WO2023186081A1 - 抗tigit-抗pvrig双特异性抗体、其药物组合物及用途 - Google Patents

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

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WO2023186081A1
WO2023186081A1 PCT/CN2023/085428 CN2023085428W WO2023186081A1 WO 2023186081 A1 WO2023186081 A1 WO 2023186081A1 CN 2023085428 W CN2023085428 W CN 2023085428W WO 2023186081 A1 WO2023186081 A1 WO 2023186081A1
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Prior art keywords
seq
amino acid
acid sequence
tigit
pvrig
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PCT/CN2023/085428
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English (en)
French (fr)
Chinese (zh)
Inventor
翟天航
戴爽
黄威峰
缪小牛
曾竣玮
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Biotheus Inc
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Biotheus Inc
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Priority to EP23778445.9A priority Critical patent/EP4506365A4/en
Priority to US18/852,935 priority patent/US20250223354A1/en
Priority to CA3255478A priority patent/CA3255478A1/en
Priority to CN202380031275.9A priority patent/CN118946592A/zh
Priority to KR1020247036669A priority patent/KR20240164815A/ko
Priority to JP2024558264A priority patent/JP2025511289A/ja
Application filed by Biotheus Inc filed Critical Biotheus Inc
Priority to AU2023247712A priority patent/AU2023247712A1/en
Priority to IL316008A priority patent/IL316008A/en
Publication of WO2023186081A1 publication Critical patent/WO2023186081A1/zh
Priority to MX2024012144A priority patent/MX2024012144A/es
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Definitions

  • the invention belongs to the field of biomedicine and relates to an anti-TIGIT-anti-PVRIG bispecific antibody, its pharmaceutical composition and uses.
  • TIGIT also known as WUCAM, Vstm3 or VSIG9
  • WUCAM WUCAM
  • Vstm3 or VSIG9 T cell immunoreceptor with Ig and ITIM domains
  • Poliovirus receptor related immunoglobulin domain-containing protein belongs to the PVR receptor family. Its extracellular region has an IgV domain and its intracellular region contains An ITIM (Immunoreceptor tyrosine-based inhibitory motif) domain, mainly expressed on natural killer (NK) cells and T cells.
  • ITIM Immunoreceptor tyrosine-based inhibitory motif
  • PVRIG or its ligand PVRL2 is highly expressed in lung cancer, renal cancer, endometrial cancer, breast cancer, skin cancer and their tumor microenvironment, and PVRIG is also highly expressed on NK cells of prostate cancer patients (Whelan S, et al. Cancer Immunol Res. 2019;7(2):257-268.).
  • PVRIG co-expressed with exhausted CD8+ T cell markers TIGIT and PD-1 molecules, suggesting that the expression of PVRIG has a certain tumor specificity and may be related to the activation/exhaustion status of TIL in the tumor (Whelan S, et al. Cancer Immunol Res. 2019;7(2):257-268.).
  • WO2021180205A1 reports that anti-PVRIG antibodies can be used for the prevention and treatment of lung cancer, breast cancer, ovarian cancer, kidney cancer, gastric cancer, endometrial cancer, head and neck cancer, etc.
  • TIGIT/PVRIG jointly participate in a complex regulatory network: CD226 (DNAM-1) is an activating receptor on T/NK cells, which mediates the activation signal of T/NK cells by binding to CD155 and CD112; while TIGIT and PVRIG is an inhibitory receptor on T/NK cells. Its expression increases after the cells are activated. It independently mediates inhibitory signals by binding to two ligands, CD155 and CD112. At the same time, they also compete with CD226 for ligands. And block the stimulatory signal transmitted by it, thereby achieving negative regulation of immune cell function.
  • CD226 DNAM-1
  • TIGIT and PVRIG is an inhibitory receptor on T/NK cells. Its expression increases after the cells are activated. It independently mediates inhibitory signals by binding to two ligands, CD155 and CD112. At the same time, they also compete with CD226 for ligands. And block the stimulatory signal transmitted by it, thereby achieving negative regulation of immune cell function.
  • TIGIT antibodies and PVRIG antibodies can increase T cell expansion or IFN- ⁇ secretion respectively, and the combination of the two can produce additive or synergistic effects to further improve T cell function (Whelan S et al., PVRIG and PVRL2 Are Induced in Cancer and Inhibit CD8+T-cell Function. Cancer Immunol Res. 2019Feb;7(2):257-268.).
  • PVRIG antibodies can reduce tumor growth in TIGIT-/- mice, and tumor growth control in double knockout mice of PVRIG and TIGIT is further improved compared with single knockout mice.
  • TIGIT/CD155 and PVRIG/CD112 are two independent and non-redundant T cell negative regulatory pathways. Targeted intervention of TIGIT and PVRIG at the same time is expected to better release CD226 in tumor treatment. T cell activation signal generated.
  • SHR-2002 developed by Hengrui Medicine is the first and only drug of its kind to enter clinical development among similar TIGIT/PVRIG dual-target antibodies. It was approved for clinical use in early December 2021 and is currently in the Phase I clinical stage for cancer. treat.
  • Bispecific antibodies can specifically bind two antigens or antigenic epitopes at the same time, and have the characteristics of specificity and bifunctionality. They have become a research hotspot in the field of antibody engineering.
  • problems encountered in the development of bispecific antibodies include complex preclinical evaluation models, low expression levels, poor stability, complex processes, and large differences in quality control, which have always affected the development progress of bispecific antibodies.
  • the inventors obtained new bispecific antibodies through in-depth research and creative work.
  • the inventors surprisingly found that the bispecific antibody of the present invention has good affinity and biological activity, and has anti-tumor potential.
  • the following invention is thereby provided:
  • One aspect of the invention relates to a bispecific antibody comprising:
  • a second protein functional region that targets a target different from PVRIG (such as TIGIT);
  • the first protein functional region is anti-PVRIG immunoglobulin or an antigen-binding fragment thereof;
  • the heavy chain variable region of the anti-PVRIG immunoglobulin includes HCDR1 with an amino acid sequence as shown in SEQ ID NO:25, HCDR2 with an amino acid sequence as shown in SEQ ID NO:26, and HCDR2 with an amino acid sequence as shown in SEQ ID NO:27 HCDR3; and
  • the light chain variable region of the anti-PVRIG immunoglobulin includes LCDR1 with an amino acid sequence as shown in SEQ ID NO:22, LCDR2 with an amino acid sequence as shown in SEQ ID NO:23 and SEQ ID NO:24 as an amino acid sequence. LCDR3.
  • the bispecific antibody is an anti-TIGIT-anti-PVRIG bispecific antibody, also represented as an anti-PVRIG-anti-TIGIT bispecific antibody, referred to as the bispecific antibody of the present invention.
  • the bispecific antibody wherein the second protein functional region is an anti-TIGIT immunoglobulin or an antigen-binding fragment thereof, wherein:
  • the heavy chain variable region of the anti-TIGIT immunoglobulin includes HCDR1 with an amino acid sequence as shown in SEQ ID NO:13, HCDR2 with an amino acid sequence as shown in SEQ ID NO:14, and HCDR2 with an amino acid sequence as shown in SEQ ID NO:15 HCDR3; and the light chain variable region of the anti-TIGIT immunoglobulin includes LCDR1 with an amino acid sequence as shown in SEQ ID NO:16, LCDR2 with an amino acid sequence as shown in SEQ ID NO:17 and an amino acid sequence as SEQ ID NO :LCDR3 shown in 18;
  • the heavy chain variable region of the anti-TIGIT immunoglobulin includes HCDR1 with an amino acid sequence as shown in SEQ ID NO:19, HCDR2 with an amino acid sequence as shown in SEQ ID NO:20, and HCDR2 with an amino acid sequence as shown in SEQ ID NO:21 HCDR3; and the light chain variable region of the anti-TIGIT immunoglobulin includes LCDR1 with an amino acid sequence as shown in SEQ ID NO:22, LCDR2 with an amino acid sequence as shown in SEQ ID NO:23 and an amino acid sequence as SEQ ID NO :LCDR3 shown in 24.
  • variable regions of the light chain and heavy chain determine the binding of antigens; the variable region of each chain contains three hypervariable regions, called complementarity-determining regions (CDRs) (the CDRs of the heavy chain (H) include HCDR1, HCDR2, and HCDR3 , the CDR of the light chain (L) includes LCDR1, LCDR2, and LCDR3.
  • CDRs complementarity-determining regions
  • H the CDRs of the heavy chain
  • L includes LCDR1, LCDR2, and LCDR3.
  • HCDR1-HCDR3 and LCDR1-LCDR3 refer to U.S. Patent Publication US20210380669A1 or the document Lu. et al (Deamidation and isomerization liability analysis of 131 clinical-stage antibodies, MABS,2019,VOL.11,NO.1,45–57,DOI:10.1080/19420862.2018.1548233) are defined or numbered.
  • the bispecific antibody wherein the antigen-binding fragment is independently a single-chain antibody or a half-molecule monovalent antibody (IgG half molecule, IgG-HM).
  • the bispecific antibody wherein,
  • the first protein functional region is anti-PVRIG immunoglobulin, and the second protein functional region is anti-TIGIT single chain antibody; wherein:
  • the heavy chain variable region of the anti-PVRIG immunoglobulin includes HCDR1 with an amino acid sequence as shown in SEQ ID NO:25, HCDR2 with an amino acid sequence as shown in SEQ ID NO:26, and HCDR2 with an amino acid sequence as shown in SEQ ID NO:27 HCDR3; and the light chain variable region of the anti-PVRIG immunoglobulin includes LCDR1 with an amino acid sequence as shown in SEQ ID NO:22, LCDR2 with an amino acid sequence as shown in SEQ ID NO:23 and an amino acid sequence as SEQ ID NO : LCDR3 shown in 24; and
  • the heavy chain variable region of the anti-TIGIT single chain antibody includes HCDR1 with an amino acid sequence such as SEQ ID NO:13, HCDR2 with an amino acid sequence such as SEQ ID NO:14 and an amino acid sequence such as SEQ ID NO:15 HCDR3 as shown; and the light chain variable region of the anti-TIGIT immunoglobulin includes LCDR1 with an amino acid sequence as shown in SEQ ID NO:16, LCDR2 with an amino acid sequence as shown in SEQ ID NO:17 and an amino acid sequence as SEQ LCDR3 shown in ID NO:18; or
  • the heavy chain variable region of the anti-TIGIT single chain antibody includes HCDR1 with an amino acid sequence as shown in SEQ ID NO:19, HCDR2 with an amino acid sequence as shown in SEQ ID NO:20, and HCDR2 with an amino acid sequence as SEQ ID NO:21 HCDR3 as shown; and the light chain variable region of the anti-TIGIT immunoglobulin includes LCDR1 with an amino acid sequence as shown in SEQ ID NO:22, LCDR2 with an amino acid sequence as shown in SEQ ID NO:23 and an amino acid sequence as SEQ LCDR3 shown in ID NO:24.
  • the bispecific antibody wherein,
  • the first protein functional region is an anti-PVRIG single chain antibody
  • the second protein functional region is an anti-TIGIT immunoglobulin; wherein:
  • the heavy chain variable region of the anti-PVRIG single chain antibody includes HCDR1 with an amino acid sequence as shown in SEQ ID NO:25, HCDR2 with an amino acid sequence as shown in SEQ ID NO:26, and HCDR2 with an amino acid sequence as shown in SEQ ID NO:27. HCDR3; and the light chain variable region of the anti-PVRIG immunoglobulin includes LCDR1 with an amino acid sequence as shown in SEQ ID NO:22, LCDR2 with an amino acid sequence as shown in SEQ ID NO:23 and an amino acid sequence as SEQ ID NO : LCDR3 shown in 24; and
  • the heavy chain variable region of the anti-TIGIT immunoglobulin includes HCDR1 with an amino acid sequence such as SEQ ID NO:13, HCDR2 with an amino acid sequence such as SEQ ID NO:14 and an amino acid sequence such as SEQ ID NO:15 HCDR3 as shown; and the light chain variable region of the anti-TIGIT immunoglobulin includes LCDR1 with an amino acid sequence as shown in SEQ ID NO:16, LCDR2 with an amino acid sequence as shown in SEQ ID NO:17 and an amino acid sequence as SEQ LCDR3 shown in ID NO:18; or
  • the heavy chain variable region of the anti-TIGIT immunoglobulin includes HCDR1 with an amino acid sequence such as SEQ ID NO:19, HCDR2 with an amino acid sequence such as SEQ ID NO:20 and an amino acid sequence such as SEQ ID NO:21 HCDR3 as shown; and the light chain variable region of the anti-TIGIT immunoglobulin includes LCDR1 with an amino acid sequence as shown in SEQ ID NO:22, LCDR2 with an amino acid sequence as shown in SEQ ID NO:23 and an amino acid sequence as SEQ LCDR3 shown in ID NO:24.
  • the bispecific antibody wherein,
  • amino acid sequence of the heavy chain variable region of the anti-PVRIG immunoglobulin or anti-PVRIG single chain antibody is shown in SEQ ID NO: 5;
  • amino acid sequence of the light chain variable region of the anti-PVRIG immunoglobulin or anti-PVRIG single-chain antibody is shown in SEQ ID NO: 4;
  • the glycine at position 44 of the heavy chain variable region of the anti-PVRIG immunoglobulin or anti-PVRIG single chain antibody is replaced with cysteine, and the anti-PVRIG immunoglobulin or anti-PVRIG single chain antibody The glycine at position 100 of the light chain variable region was replaced with cysteine.
  • the bispecific antibody wherein,
  • amino acid sequence of the heavy chain variable region of the anti-TIGIT immunoglobulin or anti-TIGIT single-chain antibody is shown in SEQ ID NO: 1; and the light chain variable region of the anti-TIGIT immunoglobulin or anti-TIGIT single-chain antibody
  • amino acid sequence of the region is shown in SEQ ID NO:2;
  • amino acid sequence of the heavy chain variable region of the anti-TIGIT immunoglobulin or anti-TIGIT single-chain antibody is shown in SEQ ID NO: 3; and the light chain variable region of the anti-TIGIT immunoglobulin or anti-TIGIT single-chain antibody
  • amino acid sequence of the region is shown in SEQ ID NO:4;
  • the glycine at position 44 of the heavy chain variable region of the anti-TIGIT immunoglobulin or anti-TIGIT single-chain antibody is replaced with cysteine, and the anti-TIGIT immunoglobulin or anti-TIGIT single-chain antibody The glycine at position 100 of the light chain variable region was replaced with cysteine.
  • the bispecific antibody wherein the first protein functional region and The second protein functional region is directly connected or connected through a connecting fragment
  • connection fragment is (GGGGS)m
  • m 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: 6.
  • the first protein functional region and the second protein functional region are independently one, two, or more than two.
  • the bispecific antibody wherein:
  • the anti-TIGIT single chain antibodies are respectively connected to the C termini of the two heavy chains of the anti-PVRIG immunoglobulin; or
  • the anti-PVRIG single chain antibodies (two molecules) are respectively connected to the C-termini of the two heavy chains of anti-TIGIT immunoglobulin.
  • the bispecific antibody wherein,
  • the constant region of the anti-PVRIG immunoglobulin or anti-TIGIT immunoglobulin is derived from a human antibody
  • the constant region is independently selected from the constant region of human IgGl, IgG2, IgG3 or IgG4.
  • the bispecific antibody wherein,
  • the heavy chain constant region of the anti-PVRIG immunoglobulin or anti-TIGIT immunoglobulin is human Ig gamma-1 chain C region or human Ig gamma-4 chain C region, and its light chain constant region is human Ig kappa chain C region;
  • the heavy chain constant region of the anti-PVRIG immunoglobulin and anti-TIGIT immunoglobulin also contains the L234A mutation and the L235A mutation (abbreviated as LALA) according to the EU numbering system.
  • LALA L235A mutation
  • the letters before the site represent the amino acid before mutation
  • the letters after the site represent the amino acid after mutation
  • the bispecific antibody is a dimer or tetramer of two peptide chains with amino acid sequences such as SEQ ID NO:7 and SEQ ID NO:8, or It is a dimer or tetramer of two peptide chains with amino acid sequences such as SEQ ID NO:9 and SEQ ID NO:10.
  • 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 conjugate, which includes a bispecific antibody and a conjugation part, wherein the bispecific antibody is the bispecific antibody according to 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 colored substance or an enzyme.
  • kits comprising the bispecific antibody according to any one of the present invention, or the conjugate of the present invention
  • the kit also includes a second antibody that can specifically bind to the bispecific antibody; optionally, the second antibody also includes a detectable label, such as a radioactive isotope, a fluorescent substance, a colored substance or enzymes.
  • a detectable label such as a radioactive isotope, a fluorescent substance, a colored substance or enzymes.
  • Another aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the bispecific antibody according to any one of the present invention and one or more pharmaceutically acceptable excipients;
  • the pharmaceutical composition further comprises at least one anti-PD-1 antibody;
  • the molar ratio of the bispecific antibody to the anti-PD-1 antibody is (1:5) to (5:1), such as 1:5, 1:4, 1:3, 1:2, 1: 1, 2:1, 3:1, 4:1 or 5:1; more preferably 1:1.
  • the pharmaceutical composition wherein the anti-PD-1 antibody includes a heavy chain variable region and a light chain variable region, and the heavy chain variable region includes HCDR1 to HCDR3, the light chain variable region comprising LCDR1 to LCDR3, wherein:
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO:36
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO:37
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO:38
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:39
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO:40
  • the amino acid sequence of LCDR3 is shown in SEQ ID NO:41;
  • amino acid sequence of the heavy chain variable region of the anti-PD-1 antibody is shown in SEQ ID NO: 34
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 35.
  • the pharmaceutical composition wherein the unit dosage of the pharmaceutical composition, calculated according to the mass of the bispecific antibody, is 100 mg-1000 mg, 200 mg-800 mg, 200mg-500mg, 300mg-600mg, 400mg-500mg or 450mg.
  • Yet another aspect of the invention relates to a combination product comprising a first product and a second product individually packaged, wherein,
  • the first product comprises a bispecific antibody according to any one of the invention.
  • the second product includes at least one anti-PD-1 antibody
  • the first product and the second product also independently contain one or more pharmaceutically acceptable excipients;
  • the combination product also contains product instructions.
  • the combination product wherein the molar ratio of the bispecific antibody to the anti-PD-1 antibody is (1:5) to (5:1), such as 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1 or 5:1; more preferably 1:1.
  • the combination product wherein the anti-PD-1 antibody includes a heavy chain variable region and a light chain variable region, the heavy chain variable region includes HCDR1 to HCDR3, the light chain variable region includes LCDR1 to LCDR3, wherein:
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO:36
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO:37
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO:38
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:39
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO:40
  • the amino acid sequence of LCDR3 is shown in SEQ ID NO:41;
  • amino acid sequence of the heavy chain variable region of the anti-PD-1 antibody is shown in SEQ ID NO: 34
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 35.
  • Another aspect of the present invention relates to the use of the bispecific antibody of any one of the present invention or the conjugate of the present invention in the preparation of drugs for treating or preventing tumors;
  • the tumor is selected from colon cancer, melanoma, lung cancer, kidney cancer, endometrial cancer, breast cancer, skin cancer, ovarian cancer, gastric cancer, head and neck cancer, liver cancer, brain tumor, urothelial cancer, bone tumor , one or variety;
  • the lung cancer is non-small cell lung cancer or small cell lung cancer.
  • bispecific antibody according to any one of the present invention, the conjugate of the present invention or the pharmaceutical composition according to any one of the present invention, which is used to treat or prevent tumors;
  • the tumor is selected from colon cancer, melanoma, lung cancer, kidney cancer, endometrial cancer, breast cancer, skin cancer, ovarian cancer, gastric cancer, head and neck cancer, liver cancer, brain tumor, urothelial cancer, bone tumor , one or variety;
  • the lung cancer is non-small cell lung cancer or small cell lung cancer.
  • Another aspect of the present invention relates to a method for treating or preventing tumors, comprising administering to a subject in need an effective amount of the bispecific antibody of any one of the present invention, the conjugate of the present invention, or the present invention.
  • the tumor is selected from colon cancer, melanoma, lung cancer, kidney cancer, endometrial cancer, breast cancer, skin cancer, ovarian cancer, gastric cancer, head and neck cancer, liver cancer, brain tumor, urothelial cancer, bone tumor , one or variety;
  • the lung cancer is non-small cell lung cancer or small cell lung cancer.
  • the single administration dose of the bispecific antibody is 0.1-100 mg per kilogram of body weight, preferably 1-10 mg (such as 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg or 10 mg); alternatively, the bispecific antibody A single dose of the antibody is 10-1000 mg (e.g., about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 600 mg, about 700 mg) per subject , about 800mg, about 900mg or about 1000mg), preferably 50-500mg, 100-400mg, 150-300mg, 150-250mg or 200mg;
  • it is administered 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 is administered in a 2-week (14-day) or 3-week (21-day) cycle, preferably with the anti-PVRIG antibody administered intravenously on the first day (D1) of each cycle.
  • the bispecific antibody is administered once every two weeks (q2w) or once every three weeks (q3w).
  • PVRIG when referring to the amino acid sequence of PVRIG (NCBI GenBank ID: NP_076975.2), it includes the full length or extracellular fragment of the PVRIG protein; also includes fusion proteins of PVRIG, such as with mouse or human A fragment fused to the Fc protein fragment of IgG (mFc or hFc).
  • fusion proteins of PVRIG such as with mouse or human A fragment fused to the Fc protein fragment of IgG (mFc or hFc).
  • mutations or variations can occur naturally or be artificially introduced without affecting its biological function. Therefore, in this invention, the term "PVRIG protein” or "PVRIG” shall include all such sequences, including those shown and natural or artificial variants thereof.
  • a sequence fragment of a PVRIG protein when describing a sequence fragment of a PVRIG protein, it includes not only the sequence fragment, but also the corresponding sequence fragment in its natural or artificial variant.
  • TIGIT when referring to the amino acid sequence of TIGIT (NCBI GenBank ID: NP_776160.2), it includes the full length or functional fragments of the TIGIT protein; also includes fusion proteins of TIGIT, such as with mouse or human IgG Fc protein fragment (mFc or hFc) fusion fragment.
  • mFc or hFc mouse or human IgG Fc protein fragment
  • a sequence fragment of a TIGIT protein when describing a sequence fragment of a TIGIT protein, it includes not only the sequence fragment, but also the corresponding sequence fragment in its natural or artificial variants.
  • EC50 refers to the concentration for 50% of maximal effect, the concentration that causes 50% of the maximal effect.
  • antibody refers to an immunoglobulin molecule typically 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 into 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 connected by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region ( VH ) and a heavy chain constant region ( CH ).
  • the heavy chain constant region consists of 3 domains ( CH1 , CH2 and CH3 ).
  • Each light chain consists of a light chain variable region (V L ) and a light chain constant region ( CL ).
  • the light chain constant region consists of one domain, CL .
  • the constant region of an antibody may mediate 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 conservative regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions ( VH and VL ) of each heavy chain/light chain pair respectively form the antigen-binding site.
  • the assignment of amino acids to each region or domain follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda Md (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 1987; 196: 901-917; Chothia et al.
  • IMGT/3Dstructure-DB and IMGT/DomainGapAlign a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF.
  • the term "antibody” is not limited to any particular method of producing the antibody. For example, they include, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies.
  • the antibodies can be antibodies of different isotypes, for example, IgG (for example, IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • IgG for example, IgG1, IgG2, IgG3 or IgG4 subtypes
  • IgA1, IgA2, IgD, IgE or IgM antibodies antibodies of different isotypes, for example, IgG (for example, IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen that the full-length antibody binds, and/or competes with the full-length antibody Specific binding to an antigen, which is also called an "antigen-binding moiety.”
  • an antigen-binding moiety which is also called an "antigen-binding moiety.
  • the antigen-binding fragments include Fab, Fab', F(ab') 2 , Fd, Fv, dAb, and complementarity determining regions (CDRs) Fragments, single chain antibodies (eg, scFv), chimeric antibodies, diabodies, and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.
  • CDRs complementarity determining regions
  • the term “Fd fragment” means an antibody fragment consisting of the VH and CH1 domains;
  • the term “Fv fragment” means a fragment consisting of the VL and VH domains of a single arm of an antibody Antibody fragments;
  • the term “dAb fragment” means an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544-546 (1989));
  • the term “Fab fragment” means an antibody fragment consisting of VL , VH , C Antibody fragment consisting of L and CH 1 domains;
  • the term “F(ab') 2 fragment” means an antibody fragment containing two Fab fragments connected by a disulfide bridge on the hinge region.
  • the antigen-binding fragment of the antibody is a single chain antibody (e.g., scFv) in which the V L and V H domains are paired to form a monovalent molecule by a linker that enables production of a single polypeptide chain (see, e.g., Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)).
  • scFv molecules may have the general structure: NH2 - VL -linker- VH -COOH or NH2 - VH -linker- VL -COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
  • a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • Other linkers useful in the present invention are provided by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, described by Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.
  • the antigen-binding fragment of an antibody is a diabody, i.e., a diabody in which the V H and V L domains are expressed on a single polypeptide chain, but a linker that is too short is used to allow the use of two linkers on the same chain. of structural domains pairing, thereby forcing the domain to pair with the complementary domain of the other chain and creating two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993 ), and Poljak RJ et al., Structure 2:1121-1123 (1994)).
  • the antigen-binding fragment of an antibody is a "bispecific antibody", which refers to a conjugate formed by a first antibody (fragment) and a second antibody (fragment) or antibody analog through a coupling arm.
  • Methods of linkage include, but are not limited to, chemical reactions, gene fusion, and enzymatic processes.
  • Antigen-binding fragments of antibodies can be "multispecific antibodies” including, for example: trispecific antibodies and tetraspecific antibodies, the former being antibodies with three different antigen-binding specificities, and the latter being antibodies with four different antigen-binding specificities. of antibodies.
  • ankyrin repeat proteins linked to IgG antibodies, scFv-Fc antibody fragments or combinations thereof, such as CN104341529A.
  • Anti-IL-17a fynomer is combined with anti-IL-6R antibody, such as WO2015141862A1.
  • a given antibody e.g., the monoclonal antibodies ADI-56127, ADI-55796, and ADI-55812 provided by the present invention
  • the terms “monoclonal antibody” and “monoclonal antibody” refer to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, except for natural mutations that may occur spontaneously. A group of identical antibody molecules. Monoclonal antibodies are highly specific for a single epitope on the antigen. Polyclonal antibodies are relative to monoclonal antibodies, which usually contain at least two or more different antibodies, and these different antibodies usually recognize different epitopes on the antigen. 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 can also be obtained using recombinant DNA technology (eg, see US Patent 4,816,567).
  • humanized antibody refers to a product in which all or part of the CDR regions of a human immunoglobulin (recipient antibody) are replaced by the CDR regions of a non-human antibody (donor antibody).
  • some amino acid residues in the framework region (FR) of the recipient antibody can also be replaced by amino acid residues of the corresponding non-human antibody, or by amino acid residues of other antibodies, to further improve or optimize the performance of the antibody.
  • the term “isolated” or “isolated” means obtained from the natural state by artificial means. If an "isolated" substance or ingredient occurs in nature, it may be that the natural environment in which it is located has changed, or that the substance has been separated from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the high purity of the same polynucleotide or polypeptide isolated from this natural state is called isolation. of.
  • isolation a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the high purity of the same polynucleotide or polypeptide isolated from this natural state is called isolation. of.
  • the term “isolated” or “isolated” does not exclude the admixture of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses, etc.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, Polyoma vacuolating viruses (such as 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 baculoviruses
  • papillomaviruses papillomaviruses
  • Polyoma vacuolating viruses such as SV40.
  • a vector can contain a variety of expression-controlling elements, including, but not limited to,
  • the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, GS 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, COS cells, NSO cells, HeLa cells, GS cells, BHK cells, HEK 293 cells or human cells.
  • an antibody that specifically binds to an antigen refers to an antibody that binds to an antigen at a concentration of 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 of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen.
  • antibodies dissociate with 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.
  • Binding antigen e.g. TIGIT protein
  • KD can be determined using methods known to those skilled in the art, such as using the Fortebio Molecular Interaction Instrument.
  • amino acids are generally represented by one-letter and three-letter abbreviations well 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, as is well known in the art (See, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers.
  • pH adjusters include but are not limited to phosphate buffer; surfactants include but are not limited to cationic, anionic or nonionic surfactants such as Tween-80; ionic strength enhancers include but are not limited to sodium chloride.
  • an effective amount refers to an amount sufficient to obtain, at least in part, the desired effect.
  • an effective amount to prevent a disease is an amount that is sufficient to prevent, prevent, or delay the occurrence of a disease (such as a tumor);
  • an effective amount to treat a disease is an amount that is sufficient to cure or at least partially prevent the occurrence of a disease in a patient who already has the disease. The amount of disease and its complications.
  • hybridoma and “hybridoma cell line” are used interchangeably, and when the terms “hybridoma” and “hybridoma cell line” are referred to, it also includes subclones of the hybridoma and progeny cells.
  • first and the "second” are for the purpose of distinction in reference or clarity of expression, and are not typical. meaning in order.
  • the present invention achieves one or more of the technical effects described in the following items (1) to (4):
  • the bispecific antibody of the present invention can bind to TIGIT and PVRIG with high affinity.
  • the antibody of the present invention can effectively treat and/or prevent tumors, such as colon cancer, melanoma, lung cancer, kidney cancer, endometrial cancer, breast cancer, skin cancer, ovarian cancer, gastric cancer, head and neck cancer, liver cancer, Brain tumors, urothelial cancer, bone tumors, cholangiocarcinoma, rectal cancer, pancreatic cancer, cervical tumors, multiple myeloma, Hodgkin lymphoma, Non-Hodgkin lymphoma, B lymphoma, plasma cell carcinoma, prostate cancer and testicular cancer, etc.
  • tumors such as colon cancer, melanoma, lung cancer, kidney cancer, endometrial cancer, breast cancer, skin cancer, ovarian cancer, gastric cancer, head and neck cancer, liver cancer, Brain tumors, urothelial cancer, bone tumors, cholangiocarcinoma, rectal cancer, pancreatic cancer, cervical tumors, multiple myeloma, Hodgkin lymphoma, Non-Ho
  • the bispecific antibody of the present invention has low toxic and side effects.
  • FIG. 1A to Figure 1C Schematic structural diagram of the bispecific antibody of the present invention.
  • Figure 2 Binding curve of the bispecific antibody of the present invention to human TIGIT overexpressed on CHO cells.
  • Figure 3 Binding curve of the bispecific antibody of the present invention to cynomolgus monkey TIGIT overexpressed on CHO cells.
  • Figure 4 Binding curve of the bispecific antibody of the present invention to mouse TIGIT overexpressed on CHO cells.
  • Figure 5 Graph of bispecific antibodies of the present invention blocking the binding of human CD155 to human TIGIT overexpressed on CHO cells.
  • Figure 6 A graph showing that bispecific antibodies of the present invention block the binding of mouse CD155 to mouse TIGIT overexpressed on CHO cells.
  • Figure 7 Binding curve of bispecific antibodies of the present invention to human PVRIG overexpressed on CHO cells.
  • Figure 8 Binding curve of the bispecific antibody of the present invention to cynomolgus monkey PVRIG overexpressed on CHO cells.
  • Figure 9 Binding curve of the bispecific antibody of the present invention to mouse PVRIG overexpressed on CHO cells.
  • Figure 10 Graph of bispecific antibodies of the present invention blocking the binding of human PVRIG to human CD112 overexpressed on CHO cells.
  • Figure 11 A graph showing that the bispecific antibody of the present invention blocks the binding of mouse CD112 to mouse PVRIG protein.
  • Figure 12 A graph showing that the bispecific antibody of the present invention simultaneously binds to human PVRIG and human TIGIT proteins.
  • Figure 13 A graph showing that the bispecific antibody of the present invention blocks the TIGIT/CD155 and PVRIG/CD112 signaling pathways.
  • Figure 14A to Figure 14B Curves showing that the bispecific antibody of the present invention combined with anti-PD-1/L1 monoclonal antibody blocks the TIGIT/CD155, PVRIG/CD112 and PD-1/PD-L1 signaling pathways.
  • Figure 15 The efficacy curve of the bispecific antibody of the present invention in the B-NDG mouse model mixed with A375 and human PBMC.
  • FIG 16A to Figure 16B The bispecific antibody of the present invention combined with anti-PD-1 monoclonal antibody is used in mixed vaccination with A375 and human Pharmacodynamic curve of B-NDG mouse model of PBMC.
  • Figure 17 Half-life curve of the bispecific antibody of the present invention in mice.
  • Figure 18 Binding activity curve of the anti-TIGIT antibody of the present invention to human TIGIT overexpressed on CHO cells.
  • Figure 19 Binding activity curve of the anti-TIGIT antibody of the present invention to cynomolgus monkey TIGIT overexpressed on CHO cells.
  • Figure 20 Binding activity curve of the anti-TIGIT antibody of the present invention to mouse TIGIT overexpressed on CHO cells.
  • Figure 21 Blocking activity curve of anti-TIGIT antibodies of the present invention blocking the binding of human CD155 to human TIGIT overexpressed on CHO cells.
  • Figure 22 Blocking activity curve of anti-TIGIT antibodies of the present invention blocking the binding of mouse CD155 to mouse TIGIT overexpressed on CHO cells.
  • Figure 23 Binding activity curve of anti-TIGIT antibodies of the present invention and TIGIT on activated human primary T cells.
  • Figure 24 Binding activity curve of anti-PVRIG antibody of the present invention to human PVRIG overexpressed on CHO cells.
  • Figure 25 Binding activity curve of the anti-PVRIG antibody of the present invention to cynomolgus monkey PVRIG overexpressed on CHO cells.
  • Figure 26 Binding activity curve of the anti-PVRIG antibody of the present invention to mouse PVRIG overexpressed on CHO cells.
  • Figure 27 Blocking activity curve of anti-PVRIG antibodies of the present invention blocking the binding of human PVRIG to human CD112 overexpressed on CHO cells.
  • Figure 28 Graph of anti-PVRIG antibody blocking the binding of mouse CD112 to mouse PVRIG protein.
  • Figure 29 Binding profile of anti-PVRIG antibodies to PVRIG on activated human primary T cells.
  • Figure 30 Pharmacodynamic diagram of anti-PVRIG antibody in NDG mouse model mixed with A375 and human PBMC.
  • Antibody Atezolizumab anti-PD-L1 monoclonal antibody, trade name Tecentriq, Roche.
  • Antibody Pembrolizumab anti-PD-1 monoclonal antibody, trade name Keytruda, Merck.
  • control antibodies were expressed and purified from HEK293 cells:
  • COM701 is an anti-human PVRIG antibody from Compugen expressed in HEK293 cells. Its light chain variable region sequence and heavy chain variable region sequence are consistent with SEQ ID NO: 1372 and SEQ ID NO: 1380 in U.S. patent US10227408B2;
  • Mab46 is an anti-human PVRIG antibody from Surface Oncology expressed by HEK293 cells. Its light chain variable region and heavy chain variable region sequences are consistent with SEQ ID NO:912 and SEQ ID NO:918 in the US patent publication US20200040081A1.
  • Tiragolumab is an anti-human TIGIT antibody from Genentech expressed by HEK293 cells. Its light chain and heavy chain sequences are derived from WHO Drug Information, Vol. 31, No. 2, 2017, Proposed INN: List 117, CAS number: 1918185- 84-8.
  • Example 1 Design and preparation of anti-TIGIT-anti-PVRIG bispecific antibodies
  • the scFv of the anti-PVRIG monoclonal antibody ADI-56127 was connected in series through the GGGGSGGGGSG (SEQ ID NO: 44) (other linkers such as GGGGSGGGGS, SEQ ID NO: 6) linkers can also be used using full gene synthesis.
  • the scFv of the anti-TIGIT antibody and the scFv of the anti-PVRIG antibody adopt the "VH-linker-VL" connection method respectively.
  • the flexible peptide linker between VH and VL is GGGGSGGGGSGGGGSGGGGSG (SEQ ID NO: 45) (other flexible peptide linkers can also be used)
  • the anti-TIGIT monoclonal antibody ADI-55796 and the anti-PVRIG monoclonal antibody ADI-56127 respectively combine the glycine at position 44 of the heavy chain variable region and the glycine at position 100 of the light chain variable region.
  • heterosexual antibodies Mated to cysteine to enhance scFv stability.
  • Three double specials The heterosexual antibodies all use human IgG1 subtype.
  • the sequences of anti-TIGIT monoclonal antibodies ADI-55796, ADI-55812 and anti-PVRIG monoclonal antibodies ADI-56127 and the formed bispecific antibodies are shown in Table 1 below.
  • the pcDNA3.1 vector carrying the peptide chain #1 and peptide chain #2 of the anti-TIGIT-anti-PVRIG bispecific antibody TP-007-008 was transferred into HEK293 cells to prepare TP-007-008 bispecific antibody using chemical transfection method.
  • Anti-TIGIT-anti-PVRIG bispecific antibody TP-003-009 bispecific antibody TP-003-009 peptide chain #1 and peptide chain #2 pcDNA3.1 vector was transferred into HEK293 cells to prepare TP-003-009 bispecific antibody.
  • the pcDNA3.1 vector containing peptide chain #1, peptide chain #2 and peptide chain #3 of anti-TIGIT-anti-PVRIG bispecific antibody TP-001-002-003 was transferred into HEK293 cells to prepare TP-001-002-003 Double resistance. After 5 days of cell culture, the supernatant was collected, and the target protein was purified using protein A magnetic beads (purchased from GenScript). Resuspend the magnetic beads in an appropriate volume of binding buffer (PBS+0.1% Tween 20, pH 7.4) (1-4 times the volume of the magnetic beads), add it to the sample to be purified, and incubate at room temperature for 1 hour, shaking gently during the period.
  • binding buffer PBS+0.1% Tween 20, pH 7.4
  • elution buffer 0.1M sodium citrate, pH 3.2
  • TP-007-008, TP-003-009, and TP-001-002-003 three strains of bisAb were obtained, replaced with PBS by ultrafiltration, and the molecular weight was confirmed by LC-MS for subsequent in vivo and in vitro activity detection.
  • Fortebio affinity assay was performed according to existing methods (Este, P et al. High throughput solution-based measurement of antibody-antigen affinity and epitope binning.
  • the sensor is equilibrated offline in the analysis buffer for 20 minutes, then detected online for 120 seconds to establish a baseline, and the complete antibody is loaded onto the AHQ sensor to the thickness 1nm for affinity detection.
  • the antibody-loaded sensor was incubated in 100 nM target antigen until plateau phase, and then the sensor was transferred to assay buffer for at least 2 minutes for off-rate measurement.
  • Kinetic analysis was performed using a 1:1 binding model.
  • the sensor was equilibrated offline in the analysis buffer for 20 minutes, then detected online for 120 seconds to establish a baseline, and the complete antibody was loaded onto the AHQ sensor to a thickness of 1nm for affinity detection.
  • Kinetic analysis was performed using a 1:1 binding model.
  • Example 3 Anti-TIGIT-anti-PVRIG bispecific antibody and overexpression of human/cynomolgus/mouse TIGIT CHO Cell binding activity and blocking activity
  • CHO-S cells overexpressing human TIGIT were generated through pressure screening by transfecting the pCHO1.0 vector (purchased from Invitrogen) of human TIGIT, cynomolgus TIGIT, and mouse TIGIT cDNA cloned into MCS. ), CHO-S cells overexpressing cynomolgus TIGIT (CHO-cynoTIGIT cells) and CHO-S cells overexpressing mouse TIGIT (CHO-muTIGIT cells). Adjust the expanded cultured overexpressing cells to a suitable cell density and add them to a 96-well flow cytometry plate.
  • the anti-TIGIT-anti-PVRIG bispecific antibody blocks the binding of human CD155 to human TIGIT overexpressed on CHO cells and the binding of mouse CD155 to mouse TIGIT overexpressed on CHO cells. Blocking activity.
  • 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 antibody to be tested with PBS, starting with 400 nM and diluting 3 times for a total of 12 points. Add 60 ⁇ L/well of the diluted sample to the above-mentioned 96-well flow plate with cells, and incubate at 4°C for 30 minutes. Then add 60 ⁇ L/well of human CD155 protein with Mouse IgG2a Fc Tag to a final concentration of 2 ⁇ g/mL, and incubate at 4°C for 30 minutes.
  • the results are shown in Table 3 and Figures 5 to 6.
  • the results show that the anti-TIGIT-anti-PVRIG bispecific antibody TP-007-008 of the present invention blocks the binding of human CD155 to human TIGIT overexpressed on CHO cells, as well as the binding between mouse CD155 and mouse TIGIT overexpressed on CHO cells.
  • the binding blocking activity is similar to that of its TIGIT-terminated monoclonal antibody molecule ADI-55796.
  • Example 4 Anti-TIGIT-anti-PVRIG bispecific antibody and overexpression of human/cynomolgus/mouse PVRIG Binding activity and blocking activity of CHO cells
  • CHO-S cells overexpressing human PVRIG were generated by pressure screening by transfecting the pCHO1.0 vector (purchased from Invitrogen) of human PVRIG, cynomolgus PVRIG, and mouse PVRIG cloned into MCS.
  • CHO-S cells overexpressing cynomolgus PVRIG (CHO-cynoPVRIG cells)
  • CHO-S cells overexpressing mouse PVRIG (CHO-muPVRIG cells). Adjust the expanded cultured overexpressing cells to a suitable cell density and add them to a 96-well flow cytometry plate.
  • the results show that the binding activity of the anti-TIGIT-anti-PVRIG bispecific antibody TP-003-009 of the present invention to human/cynomolgus/mouse PVRIG overexpressed on CHO cells is similar to its PVRIG-terminal monoclonal antibody molecule ADI-56127;
  • the binding activity of the anti-TIGIT-anti-PVRIG bispecific antibody TP-007-008 of the present invention to human PVRIG overexpressed on CHO cells is similar to its PVRIG-terminal monoclonal antibody molecule ADI-56127.
  • CHO-S cells overexpressing human CD112 were generated by pressure screening by transfecting the pCHO1.0 vector (purchased from Invitrogen) of human CD112 cloned into MCS. Dilute the purified antibody to be tested with PBS, and add 60 ⁇ L/well of the diluted sample into a 96-well flow plate. Then add 1 ⁇ g/mL human PVRIG protein with Mouse IgG2a Fc Tag at 60 ⁇ L/well, mix and incubate at 4°C for 30 minutes.
  • the results are shown in Table 4 and Figure 10.
  • the anti-TIGIT-anti-PVRIG bispecific antibodies TP-007-008 and TP-003-009 of the present invention block the binding of human PVRIG to human CD112 overexpressed on CHO cells.
  • the activity is similar to its PVRIG-terminal monoclonal antibody molecule ADI-56127.
  • the mouse CD112-his protein was dissolved according to the instructions, diluted to 1 ⁇ g/mL with 1 ⁇ ELISA coating solution, coated at 100 ⁇ L/well in a 96-well ELISA plate, and covered overnight at 4°C. Discard the coating solution, wash 3 times with 1 ⁇ PBST, 200 ⁇ L/well was added with 5% BSA/PBS for blocking at room temperature for 2 hours. During the blocking period, the purified antibody to be tested was serially diluted with 1% BSA/PBS to a final volume of 60 ⁇ L/well. Then add 60 ⁇ L/well of biotinylated mouse PVRIG protein with Mouse IgG2a Fc Tag, mix and incubate at room temperature for 1 hour.
  • Example 5 Co-binding activity of anti-TIGIT-anti-PVRIG bispecific antibodies with human TIGIT and human PVRIG
  • the co-binding activity of the anti-TIGIT-anti-PVRIG bispecific antibody of the present invention with human TIGIT and human PVRIG proteins is detected based on an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • the human PVRIG protein was dissolved according to the instructions, diluted to 1 ⁇ g/mL with 1 ⁇ ELISA coating solution, coated in a 96-well ELISA plate at 100 ⁇ L/well, and covered overnight at 4°C. Discard the coating solution, wash 3 times with 1 ⁇ PBST, and add 200 ⁇ L/well of 5% BSA/PBS for blocking 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 results are shown in Figure 12.
  • the anti-TIGIT-anti-PVRIG bispecific antibodies TP-007-008, TP-003-009, and TP-001-002-003 of the present invention can all bind to human TIGIT and human PVRIG proteins simultaneously.
  • Example 6 Anti-TIGIT-anti-PVRIG bispecific antibody in luciferase reporter gene system Blocking activity of TIGIT/CD155 and PVRIG/CD112 signaling pathways
  • this example constructed the following luciferase reporter gene system. Briefly, lentivirus was used to transfect cells to construct a CHO-K1 cell line (CHO-K1-CD155-CD112-aAPC) overexpressing human CD155, human CD112 and anti-CD3scFv, and a CHO-K1 cell line overexpressing human TIGIT, human Jurkat cell line (Jurkat-TIGIT-PVRIG-NF-AT-luc) of PVRIG and NF-AT luciferase reporter genes (purchased from promega), and subsequently used this reporter gene system to carry out relevant experiments.
  • CHO-K1 cell line CHO-K1-CD155-CD112-aAPC
  • human CD155 human CD112 and anti-CD3scFv
  • a CHO-K1 cell line overexpressing human TIGIT
  • human Jurkat cell line Jurkat-TIGIT-PVRIG-NF-AT-luc
  • CHO-K1-CD155-CD112-aAPC functional cells were obtained by digestion, the cell density was adjusted, 100 ⁇ L/well was added to a 96-well white bottom plate, and the cells were adherent and cultured overnight. The next day, prepare a Jurkat-TIGIT-PVRIG-NF-AT-luc effector cell suspension, and gradiently dilute the sample to be tested with reaction medium.
  • the results are shown in Figure 13.
  • the results show that the anti-TIGIT-anti-PVRIG bispecific antibodies TP-007-008, TP-003-009, and TP-001-002-003 of the present invention can respectively relieve the CD155-mediated TIGIT downstream inhibitory signaling pathway and CD112
  • the PVRIG-mediated downstream inhibition signaling pathway upregulates the expression of reporter gene luciferase, and the blocking activity of TP-007-008 is similar to the combined application of two single-end antibody molecules.
  • Example 7 Luciferase reporter gene system detects anti-TIGIT-anti-PVRIG bispecific antibodies and anti-PD- 1/L1 antibody combined blocking effect
  • this example constructed the following luciferase reporter gene system, which is briefly described as: On the basis of Example 6, CHO-K1-CD155-CD112-aAPC was infected with lentivirus to overexpress PD-L1 to obtain CHO-K1-CD155-CD112-PD-L1-aAPC functional cells, and Jurkat-TIGIT-PVRIG was infected with lentivirus. -NF-AT-luc overexpressed PD-1 to obtain Jurkat-TIGIT-PVRIG-PD-1-NF-AT-luc effector cells, and subsequently used this reporter gene system to carry out relevant experiments.
  • CHO-K1-CD155-CD112-PD-L1-aAPC functional cells were obtained by digestion, the cell density was adjusted, 100 ⁇ L/well was added to a 96-well white bottom plate, and the cells were adherent and cultured overnight. The next day, prepare the Jurkat-TIGIT-PVRIG-PD-1-NF-AT-luc effector cell suspension, and gradiently dilute the sample to be tested with the reaction medium. Take out the white bottom plate, aspirate the culture supernatant, add 40 ⁇ L/well of the above diluted sample to the white bottom plate, and add 40 ⁇ L/well at the same time.
  • Jurkat-TIGIT-PVRIG-PD-1-NF-AT-luc effector cell suspension was cultured in a 37°C, 5% CO2 incubator for 6 hours. After the culture is completed, add 80 ⁇ L/well of Bio-GloTM reagent, and use a multifunctional microplate reader to read the fluorescence signal value.
  • Example 8 Anti-TIGIT-anti-PVRIG bispecific antibody mixed with A375 and human in B-NDG mice In vivo pharmacodynamic study of PBMC
  • B-NDG mice were mixedly inoculated with A375 (purchased from Addexbio, C0020004, a human malignant melanoma cell) and human PBMC cells (Shanghai Miaoshun, A10S033014/PB100C) in a model (A375huPBMC model).
  • A375 purchased from Addexbio, C0020004, a human malignant melanoma cell
  • human PBMC cells Strestructure the human immune system
  • PBMC human immune cells
  • A375 cells and human PBMC were first mixed into 0.1 mL cell suspension in an equal volume of 1:1, and the A375huPBMC model was established by subcutaneous injection in the right abdominal groin of mice. Grouping was performed when the average tumor volume grew to about 75 mm. Different doses and the same administration volume of PBS or antibody treatment were administered intraperitoneally, with 6 mice in each group. Monitor the changes in tumor volume and body weight of mice in each group. The monitoring frequency is 2-3 days/time, and continuous monitoring is performed for 2 to 3 weeks. The dosage and method of administration are as shown in Table 5.
  • the molecular weights of TP-001-002-003, TP-003-009, TP-007-008, ADI-55796, and ADI-56127 are approximately 150KD, 200KD, 200KD, 150KD, and 150KD respectively.
  • the dosage is based on each component. Equimolar doses were administered.
  • Example 9 Anti-TIGIT-anti-PVRIG bispecific antibody combined with anti-PD-1 monoclonal antibody in B-NDG mice In vivo pharmacodynamic study of co-vaccination of A375 and human PBMC
  • This experiment uses subcutaneous mixed inoculation to establish the A375huPBMC model (the modeling steps are the same as in Example 8).
  • the average tumor volume grows to about 200 mm, they are divided into groups, and different doses and the same volume of PBS or antibody treatment are given by intraperitoneal injection. , 6 mice per group. Monitor the changes in tumor volume and body weight of mice in each group. The monitoring frequency is 2-3 days/time, and continuous monitoring is performed for 2 to 3 weeks.
  • the dosage and method of administration are as shown in Table 6.
  • A375huPBMC model was established by subcutaneous mixed inoculation ( The modeling steps are the same as those in Example 8). When the average tumor volume reaches about 300 mm, they are divided into groups. Different doses of TP-007-008 and a fixed dose of anti-PD-1 antibody are administered by intraperitoneal injection. Each group has 6 mice. mouse. Monitor the changes in tumor volume and body weight of mice in each group. The monitoring frequency is 2-3 days/time, and continuous monitoring is performed for 2 to 3 weeks. The dosage and method of administration are as shown in Table 7.
  • the sequence of the anti-PD-1 monoclonal antibody used in this example is as follows:
  • the CDRs of the above anti-PD-1 antibodies are as follows:
  • the anti-PD-1 monoclonal antibody heavy chain constant region used in this example uses the IgG1 heavy chain constant region modified by L234A and L235A:
  • the anti-PD-1 monoclonal antibody heavy chain constant region used in this example uses the human kappa light chain constant region:
  • Example 10 In vivo half-life study of anti-TIGIT-anti-PVRIG bispecific antibody in mice
  • the tail vein single injection method was used to detect the half-life of the anti-TIGIT-anti-PVRIG bispecific antibody TP-007-008 of the present invention in mice.
  • the experiment used Balb/c mice, 3 males and 3 males, with 12/12 hours of light/dark regulation, a temperature of 24°C ⁇ 2°C, a humidity of 40%-70%, and free access to water and food.
  • Balb/c mice were given a single tail vein injection of monoclonal antibody molecules at a dose of 10 mg/kg.
  • Blood collection time points Collect blood from the mouse orbits 5 minutes, 0.5 hours, 2 hours, 6 hours, 24 hours, 48 hours, 96 hours, 168 hours, 336 hours, and 504 hours after administration.
  • the whole blood sample was placed at 2°C-8°C for 30 minutes, centrifuged at 12000rpm for 5 minutes to collect the serum.
  • the obtained serum was centrifuged at 2°C-8°C, 12000rpm for 5 minutes, and stored at -80°C.
  • the content of bispecific antibody molecules in the serum was detected by ELISA ( The ELISA detection method is the same as Example 5).
  • Each heavy chain variable region of the anti-TIGIT monoclonal antibody (the amino acid sequence is shown in SEQ ID NO: 1-2 respectively) was recombined into the human IgG1 heavy chain constant region, and the human IgG1 heavy chain L234A, L235A modified constant region. . also, Each light chain variable region (the amino acid sequence is shown in SEQ ID NO: 3-4 respectively) was recombined into the human kappa light chain constant region, and the anti-TIGIT monoclonal antibodies were named 55796-G1, 55796-G1LALA, and 55812-G1. ,55812-G1LALA. Transient expression and purification via HEK293 expression system.
  • the specific operation is as follows: use chemical transfection method to transfer the pcDNA3.1 vector with antibody heavy chain and light chain into HEK293 cells, and culture at 37°C, 8% CO2 for 7 days. Collect the cell fluid and centrifuge at 13,000 rpm for 20 minutes. Take the supernatant, purify the supernatant with Protein A, and detect the antibody purity by SEC while controlling the endotoxin content.
  • Anti-TIGIT monoclonal antibodies 55796-G1, 55796-G1LALA, 55812-G1, and 55812-G1LALA were thus prepared.
  • Test Example 1 Affinity detection of anti-TIGIT monoclonal antibody
  • Biofilm layer optical interference technology (ForteBio) was used to determine the binding dissociation constant (K D ) of the anti-TIGIT monoclonal antibody prepared in Preparation Example 1 that binds to human, cynomolgus monkey, and mouse TIGIT.
  • Fortebio affinity measurement was performed according to existing methods (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 cynomolgus TIGIT, and the amino acid sequence of the extracellular segment of mouse TIGIT are shown in Table 1 above.
  • Test Example 2 Binding activity of anti-TIGIT monoclonal antibody to overexpressing human/cynomolgus/mouse TIGIT CHO cells and blocking activity
  • CHO-S cells overexpressing human TIGIT were generated through pressure screening by transfecting the pCHO1.0 vector (purchased from Invitrogen) of human TIGIT, cynomolgus TIGIT, and mouse TIGIT cDNA cloned into MCS. ), CHO-S cells overexpressing cynomolgus TIGIT (CHO-cynoTIGIT cells) and CHO-S cells overexpressing mouse TIGIT (CHO-muTIGIT cells). Adjust the expanded cultured overexpressing cells to a suitable cell density and add them to a 96-well flow cytometry plate.
  • 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 with 3-fold dilution at 400 nM, for a total of 12 points. Add 60 ⁇ L/well of the diluted sample to the above-mentioned 96-well flow plate with cells, and incubate at 4°C for 30 minutes.
  • Table 9 Summary table of anti-TIGIT monoclonal antibody overexpression cell binding blocking activity Note: “NB”:no binding; and “NA”:not available.
  • the anti-TIGIT monoclonal antibody of the present invention (1) has better binding activity with the human TIGIT protein overexpressed on the surface of CHO cells than the control molecule Tiragolumab; (2) with the overexpressed human TIGIT protein on the surface of CHO cells The binding activity of cynomolgus monkey TIGIT protein is better than the control molecule Tiragolumab; (3) 55796-G1 and 55796-G1LALA significantly bind to the mouse TIGIT protein overexpressed on the surface of CHO cells.
  • the anti-TIGIT monoclonal antibody of the present invention (1) blocks the binding of human CD155 to the overexpressed human TIGIT protein on the surface of CHO cells better than the control molecule Tiragolumab; (2) binds to CHO cells
  • the anti-TIGIT antibody molecules 55796-G1 and 55796-G1LALA bound to the mouse TIGIT protein overexpressed on the cell surface can significantly block the binding of mouse CD155 to the mouse TIGIT protein overexpressed on the surface of CHO cells.
  • Test Example 3 Anti-TIGIT monoclonal antibody binds to TIGIT on the surface of primary T cells
  • the binding activity of the anti-TIGIT antibody of the invention to TIGIT on the surface of activated T cells is detected based on the flow cytometry detection method.
  • human PBMC were sorted according to the experimental protocol provided by STEMCELL (stemcell, product number: #17951C) to obtain human total T cells.
  • STEMCELL stemcell, product number: #17951C
  • CD3/CD28 Dynabeads purchased from gibco, product number: 11132D
  • After centrifugation add gradient dilutions of the sample to be tested and incubate 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, and wash twice with PBS. Add PBS to resuspend the cells, detect on the CytoFlex flow cytometer and calculate the corresponding MFI.
  • the heavy chain variable region of the anti-PVRIG monoclonal antibody (the amino acid sequence is shown in SEQ ID NO: 5) was recombined into the human IgG1 heavy chain constant region and the human IgG1 heavy chain L234A and L235A modified constant regions respectively.
  • the light chain variable region (the amino acid sequence is shown in SEQ ID NO: 4) was recombined into the human kappa light chain constant region, and the anti-PVRIG monoclonal antibodies were named ADI-56127-G1 and ADI-56127-G1LALA respectively.
  • the specific operation is as follows: use chemical transfection method to transfer the pcDNA3.1 vector with antibody heavy chain and light chain into HEK293 cells, and culture at 37°C, 8% CO2 for 7 days. Collect the cell fluid and centrifuge at 13,000 rpm for 20 minutes. Take the supernatant, purify the supernatant with Protein A, and detect the antibody purity by SEC while controlling the endotoxin content.
  • Anti-PVRIG monoclonal antibodies ADI-56127-G1 and ADI-56127-G1LALA were thus prepared.
  • Test Example 4 Binding activity of anti-PVRIG antibodies to overexpressing human/cynomolgus/mouse PVRIG CHO cells and blocking activity
  • CHO cells overexpressing human PVRIG were generated through pressure screening by transfecting the pCHO1.0 vector (purchased from Invitrogen) of human PVRIG, cynomolgus PVRIG, and mouse PVRIG cDNA cloned into MCS, CHO cells overexpressing cynomolgus PVRIG (CHO-cynoPVRIG cells) and CHO cells overexpressing mouse PVRIG (CHO-muPVRIG cells). Adjust the expanded cultured overexpressing cells to a suitable cell density and add them to a 96-well flow cytometry plate. After centrifugation, add gradient dilutions of the test sample and incubate at 4°C for 30 minutes.
  • CHO cells overexpressing human CD112 were generated by pressure screening by transfecting the pCHO1.0 vector (purchased from Invitrogen) of human CD112 cDNA cloned into MCS. Adjust the cell density of the expanded cultured CHO-huCD112 cells to 2 ⁇ 10 6 cells/mL, add 100 ⁇ L/well to a 96-well flow plate, and centrifuge for later use.
  • mouse CD112-his protein was diluted with 1 ⁇ coating buffer to a final concentration of 1 ⁇ g/mL, 100 ⁇ L/well was added to the enzyme plate, covered with film, and left to coat at 4°C overnight. Discard the coating solution in the enzyme plate, wash three times with 1 ⁇ PBST, and add 200 ⁇ L/well of 5% BSA/PBS to block for 2 hours at room temperature. During the blocking period, the sample to be tested was diluted with 1% BSA/PBS gradient to a final volume of 60 ⁇ L/well.
  • Table 10 Summary table of cell binding blocking activity of overexpressed anti-PVRIG antibody molecules Note: “NB”: no binding; “NA”: not available (not detected).
  • ADI-56127-G1 and ADI-56127-G1LALA can significantly block the binding of mouse PVRIG to mouse CD112 protein, and the blocking activity is better than the control molecules Mab46-G1 and Mab46-G1LALA.
  • Test Example 5 Anti-PVRIG antibody binds to PVRIG on the surface of primary T cells
  • the binding activity of the anti-PVRIG antibody of the invention to PVRIG on the surface of activated T cells is detected based on the flow cytometry detection method.
  • human PBMC were sorted according to the experimental protocol provided by STEMCELL (stemcell, product number: #17951C) to obtain human total T cells.
  • STEMCELL stemcell, product number: #17951C
  • CD3/CD28 Dynabeads purchased from gibco, product number: 11132D
  • Adjust activated T cells to the appropriate cells Add the cell density to a 96-well flow plate, centrifuge, add gradient dilutions of the test sample, and incubate 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, and wash twice with PBS. Add PBS to resuspend the cells, detect on the CytoFlex flow cytometer and calculate the corresponding MFI.
  • Test Example 6 In vivo pharmacodynamic study of anti-PVRIG antibody in NDG mice mixed with A375 and human PBMC study
  • B-NDG mice were mixedly inoculated with A375 (purchased from Addexbio, C0020004, a human malignant melanoma cell) and human PBMC cells (Shanghai Miaoshun, A10S033014/PB100C) in a model (A375huPBMC model).
  • A375 purchased from Addexbio, C0020004, a human malignant melanoma cell
  • human PBMC cells Strestructure the human immune system
  • PBMC human immune cells
  • A375 cells and human PBMC were first mixed into 0.1 mL cell suspension at a 1:1 equal volume, and the A375huPBMC model was established by subcutaneous injection in the right abdominal groin of mice.
  • the average tumor volume reaches about 30-50 mm, they are divided into groups, and different doses and the same volume of PBS or antibody treatment are administered intraperitoneally, with 6 mice in each group.
  • the monitoring frequency is 2-3 days/time, and continuous monitoring is performed for 2 to 3 weeks.
  • the dosage and method of administration are as shown in Table 11.

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PCT/CN2023/085428 2022-04-02 2023-03-31 抗tigit-抗pvrig双特异性抗体、其药物组合物及用途 Ceased WO2023186081A1 (zh)

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CA3255478A CA3255478A1 (en) 2022-04-02 2023-03-31 Anti-tigit-anti-pvrig bispecific antibody, pharmaceutical composition thereof and use thereof
CN202380031275.9A CN118946592A (zh) 2022-04-02 2023-03-31 抗tigit-抗pvrig双特异性抗体、其药物组合物及用途
KR1020247036669A KR20240164815A (ko) 2022-04-02 2023-03-31 항-tigit/항-pvrig 이중특이적 항체, 이의 약제학적 조성물 및 이의 용도
JP2024558264A JP2025511289A (ja) 2022-04-02 2023-03-31 抗ｔｉｇｉｔ－抗ｐｖｒｉｇ二重特異的抗体、その医薬組成物及びその使用
EP23778445.9A EP4506365A4 (en) 2022-04-02 2023-03-31 BI-SPECIFIC ANTIBODY ANTI-TIGIT/ANTI-PVRIG, PHARMACEUTICAL COMPOSITION AND ASSOCIATED USES
AU2023247712A AU2023247712A1 (en) 2022-04-02 2023-03-31 Anti-TIGIT-anti-PVRIG bispecific antibody, pharmaceutical composition thereof and use thereof
IL316008A IL316008A (en) 2022-04-02 2023-03-31 Anti-tigit-anti-pvrig bispecific antibody, pharmaceutical composition thereof and use thereof
MX2024012144A MX2024012144A (es) 2022-04-02 2024-09-30 Anticuerpo biespecifico anti-tigit y anti-pvrig, composicion farmaceutica del mismo, y uso del mismo

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CN119431583B (zh) * 2024-12-19 2025-09-30 重庆国际免疫创新中心有限公司 靶向cd155的抗体或抗原结合片段及其应用

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