT/PD-L1双抗 6mg/kg Q3-4d×3
抗TIGIT/PD-L1双抗 30mg/kg Q3-4d×3
结果如图8所示，本发明的抗TIGIT-抗PD-L1双特异性抗体能够显著抑制小鼠肿瘤的生长，且呈一定的剂量依赖关系。
实施例10：抗TIGIT-抗PD-L1双特异性抗体在huPD-L1/PD-1/TIGIT KI小鼠体 内药效学研究
本实验采用在人PD-L1/PD-1/TIGIT转基因小鼠(huPD-L1/PD-1/TIGIT KI小鼠)体内移植CT-26-huPD-L1肿瘤细胞(人PD-L1敲入CT26小鼠结肠癌细胞，购自江苏集萃药康)测定本发明的抗TIGIT-抗PD-L1双特异性抗体的抗肿瘤作用。
具体地，首先制备CT-26-huPD-L1细胞悬液，在小鼠右腹沟皮下注射0.1mL约5×10 5细胞建立CT-26-huPD-L1荷瘤小鼠模型，待平均肿瘤体积长至80-120mm 3时进行分组，腹腔注射给予不同剂量、相同给药体积的PBS或抗体治疗，每组6只小鼠。监测各组小鼠瘤体积和体重变化，监测频率均为2-3天/次，连续监测2到3周，给药剂量和方式如表6。
表6：抗TIGIT-抗PD-L1双抗huPD-L1/PD-1/TIGIT KI小鼠模型给药方案
组别 给药剂量 给药频率/次数
PBS N/A Q2d×5
Atezolizumab 10mg/kg Q2d×5
Tiragolumab 10mg/kg Q2d×5
Atezolizumab+Tiragolumab 10mg/kg+10mg/kg Q2d×5
抗TIGIT/PD-L1双抗 12mg/kg Q2d×5
结果如图9所示，本发明的抗TIGIT-抗PD-L1双特异性抗体与临床上的用药组合(Atezolizumab+Tiragolumab)具有相似的药效，且抗肿瘤活性优于两个单抗用药组。
实施例11：抗TIGIT-抗PD-L1双特异性抗体小鼠体内半衰期研究
采用尾静脉单次注射法，检测本发明的抗TIGIT-抗PD-L1双特异性抗体在小鼠体内半衰期。
具体地，实验用Balb/c小鼠，雌雄各3只，12/12小时光/暗调节，温度24℃±2℃，湿度40％-70％，自由进水饮食。实验当天对Balb/c小鼠单次尾静脉注射单克隆抗体分子， 注射剂量为10mg/kg。取血时间点：给药后5分钟、0.5小时、2小时、6小时、24小时、48小时、96小时、168小时、336小时、504小时于小鼠眼眶采血。全血样品2℃-8℃放置30分钟，12000rpm离心5分钟收集血清，所得血清再于2℃-8℃，12000rpm离心5分钟，-80℃保存，ELISA检测血清中双特异性抗体分子含量。
结果如图10所示。结果表明，本发明的抗TIGIT-抗PD-L1双特异性抗体单次注射在小鼠体内半衰期为189小时。
制备例1：抗TIGIT单抗的制备
将抗TIGIT单抗的重链可变区(氨基酸序列如SEQ ID NO:19所示)分别重组到人IgG1重链恒定区，以及人IgG1重链L234A、L235A改造的恒定区中。此外，将轻链可变区(氨基酸序列如SEQ ID NO:20所示)重组到人kappa轻链恒定区，将抗TIGIT单抗分别命名为55796-G1、55796-G1LALA。经由HEK293表达系统瞬时表达纯化。具体操作如下：使用化学转染的方法将带有抗体重链和轻链的pcDNA3.1载体转入HEK293细胞中，在37℃，8％CO 2条件下，培养7天。收集细胞液，13000rpm离心20分钟。取上清液，Protein A纯化上清液，SEC检测抗体纯度，同时控制内毒素含量。
制得的抗TIGIT单抗55796-G1、55796-G1LALA，用于下面的试验例1-3。
试验例1：抗TIGIT单抗的亲和力检测
采用生物膜层光学干涉技术(ForteBio)测定制备例1制得的抗TIGIT单抗结合人、食蟹猴、小鼠TIGIT的结合解离常数(K D)。Fortebio亲和力测定按照现有方法(Este,P等人High throughput solution-based measurement of antibody-antigen affinity and epitope binning.Mabs,2013.5(2):p.270-8)进行。其中人TIGIT的胞外段氨基酸序列、食蟹猴TIGIT的胞外段氨基酸序列、小鼠TIGIT的胞外段氨基酸序列分别如SEQ ID NOs:8-9和21所示。
测量完整抗体(即，通过Adimab原始获得的全长IgG)与人、食蟹猴、小鼠TIGIT-his蛋白的单价亲和力：传感器在分析缓冲液中线下平衡20分钟，然后线上检测120s建立基线，加载完整的TIGIT抗体至AHQ传感器至厚度1nm进行亲和力检测。将已加载抗体的传感器于100nM TIGIT-his抗原中作用至平台期，然后将传感器转移到分析缓冲液中至少2分钟用于解离速率测量。使用1:1结合模型进行动力学分析。
在如上测定方法中，测得的K D值如下表7所示：
表7：抗TIGIT单抗的K D值
Figure PCTCN2022132422-appb-000012
注："NA":not available。
由表7结果可见：(1)抗TIGIT单抗与人TIGIT-his蛋白的单价亲和力高于对照分子Tiragolumab；(2)抗TIGIT单抗与食蟹猴TIGIT-his蛋白的单价亲和力与对照分子Tiragolumab相当；(3)抗TIGIT单抗和小鼠TIGIT有交叉结合活性。
试验例2：抗TIGIT单抗与过表达人/食蟹猴/小鼠TIGIT CHO细胞的结合活性及 阻断活性
2.1基于流式细胞术检测法检测抗TIGIT单抗与过表达在CHO细胞上人/食蟹猴/小鼠TIGIT的结合活性。
具体地，通过转染克隆到MCS的人TIGIT、食蟹猴TIGIT、小鼠TIGIT cDNA的pCHO1.0载体(购自Invitrogen)加压筛选产生过表达人TIGIT的CHO-S细胞(CHO-huTIGIT细胞)，过表达食蟹猴TIGIT的CHO-S细胞(CHO-cynoTIGIT细胞)以及过表达小鼠TIGIT的CHO-S细胞(CHO-muTIGIT细胞)。将扩大培养的过表达细胞调整至合适细胞密度加入96孔流式板，离心后加入梯度稀释的待测样品，4℃孵育30分钟。PBS清洗两次，加入对应稀释至合适浓度的荧光二抗，4℃孵育30分钟，PBS清洗两次。加入PBS重悬细胞，在CytoFlex流式细胞仪上进行检测并计算对应的MFI。采用Graphpad软件作图分析获得EC50数值。结果如表8和图11至图13所示。
2.2基于流式细胞术检测法检测抗TIGIT单抗，阻断人CD155与过表达在CHO细胞上人TIGIT的结合，阻断小鼠CD155与过表达在CHO细胞上小鼠TIGIT的结合的阻断活性。
具体地，将扩大培养的CHO-huTIGIT细胞调整细胞密度至2×10 6细胞/mL，100μL/孔加入96孔流式板，离心备用。将纯化的单克隆抗体用PBS稀释，400nM开始3倍稀释共12个点。将稀释好的样品60μL/孔加入上述带有细胞的96孔流式板中，4℃共孵育30分钟。然后60μL/孔加入带有Mouse IgG2a Fc Tag的人CD155蛋白，终浓度为2μg/mL， 4℃共孵育30分钟，PBS清洗两次。100μL/孔加入用PBS稀释100倍的APC羊抗鼠IgG抗体，4℃孵育30分钟，PBS清洗两次。100μL/孔加入PBS重悬细胞，在CytoFlex流式细胞仪上进行检测并计算对应的MFI。
将扩大培养的CHO-muTIGIT细胞调整细胞密度至2×10 6细胞/mL，100μL/孔加入96孔流式板，离心备用。将纯化的单克隆抗体用PBS稀释，400nM开始3倍稀释共12个点。将稀释好的样品60μL/孔加入上述带有细胞的96孔流式板中，4℃共孵育30分钟。然后60μL/孔加入带有Mouse IgG2a Fc Tag的小鼠CD155蛋白，终浓度为2μg/mL，4℃共孵育30分钟,PBS清洗两次。100μL/孔加入用PBS稀释100倍的APC羊抗鼠IgG抗体，4℃孵育30分钟，PBS清洗两次。100μL/孔加入PBS重悬细胞，在CytoFlex流式细胞仪上进行检测并计算对应的MFI。采用Graphpad软件作图分析获得IC 50数值。结果如表8和图14至图5所示。
表8：抗TIGIT单抗过表达细胞结合阻断活性汇总表
Figure PCTCN2022132422-appb-000013
注："N.B.":no binding；and"NA":not available.
由表8和图11至图13可见：本发明的抗TIGIT单抗，(1)与CHO细胞表面过表达的人TIGIT蛋白结合活性优于对照分子Tiragolumab；(2)与CHO细胞表面过表达的食蟹猴TIGIT蛋白结合活性优于对照分子Tiragolumab；(3)与CHO细胞表面过表达的小鼠TIGIT蛋白有明显结合。
由表8和图14至图15可见：本发明的抗TIGIT单抗，(1)阻断人CD155与CHO细胞表面过表达的人TIGIT蛋白结合的能力优于对照分子Tiragolumab；(2)与CHO细胞表面过表达的小鼠TIGIT蛋白有结合的抗TIGIT抗体分子可以明显阻断小鼠CD155与CHO细胞表面过表达的小鼠TIGIT蛋白的结合。
试验例3：抗TIGIT单抗与原代T细胞表面TIGIT结合
基于流式细胞术检测法检测发明抗TIGIT抗体与激活T细胞表面的TIGIT结合活性。
具体地，将人PBMC按照STEMCELL公司提供的试验方案(stemcell,货号：#17951C)进行分选获得human total T细胞。用X-VIVO15培养基(购自lonza，货号：04-418Q)调整T细胞浓度至1.0×10 6cells/mL，加入1μLIL-2储备液(100万IU)，同时1:1(bead-to-cell)加入CD3/CD28 Dynabeads(购自gibco，货号：11132D)，于37℃，5％CO 2培养箱内培养48小时。将活化后的T细胞调整至合适细胞密度加入96孔流式板，离心后加入梯度稀释的待测样品，4℃孵育30分钟。PBS清洗两次，加入对应稀释至合适浓度的荧光二抗，4℃孵育30分钟，PBS清洗两次。加入PBS重悬细胞，在CytoFlex流式细胞仪上进行检测并计算对应的MFI。
结果如图16所示。结果表明，本发明抗TIGIT抗体55796-G1、55796-G1LALA能够结合T细胞表面的TIGIT分子，且结合活性优于对照分子Tiragolumab。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (22)

  1. 一种双特异性抗体，其包括：
    靶向TIGIT的第一蛋白功能区，和
    靶向不同于TIGIT的靶点(例如PD-L1)的第二蛋白功能区；
    其中：
    所述第一蛋白功能区为抗TIGIT免疫球蛋白或其抗原结合片段；
    所述抗TIGIT免疫球蛋白的重链可变区包含氨基酸序列如SEQ ID NO:10所示的HCDR1、氨基酸序列如SEQ ID NO:11所示的HCDR2以及氨基酸序列如SEQ ID NO:12所示的HCDR3；并且
    所述抗TIGIT免疫球蛋白的轻链可变区包含氨基酸序列如SEQ ID NO:13所示的LCDR1、氨基酸序列如SEQ ID NO:14所示的LCDR2以及氨基酸序列如SEQ ID NO:15所示的LCDR3。
  2. 根据权利要求1所述的双特异性抗体，其中，所述第一蛋白功能区和第二蛋白功能区直接连接或者通过连接片段连接；
    优选地，所述连接片段为(GGGGS)m，m为正整数，例如1、2、3、4、5或6；
    优选地，所述连接片段为(GGGGS)nG，n为正整数，例如1、2、3、4、5或6；
    优选地，所述连接片段的氨基酸序列如SEQ ID NO:2所示。
  3. 根据权利要求1至2中任一权利要求所述的双特异性抗体，其中，所述第一蛋白功能区和第二蛋白功能区独立地为1个、2个或者2个以上。
  4. 根据权利要求1至3中任一权利要求所述的双特异性抗体，其中，
    所述第一蛋白功能区为抗TIGIT免疫球蛋白或其抗原结合片段，并且所述第二蛋白功能区为靶点不同于TIGIT的单域抗体或单链抗体；
    优选地，所述单域抗体为抗PD-L1单域抗体；
    优选地，所述单链抗体为抗PD-L1单链抗体。
  5. 根据权利要求1至4中任一权利要求所述的双特异性抗体，其中，
    所述第一蛋白功能区为抗TIGIT单链抗体，并且所述第二蛋白功能区为靶点不同于TIGIT的免疫球蛋白或其抗原结合片段；
    优选地，靶点不同于TIGIT的免疫球蛋白为抗PD-L1免疫球蛋白。
  6. 根据权利要求5所述的双特异性抗体，其中，
    所述抗PD-L1单域抗体包含一个重链可变区，所述重链可变区包含氨基酸序列如SEQ ID NO:16所示的HCDR1、氨基酸序列如SEQ ID NO:17所示的HCDR2以及氨基酸序列如SEQ ID NO:18所示的HCDR3；
    优选地，所述抗PD-L1单域抗体的氨基酸序列如SEQ ID NO:3所示。
  7. 根据权利要求1至6中任一权利要求所述的双特异性抗体，其中，
    所述抗TIGIT免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:19所示，并且其轻链可变区的氨基酸序列如SEQ ID NO:20所示；
    优选地，
    所述抗TIGIT免疫球蛋白的重链的氨基酸序列如SEQ ID NO:1所示，其轻链的氨基酸序列如SEQ ID NO:5所示。
  8. 根据权利要求1至7中任一权利要求所述的双特异性抗体，其中，
    所述抗TIGIT免疫球蛋白或者靶点不同于TIGIT的免疫球蛋白的恒定区来自人抗体；
    优选地，所述恒定区选自人IgG1、IgG2、IgG3或IgG4的恒定区。
  9. 根据权利要求1至8中任一权利要求所述的双特异性抗体，其中，
    所述抗TIGIT免疫球蛋白或者靶点不同于TIGIT的免疫球蛋白的重链恒定区为人Ig gamma-1 chain C region(例如NCBI ACCESSION:P01857)或人Ig gamma-4 chain C region(例如NCBI ACCESSION:P01861.1)，并且其轻链恒定区为人Ig kappa chain C region(例如NCBI ACCESSION:P01834)；
    优选地，所述抗TIGIT免疫球蛋白的重链恒定区还包含按照EU编号系统的L234A突变和L235A突变。
  10. 根据权利要求4和6至9中任一权利要求所述的双特异性抗体，其中，所述单域抗体或单链抗体连接在抗TIGIT免疫球蛋白的C末端或N末端，例如，所述单域抗体或单链抗体为两个，每个单域抗体或单链抗体的一端分别连接在抗TIGIT免疫球蛋白的两条重链的C末端或N末端。
  11. 根据权利要求4和6至10中任一权利要求所述的双特异性抗体，其中，所述单域抗体为抗PD-L1单域抗体，单域抗体连接在抗TIGIT免疫球蛋白得到的肽链的氨基酸序列如SEQ ID NO:4所示。
  12. 根据权利要求4和6至11中任一权利要求所述的双特异性抗体，其为由两条相同的第一肽链和两条相同的第二肽链形成的四聚体，其中，
    所述第一肽链的氨基酸序列如SEQ ID NO:4所示；并且所述第二肽链的氨基酸序列如SEQ ID NO:5所示。
  13. 分离的核酸分子，其编码权利要求1至12中任一权利要求所述的双特异性抗体。
  14. 一种载体，其包含权利要求13所述的分离的核酸分子。
  15. 一种宿主细胞，其包含权利要求13所述的分离的核酸分子，或者权利要求14所述的载体。
  16. 制备权利要求1至12中任一权利要求所述的双特异性抗体的方法，其包括在合适的条件下培养权利要求15的宿主细胞，以及从细胞培养物中回收所述双特异性抗体的步骤。
  17. 偶联物，其包括双特异性抗体以及偶联部分，其中，所述双特异性抗体为权利要求1至12中任一权利要求所述的双特异性抗体，所述偶联部分为可检测的标记；优选地，所述偶联部分为放射性同位素、荧光物质、发光物质、有色物质或酶。
  18. 试剂盒，其包含权利要求1至12中任一权利要求所述的双特异性抗体，或者包 含权利要求17所述的偶联物；
    优选地，所述试剂盒还包含第二抗体，其能够特异性结合所述双特异性抗体；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、发光物质、有色物质或酶。
  19. 一种药物组合物，其包含权利要求1至12中任一权利要求所述的双特异性抗体或者包含权利要求17所述的偶联物；可选地，其还包括药学上可接受的辅料。
  20. 权利要求1至12中任一权利要求所述的双特异性抗体或者权利要求17所述的偶联物在制备治疗或预防恶性肿瘤的药物中的用途；优选地，所述恶性肿瘤选自黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、结肠癌、乳腺癌、食道癌和头颈癌。
  21. 根据权利要求1至12中任一权利要求所述的双特异性抗体或者权利要求17所述的偶联物，其用于治疗或预防恶性肿瘤的药物中的用途；优选地，所述恶性肿瘤选自黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、结肠癌、乳腺癌、食道癌和头颈癌。
  22. 一种治疗或预防恶性肿瘤的方法，包括给予有需求的受试者以有效量的根据权利要求1至12中任一权利要求所述的双特异性抗体或者权利要求17所述的偶联物的步骤；优选地，所述恶性肿瘤选自黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、结肠癌、乳腺癌、食道癌和头颈癌。
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JP2024529415A JP2024542192A (ja) 2021-11-17 2022-11-17 Ｔｉｇｉｔおよびｐｄ－ｌ１に対する二重特異性抗体、ならびにその医薬組成物およびその使用
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