WO2023046097A1 - Anticorps humanisé anti-tigit ou fragment de liaison à l'antigène de celui-ci et son application - Google Patents

Anticorps humanisé anti-tigit ou fragment de liaison à l'antigène de celui-ci et son application Download PDF

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WO2023046097A1
WO2023046097A1 PCT/CN2022/120984 CN2022120984W WO2023046097A1 WO 2023046097 A1 WO2023046097 A1 WO 2023046097A1 CN 2022120984 W CN2022120984 W CN 2022120984W WO 2023046097 A1 WO2023046097 A1 WO 2023046097A1
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seq
antibody
amino acid
antigen
acid sequence
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Chinese (zh)
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芦迪
胡佩佩
欧颖烨
霍永庭
刘云鹏
罗甜
路力生
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广东菲鹏制药股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of biomedicine, in particular to an anti-TIGIT humanized antibody or an antigen-binding fragment thereof and applications thereof.
  • TIGIT T cell Ig and ITIM domain, also known as WUCAM, Vstm3, VSIG9 contains ITT domain (immunoglobulin tyrosine tail motif domain) and ITIM domain (immunoreceptor tyrosine inhibitory motif Domain) common inhibitory receptors for T cells and NK cells, belonging to type I transmembrane proteins, including IgV extracellular segment and immunoglobulin tyrosine tail-like phosphorylated fragments. The gene was discovered in 2008 by the research group of Genentech.
  • the research team searched the genome for genes that meet specific conditions (1expressed on immune cells; 2belong to type I transmembrane proteins; 3extracellular with immunoglobulin domains; 4intracellular with immune regulatory domains) genes And find this gene. Through sequence comparison, it was found that this protein belongs to a large PVR protein family, and its members include activated receptors CD226 and CD96, which compete with TIGIT for ligands, and their ligands PVR, and so on. In 2012, the crystal structure of the TIGIT molecule was analyzed by X-ray diffraction. It was found that TIGIT expressed on immune cells first formed a cis homodimer on the same cell, and then the dimer passed through a TIGIT molecule on one side.
  • TIGIT molecules are relatively conservative, and human TIGIT molecules have 88% and 58% sequence homology with monkey and mouse TIGIT molecules, respectively.
  • TIGIT is expressed on the surface of immune cells such as T cells, NK cells and dendritic cells, and binds to PVR (poliomyelitis virus receptor, CD155) on the surface of cancer cells to inhibit the activity of immune cells.
  • the main ligands of TIGIT are CD155 (Necl-5, PVR poliovirus receptor) and CD112 (PVRL2, Nectin-2).
  • the binding affinity to CD155 is the highest (3.15nM, Kd), and two TIGIT molecules form a tetramer "lock-and-key structure" with two CD155 molecules (Try113 of TIGIT and AX6G of CD155, and Phe128 of CD155 and AX6G of TIGIT respectively form a "key-lock”).
  • These two ligands are also ligands for CD226 (DNAM-1), which competes with TIGIT to stimulate T cell activity.
  • the interaction between the ligand and TIGIT defeats CD226, suppresses immune activity, and tumor cells upregulate CD155 and CD122 to escape immune-mediated destruction.
  • antagonistic antibodies specific to TIGIT can inhibit T cell responses induced by CD155 and CD112 and enhance anti-tumor immunity.
  • the development of antibodies that can specifically bind to TIGIT is of great significance for the prevention or treatment of immune diseases or tumor-related diseases .
  • the technical problem to be solved in the present invention is to develop an anti-human TIGIT antibody that binds to TIGIT protein with high affinity and specificity, and carry out humanization transformation to reduce the risk of antibody immunogenicity while maintaining the functional activity of the antibody.
  • the present invention provides an anti-TIGIT humanized antibody or an antigen-binding fragment thereof, the antibody comprises a light chain CDR region and a heavy chain CDR region, the light chain CDR region consists of LCDR1, LCDR2, LCDR3, and the heavy chain CDR region consists of HCDR1, HCDR2 , HCDR3, the amino acid sequences of LCDR1, LCDR2, and LCDR3 are sequentially selected from SEQ ID NO:29 ⁇ 31 or SEQ ID NO:32 ⁇ 34, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 are sequentially selected from SEQ ID NO:35 ⁇ 37 or SEQ ID NO: 38-40, the amino acid sequence of the heavy chain variable region of the antibody is shown in any one of SEQ ID NO: 5-15 or SEQ ID NO: 21-28.
  • the invention also provides nucleic acids encoding said antibodies or antigen-binding fragments thereof.
  • the present invention also provides a vector comprising the nucleic acid.
  • the present invention also provides cells carrying the nucleic acid, containing the vector or expressing the antibody or antigen-binding fragment thereof.
  • the present invention also provides a method of producing said antibody or antigen-binding fragment thereof, comprising: culturing said cells in a culture medium; and recovering the antibody or antigen-binding fragment thereof thus produced from the culture medium or from the cultured cells .
  • the present invention also provides a pharmaceutical composition containing the antibody or antigen-binding fragment thereof, or the nucleic acid, or the carrier or the cell.
  • the present invention also provides the use of the antibody or its antigen-binding fragment, the nucleic acid, the carrier, the cell, and the pharmaceutical composition in the preparation of drugs for preventing or treating immune diseases or tumor-related diseases application.
  • the present invention also provides any of the aforementioned antibodies or antigen-binding fragments thereof, nucleic acids, vectors, cells, or pharmaceutical compositions used as medicines; in some technical solutions, the medicines are used for preventing or treating immune diseases, or tumor-related diseases.
  • the present invention also provides a method for preventing or treating a disease, the method comprising administering a therapeutically effective amount of any of the aforementioned antibodies or antigen-binding fragments thereof, nucleic acids, vectors, cells or pharmaceutical compositions to individuals in need;
  • the disease is an immune disease or a tumor-related disease.
  • the above-mentioned anti-TIGIT humanized antibody has high affinity with TIGIT and has various functional properties, and can be used alone or in combination with other agents for the prevention or treatment of immune diseases or tumor-related diseases.
  • Figure 1 is a graph showing the results of different subtypes of anti-TIGIT antibodies reducing the average tumor volume of CT26 colon cancer in Balb/c mice.
  • Figure 2 is a graph showing the effects of different subtypes of anti-TIGIT antibodies on the survival of Balb/c mice.
  • Figure 3 is a graph showing the results of TIGIT-5 mouse monoclonal antibody and 3TIGIT-16 mouse monoclonal antibody reducing the average tumor volume of CT26 colon cancer in hTIGIT knock in mice.
  • Figure 4 is a graph showing the effects of TIGIT-5 mouse monoclonal antibody and 3TIGIT-16 mouse monoclonal antibody on the survival period of hTIGIT knock in mice.
  • Figure 5 is a graph showing the results of reducing the average tumor volume of CT26 colon cancer in hTIGIT knock in mice by anti-TIGIT humanized antibodies of hIgG1 subtype.
  • Figure 6 is a graph showing the average tumor volume of CT26 colon cancer in hTIGIT knockin mice reduced by anti-TIGIT humanized antibodies of the mIgG2a subtype.
  • Figure 7 is a graph showing the effect of anti-TIGIT humanized antibodies of mIgG2a subtype on the survival period of hTIGIT knock in mice.
  • Figure 8a is the result of single-tube staining; Figure 8b is the analysis steps; Figure 8c is the ratio diagram of CD4+/CD8+T cells; Figure 8d is the ratio diagram of Treg cells; Figure 8e is the target of human peripheral blood mononuclear cells ADCC result graph of cells.
  • Figure 9 is a graph of the detection results of more samples.
  • Figure 10a is a graph showing the effect of anti-TIGIT humanized antibody on T cells in tumor cell model mouse tissue
  • Figure 10b is the effect of anti-TIGIT humanized antibody tumor cell model mouse tissue on CD3+T cells and CD4+T cells Affect the result graph.
  • Figure 11a is the SEC-HPLC detection result of anti-TIGIT humanized antibody for acid;
  • Figure 11b is the result of SEC-HPLC detection of anti-TIGIT humanized antibody for alkaline treatment;
  • Figure 11c is the CE-SDS of anti-TIGIT humanized antibody - NR detection acid results;
  • Figure 11d is the CE-SDS-NR detection alkali treatment results of anti-TIGIT humanized antibody;
  • Figure 11e is the anti-TIGIT humanized antibody cIEF detection acid peak change results after acid treatment;
  • 11f is the cIEF detection result of the main peak change after acid treatment of the anti-TIGIT humanized antibody
  • Figure 11g is the cIEF detection result of the anti-TIGIT humanized antibody base peak change after acid treatment
  • Figure 11i is the result of cIEF detection of the main peak change after alkali treatment of anti-TIGIT humanized antibody
  • Figure 11j is the result of cIEF detection of anti-TIGIT humanized antibody after alkali treatment picture.
  • Figure 12a is the result of SEC-HPLC detection of anti-TIGIT humanized antibody after repeated freeze-thaw treatment
  • Figure 12b is the result of repeated freeze-thaw treatment of CE-SDS-NR detection of anti-TIGIT humanized antibody
  • Figure 12c is the result of repeated freeze-thaw treatment of anti-TIGIT humanized antibody
  • Figure 12d is the result of cIEF detection of anti-TIGIT humanized antibody after repeated freezing and thawing of the main peak change results of cIEF detection of humanized antibody
  • Figure 12e is the cIEF of anti-TIGIT humanized antibody The results of detecting the change of the basic peak after repeated freezing and thawing treatments.
  • Figure 13a is the result of SEC-HPLC detection of anti-TIGIT humanized antibody at high temperature
  • Figure 13b is the result of SEC-HPLC detection of anti-TIGIT humanized antibody at 4°C
  • Figure 13c is the result of anti-TIGIT humanized antibody CE-SDS-NR detection results of high temperature treatment
  • Figure 13d is the result of CE-SDS-NR detection of anti-TIGIT humanized antibody at 4 °C control treatment
  • Figure 13e is the cIEF detection of anti-TIGIT humanized antibody acidity after high temperature treatment
  • Figure 13f is the result of cIEF detection of anti-TIGIT humanized antibody after high-temperature treatment of the main peak change results
  • Figure 13g is the result of cIEF detection of anti-TIGIT humanized antibody base peak change after high-temperature treatment
  • Figure 13h is the result of anti-TIGIT humanized antibody
  • Figure 13i is the cIEF detection result of the humanized anti-TIGIT antibody after the control treatment at 4
  • Figure 14a is a graph of the in vitro binding activity of the humanized anti-TIGIT antibody 316H1L1 of the hIgG1 subtype after the stress test;
  • Figure 14b is a graph of the in vitro binding activity of the humanized anti-TIGIT antibody 15H9L2 of the hIgG1 subtype after the stress test;
  • Figure 14c It is the in vitro binding activity of hIgG1 subtype anti-TIGIT humanized antibody 15H10L3 after the stress test; wherein, R0750 represents 316H1L1, R0771 represents 15H9L2, and R0774 represents 15H10L3.
  • the present invention relates to an anti-TIGIT humanized antibody or an antigen-binding fragment thereof.
  • the antibody comprises a light chain CDR region and a heavy chain CDR region.
  • the light chain CDR region consists of LCDR1, LCDR2, LCDR3, and the heavy chain CDR region consists of HCDR1, HCDR2, Composed of HCDR3, the amino acid sequences of LCDR1, LCDR2, and LCDR3 are sequentially selected from SEQ ID NO: 29-31 or SEQ ID NO: 32-34, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 are sequentially selected from SEQ ID NO: 35-37 or SEQ ID NO: 35-37 or SEQ ID NO: 38-40, the amino acid sequence of the heavy chain variable region of the antibody is shown in any one of SEQ ID NO: 5-15 or SEQ ID NO: 21-28.
  • the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are respectively SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:31, and the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are respectively SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37, and the amino acid sequence of the heavy chain variable region of the antibody is shown in any of SEQ ID NO: 5-15; in some optional embodiments , the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 14.
  • the amino acid sequences of LCDR1, LCDR2 and LCDR3 are SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34 respectively, and the amino acid sequences of HCDR1, HCDR2 and HCDR3 are respectively It is SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40, and the amino acid sequence of the heavy chain variable region of the antibody is as shown in any of SEQ ID NO: 21-28; in some optional embodiments Among them, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 21.
  • An important advantage of the antibody or its antigen-binding fragment is that it has high in vitro binding activity and species cross-binding activity with TIGIT.
  • An important advantage of the antibody or antigen-binding fragment thereof is that it has the activity of blocking the binding of PVR to TIGIT.
  • An important advantage of the antibody or its antigen-binding fragment is that it has a significant anti-tumor effect.
  • An important advantage of the antibody or antigen-binding fragment thereof is its ability to reduce the proportion of Treg cells.
  • the antibody or its antigen-binding fragment can be used as an antibody drug.
  • antibody or antigen-binding fragment thereof refers to a protein that binds to a specific antigen, and generally refers to all proteins and protein fragments including complementarity determining regions (CDR regions).
  • Antibody specifically refers to a full-length antibody.
  • full-length antibody includes polyclonal as well as monoclonal antibodies, and the term “antigen-binding fragment” is a substance comprising part or all of the CDRs of an antibody, which lacks at least some of the amino acids present in the full-length chain but is still capable of specificity. Bind to antigen.
  • Such fragments are biologically active in that they bind to the target antigen and can compete with other antigen binding molecules, including intact antibodies, for binding to a given epitope.
  • the antigen-binding fragment has the effect of specifically recognizing and binding to TIGIT.
  • a “humanized antibody” is an antibody that retains the reactivity of a non-human antibody, while being less immunogenic in humans. For example, they can be constructed by retaining the CDR regions of a non-human antibody and replacing the remainder of the antibody with their human antibody counterparts (ie, the constant regions and the framework portions of the variable regions).
  • the term "specific binding” or similar expressions thereof refers to the binding of an antibody or antigen-binding fragment thereof to a predetermined epitope on an antigen.
  • the antibody or antigen-binding fragment thereof binds with an affinity (KD) of less than about 10 "6M , eg, about less than 10 "7M , 10 "8M , 10 “9M or 10 "10M or less.
  • KD refers to the ratio of the off-rate to the on-rate (koff/kon), which quantity can be measured by methods familiar to those skilled in the art.
  • CDR complementarity determining region
  • CDR complementarity determining region
  • CDR and CDRs are used to mean comprising one or more or even all of the major amino acids that contribute to the binding affinity of an antibody or antigen-binding fragment thereof to the antigen or epitope it recognizes. region of residues.
  • the complementarity determining region of the heavy chain is represented by HCDR
  • the complementarity determining region of the light chain is represented by LCDR.
  • CDR notation methods commonly used in the field include: Kabat numbering scheme, Chothia and Lesk numbering scheme, and a new standardized numbering system introduced by Lefranc et al. in 1997 for all protein sequences of the immunoglobulin superfamily. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions.
  • sequences of Proteins of Immunological Interest the amino acid sequences of the light chain ( ⁇ , ⁇ ) variable regions and antibody heavy chains, and the variable region of the T cell receptor ( ⁇ , ⁇ , ⁇ , ⁇ ) are aligned and numbered.
  • the accumulation of sequences over the past few decades has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered the widely adopted standard for numbering antibody residues.
  • the present invention uses the Kabat annotation standard to mark the CDR region, but the CDR region marked by other methods also belongs to the protection scope of the present invention.
  • the terms “specifically recognize”, “selectively bind”, “selectively bind” and “specifically bind” or similar expressions refer to the binding of an antibody or antigen-binding fragment thereof to a predetermined antigen. Epitope binding. Typically, the antibody or antigen-binding fragment thereof binds with an affinity ( KD ) of about less than 10 "6M , eg, about less than 10 "7M , 10 "8M , 10 "9M or 10" 10M or less.
  • the provided antibodies or antigen-binding fragments thereof can specifically recognize TIGIT from various genera, such as human, mouse, and monkey (such as cynomolgus monkey).
  • the amino acid sequence of the light chain variable region of any of the above antibodies is shown in any one of SEQ ID NO: 1-4 or SEQ ID NO: 16-20.
  • the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO: 14, or as shown in any of SEQ ID NO: 5-13, 15, and the light chain of the antibody
  • the amino acid sequence of the variable region is as shown in SEQ ID NO: 3, or as shown in any of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4; or the heavy chain variable region of the antibody
  • the amino acid sequence is as shown in any of SEQ ID NO:21-28, and the amino acid sequence of the light chain variable region of the antibody is as shown in any of SEQ ID NO:16-20;
  • amino acid sequences of the light chain variable region and the heavy chain variable region of the antibody are shown in Table 1 below:
  • serial number light chain variable region heavy chain variable region 1 SEQ ID NO:3 SEQ ID NO:14 2 SEQ ID NO:16 SEQ ID NO:21 3 SEQ ID NO:1 SEQ ID NO:5 4 SEQ ID NO:1 SEQ ID NO:12 5 SEQ ID NO:4 SEQ ID NO:11 6 SEQ ID NO:4 SEQ ID NO:15 7 SEQ ID NO:2 SEQ ID NO:12 8 SEQ ID NO:2 SEQ ID NO:13 9 SEQ ID NO:2 SEQ ID NO:14 10 SEQ ID NO:2 SEQ ID NO:15 11 SEQ ID NO:4 SEQ ID NO:14
  • the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 2
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13
  • the light chain variable region of the antibody The amino acid sequence of the chain variable region is shown in SEQ ID NO: 3
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 14
  • the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO Shown in: 16
  • the aminoacid sequence of heavy chain variable region is shown in SEQ ID NO: 21.
  • the antibody contains a heavy chain constant region and a light chain constant region, and the heavy chain constant region sequence is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD Constant region sequence. In some embodiments, the heavy chain constant region sequence is selected from IgG1.
  • the light chain constant region is a kappa or lambda chain. In some embodiments, the light chain constant region sequence is selected from a kappa chain.
  • the species sources of the heavy chain constant region and the light chain constant region are selected from cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer , mink, chicken, duck, goose or human.
  • the species origin of the heavy chain constant region and the light chain constant region is human.
  • the amino acid sequence of the heavy chain constant region is as shown in any one of SEQ ID NO:45-48, and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO:49 or SEQ ID NO:50 Show.
  • the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:53, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:50.
  • the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:45, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:49.
  • amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:45 or SEQ ID NO:48. In some embodiments, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:53.
  • amino acid sequence of the heavy chain constant region of the antibody described in any one of the foregoing is shown in SEQ ID NO:53, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:50.
  • amino acid sequence of the heavy chain constant region of the antibody described in any one of the foregoing is shown in SEQ ID NO:45, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:50.
  • the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 50; and the heavy chain of the antibody
  • the amino acid sequence of the chain variable region is shown in SEQ ID NO: 14
  • the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 53 or SEQ ID NO: 45.
  • the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 16, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 50; and the heavy chain of the antibody
  • the amino acid sequence of the chain variable region is shown in SEQ ID NO:21, and the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:53 or SEQ ID NO:45.
  • the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 50; and the heavy chain of the antibody
  • the amino acid sequence of the chain variable region is shown in SEQ ID NO: 13
  • the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 53 or SEQ ID NO: 45.
  • the amino acid sequence of the heavy chain of the antibody is set forth in SEQ ID NO:51, and the amino acid sequence of the light chain is set forth in SEQ ID NO:52.
  • the antigen-binding fragment is any one of F(ab') 2 , Fab, scFv and bispecific antibody.
  • the term "F(ab') 2" contains two light chains and two heavy chains containing part of the constant region between the CH1 and CH2 domains, so that an interchain chain is formed between the two heavy chains. disulfide bond.
  • the F(ab') 2 fragment thus consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.
  • bispecific antibody is a multispecific antigen binding protein or multispecific antibody, and can be produced by various methods including, but not limited to fusion of hybridomas or linking of Fab' fragments .
  • the two binding sites of a bispecific antigen binding protein or antibody will bind two different epitopes, present on the same or different protein targets.
  • the invention also relates to nucleic acids encoding said antibodies or antigen-binding fragments thereof.
  • the nucleic acid is usually RNA or DNA, and the nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence.
  • DNA is preferably used when it is ligated into a vector.
  • the nucleic acid since antibodies are membrane proteins, the nucleic acid usually carries a signal peptide sequence.
  • the nucleic acid comprises: a first nucleic acid encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof, and/or a first nucleic acid encoding the light chain variable region of the antibody or antigen-binding fragment thereof second nucleic acid.
  • the invention also relates to a vector comprising said nucleic acid.
  • the term "vector" is a nucleic acid delivery tool into which a polynucleotide can be inserted.
  • the vector is capable of achieving expression of the protein encoded by the inserted polynucleotide, 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 adeno-associated viruses
  • herpesviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • papillomaviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • the invention also relates to cells carrying said nucleic acid, containing said vector or expressing said antibody or antigen-binding fragment thereof.
  • the invention also relates to methods of producing said antibodies or antigen-binding fragments thereof, comprising: culturing said cells in a culture medium; and recovering the antibodies or antigen-binding fragments thereof so produced from the culture medium or from the cultured cells.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising said antibody or antigen-binding fragment thereof, or said nucleic acid, or said vector or said cell.
  • the term "pharmaceutical composition” is in a form that allows the biological activity of the active ingredients to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to whom the composition will be administered.
  • the pharmaceutical composition further includes pharmaceutically acceptable carriers and/or excipients.
  • the term "pharmaceutically acceptable carrier” may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible, used for Extend the shelf life or potency of antibodies.
  • the present invention also relates to the application of the antibody or its antigen-binding fragment, the nucleic acid, the carrier, the cell, and the pharmaceutical composition in the preparation of drugs for preventing or treating immune diseases or tumor-related diseases .
  • the present invention also provides any of the aforementioned antibodies or antigen-binding fragments thereof, nucleic acids, vectors, cells, or pharmaceutical compositions used as medicines; in some technical solutions, the medicines are used for preventing or treating immune diseases, or tumor-related diseases.
  • the present invention also provides a method for preventing or treating diseases, said method comprising administering a therapeutically effective amount of any of the antibodies or antigen-binding fragments thereof, nucleic acids, vectors, cells or pharmaceutical compositions described above to individuals in need ;
  • the disease is an immune disease or a tumor-related disease;
  • any one of the diseases described above is a disease related to human TIGIT, more preferably a T cell dysfunction disease.
  • the present invention also provides a reagent for detecting or measuring human TIGIT, said reagent comprising any one of the anti-TIGIT antibodies or antigen-binding fragments thereof.
  • the anti-human TIGIT humanized antibody of the present invention has good binding activity and blocking activity, significant anti-tumor effect, effectively prolongs the survival period of mice, significantly reduces the proportion of Treg cells in healthy human PBMC and tumor infiltrating lymphocytes, and has good stability. It has good druggability and can be widely used in the prevention or treatment of immune diseases or tumor-related diseases.
  • the TIGIT-5 mouse monoclonal antibody (VL, VH sequences shown in SEQ ID NO: 41, SEQ ID NO: 42) and 3TIGIT-16 mouse monoclonal antibody (VL, VH sequences shown in SEQ ID NO: 43, SEQ ID NO: 44) have good binding affinity to CHO-hTIGIT, healthy PBMC, and cynoTIGIT, block the combination of CHO-hTIGIT, CHO-hPVR and hPVR-hFc, block tumor cells,
  • the combination of CHO-CD112 and TIGIT, and the binding kinetic constants of human TIGIT recombinant protein are all at the pM level, and it has a better ability to activate NK92-hTIGIT to kill U2-OS;
  • Anti- and 3TIGIT-16 mouse monoclonal antibody was humanized, the specific method is as follows:
  • CDR-grafting Humanization was carried out by the method of complementarity determining region grafting (CDR-grafting).
  • MOE Molecular Operating Environment
  • the humanized VL sequence (VL1-VL4) is shown in Table 2-1 below, and the humanized VH sequence (VH1-VH11) is shown in Table 2-2 below:
  • VL5-VL9 the humanized VL sequence (VL5-VL9) is shown in Table 3 below, and the humanized VH sequence (VH12-VH19) is shown in Table 4 below:
  • Combining different humanized VL and VH can produce different humanized antibody molecules.
  • the main consideration of different combinations is the balance between the degree of humanization and the maintenance of antibody activity after humanization. Simply put, pairing the VL and VH with the highest degree of humanization can obtain the antibody molecule with the highest degree of humanization, which means that the immunogenicity may be lower, but the high degree of humanization may lead to The activity of the molecule is reduced (key amino acid residues that affect the activity of the parental antibody are replaced with humanized amino acids, resulting in changes in the antigen-binding region of the engineered antibody).
  • the specific numbered humanized antibody (such as R0774 (also identified as 15H10L3)) listed in the above-mentioned Table 5 and Table 6, its heavy chain constant region is hIgG1 containing K214R/D356E/L358M mutation (amino acid sequence such as SEQ ID NO : shown in 53), the light chain constant region is Hu-CL- ⁇ (amino acid sequence shown in SEQ ID NO: 50).
  • R0774 also identified as 15H10L3
  • R0774 heavy chain amino acid sequence (SEQ ID NO:51):
  • the antibody was prepared by conventional methods, and the expression supernatant was purified by ProA affinity chromatography. The process is as follows:
  • AKTA york 150 chromatographic equipment, equilibrate the chromatographic column (such as MabSelectSuRe LX, GE) with at least 5CV equilibration buffer (10mM PBS), load the sample to the chromatographic column, so that the target protein is adsorbed on the chromatographic column while other impurities pass through. Permeable separation.
  • chromatographic column such as MabSelectSuRe LX, GE
  • 5CV equilibration buffer 10mM PBS
  • the equipment detects the absorbance value of the sample, and calculates the concentration of anti-TIGIT humanized antibody according to Lambert-Beer law. If the concentration of the sample is too low, it needs to be concentrated by ultrafiltration.
  • Ultra-15 Centrifugal Filter Devices 30kD
  • a 0.22um sterile syringe filter Cobet, PES, 0.22um , 13 mm in diameter
  • the humanized degree and purity results of the anti-TIGIT humanized antibody are shown in Table 8 and Table 9. The results show that the humanized degree, titer and purity of the anti-TIGIT humanized antibody prepared in this example are very good. good.
  • the leftmost value is the percentage of aggregates
  • the rightmost value is the percentage of fragments
  • the middle value is the percentage of antibody monomers, such as "4.54%/94.62%/0.84%" , which means that in the antibody solution, the aggregate percentage is 4.54%, the antibody monomer purity percentage is 94.62%, and the fragment percentage is 0.84%";
  • Embodiment 3 anti-TIGIT humanized antibody verification
  • the anti-TIGIT humanized antibody obtained above was tested for in vitro binding activity, species cross-binding activity and blocking activity, the method is as follows:
  • FCM buffer to test samples including positive control group, TIGIT-5 mouse monoclonal antibody (R0435), 3TIGIT-16 mouse monoclonal antibody (R0518), anti-TIGIT humanized antibody; wherein R0300-CH2a comes from patent WO2017053748 (A2 ) in tiragolumab, R0300-CH2a is to replace the Fc region with the Fc region of mIgG1) diluted to 20 ⁇ g/mL, and then use FCM buffer to perform 3-fold serial dilution of 12 concentration points.
  • Centrifuge 300g/5min
  • discard the supernatant add 200 ⁇ L FCM buffer to wash once
  • centrifuge 300g/5min
  • discard the supernatant Add 100 ⁇ L 1 ⁇ PBS to resuspend, and test on the machine.
  • Centrifuge 300g/5min
  • discard the supernatant add 200 ⁇ L FCM buffer to wash once
  • centrifuge 300g/5min
  • discard the supernatant Add 100 ⁇ L 1 ⁇ PBS to resuspend, and test on the machine.
  • the following experiments were specially carried out: the CHO engineered cells CHO-hTIGIT cells to be tested for recombinant expression of full-length human TIGIT were counted and centrifuged at 300g After 5 minutes, resuspend in FCM buffer and adjust the cell density to 4E+06 cells/mL for later use.
  • FCM buffer to dilute the sample to be tested (including the positive control group R0300-CH2a, R0435, R0518, anti-TIGIT humanized antibody) to 20 ⁇ g/mL, and then use FCM buffer to perform 3-fold serial dilution of 12 concentration points; use FCM buffer
  • the ligand protein hPVR-hFc was diluted to 20 ⁇ g/mL.
  • 50 ⁇ L of antibody and 50 ⁇ L of cells were added to each well of the 96-well V-type plate (the amount of each cell strain added to the 96-well V-type plate was 2E+05 cells/well), and incubated on ice for 30 minutes in the dark.
  • test results of in vitro binding activity, species cross-binding activity and blocking activity of anti-TIGIT humanized antibodies are shown in Table 10 and Table 11. The results show that most of the anti-TIGIT humanized antibodies can maintain similar In vitro binding activity, species cross-binding activity and blocking activity.
  • N/A The activity is too weak to be calculated.
  • N/A The activity is too weak to be calculated.
  • R0226 (mIgG2a) [R0226 is derived from Oncomed, OMP-313R12, WO2016191643 (A2)], R0564 (mIgG1) in the CT26 mouse colon cancer subcutaneous xenograft model [R0564 is the mIgG1 subtype of R0226], the anti-tumor effect of R0565 (mIgG2a DLE) [R0565 is the mIgG2a-DLE subtype of R0226], and set the isotype control group R0513 (Isotype-mIgG2a).
  • C26 Mouse colon cancer cells (CT26) were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (1:1).
  • CT26 cells in logarithmic growth phase were collected, washed twice with pre-cooled DPBS, and the cell concentration was adjusted to 1 ⁇ 10 6 /mL.
  • Female Balb/c mice were taken and CT26 cells were subcutaneously inoculated with an inoculation volume of 0.1 mL/mouse, that is, 1 ⁇ 10 5 /mouse.
  • the administration volume is adjusted according to the animal's body weight (10L/g); if the body weight drops more than 15% during the administration period, the administration regimen will be adjusted.
  • i.p. means intraperitoneal injection
  • Q3D*4 means 1 administration every 3 days, a total of 4 administrations.
  • the tumor volume was measured and the body weight of the mice was recorded. After that, the body weight was recorded twice a week and the long diameter and short diameter of the tumor were measured with a vernier caliper.
  • the tumor volume was calculated by the formula: (1/2) ⁇ long diameter ⁇ (short diameter) 2 .
  • the independent sample T test was used for comparison between two groups. More than 3 groups were compared using One-Way ANOVA. If the F value shows a significant difference, post hoc analysis among multiple groups can be performed. Data were processed using Prism GraphPad, when p ⁇ 0.05 indicated a statistically significant difference.
  • Tumor volume V 0.5a ⁇ b 2 , where a and b are the long and short diameters of the tumor, respectively.
  • TGI (%) [1-(Ti-T0)/(Vi-V0)] ⁇ 100
  • Ti is the average tumor volume of the treatment group on day i
  • T0 is the average tumor volume of the treatment group at the beginning of treatment
  • Vi is the average tumor volume of the vehicle control group on day i
  • V0 is the average tumor volume of the vehicle control group at the beginning of treatment.
  • results of different subtypes of anti-TIGIT antibodies reducing the average tumor volume of CT26 colon cancer in Balb/c mice are shown in Table 13 and Figure 1.
  • Isotype represents the isotype control group R0513 (Isotype-mIgG2a)
  • R0226 represents R0226 (mIgG2a)
  • R0564 represents R0564 (mIgG1)
  • R0565 represents R0565 (mIgG2a DLE).
  • mice Female, 6-8 weeks old (Balb/c background, source: Jiangsu Jicui Yaokang Biotechnology Co., Ltd., certificate number: 202008518); CT26 cells (Shanghai Cell Bank) RPMI 1640 medium (Gibco, 11875085), FBS (Gibco, 10091-148), 0.25% trypsin-EDTA (Gibco, 25200056), penicillin-streptomycin (Gibco, 15140122), DMSO (Sigma, D2650), DPBS (Hyclone, SH30028.02).
  • C26 Mouse colon cancer cells (CT26) were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (1:1).
  • CT26 cells in logarithmic growth phase were collected, washed twice with pre-cooled DPBS, and the cell concentration was adjusted to 2 ⁇ 10 6 /mL.
  • Female hTIGIT mice were taken and CT26 cells were subcutaneously inoculated with an inoculation volume of 0.1 mL/mouse, that is, 2 ⁇ 10 5 /mouse.
  • Dosing frequency Isotype-m 10 10 i.p. Q3D*4 R0300-CH2a 10 10 i.p. Q3D*4 R0435 10 10 i.p. Q3D*4 R0518 10 10 i.p. Q3D*4
  • the administration volume is adjusted according to the animal's body weight (10 L/g); if the body weight decreases by more than 15% during the administration period, the administration regimen will be adjusted.
  • Isotype-m represents the isotype control group (R0513).
  • Q3D*4 means 1 administration every 3 days, a total of 4 administrations.
  • the tumor volume was measured and the body weight of the mice was recorded. After that, the body weight was recorded twice a week and the long diameter and short diameter of the tumor were measured with a vernier caliper. The tumor volume was calculated by the formula: (1/2) ⁇ long diameter ⁇ (short diameter) 2 . When each mouse reached the end point of the experiment (the body weight decreased by 15% or the tumor volume exceeded 2000 mm 3 to reach the benevolent end point), the mice were killed by CO 2 asphyxiation, and the survival curve was recorded.
  • TGI (%) [1-(Ti-T0)/(Vi-V0)] ⁇ 100
  • Ti is the average tumor volume of the treatment group on day i
  • T0 is the average tumor volume of the treatment group at the beginning of treatment
  • Vi is the average tumor volume of the vehicle control group on day i
  • V0 is the average tumor volume of the vehicle control group at the beginning of treatment.
  • TIGIT-5 mouse monoclonal antibody and 3TIGIT-16 mouse monoclonal antibody reduce the average tumor volume of CT26 colon cancer in hTIGIT knock in mice
  • the effect on the survival period of hTIGIT knock in mice is shown in Figure 4.
  • the results showed that compared with the isotype control group, 3TIGIT-16 mouse monoclonal antibody (R0518) had a significant inhibitory effect on the tumor growth of hTIGIT knock in mouse CT26 model.
  • TGI tumor effect
  • R0518 significantly prolongs the survival period of mice
  • the anti-tumor effect of TIGIT-5 mouse monoclonal antibody (R0435) is comparable to that of the positive control group R0300-CH2a (TGI is 76.5%, 86.7%, respectively).
  • Isotype represents the isotype control group (R0513).
  • mice Female, 6-8 weeks old (Balb/c background, source: Jiangsu Jicui Yaokang Biotechnology Co., Ltd., certificate number: 202102514); CT26 cells (Shanghai Cell Bank) RPMI 1640 medium (Gibco, 11875085), FBS (Gibco, 10091-148), 0.25% trypsin-EDTA (Gibco, 25200056), penicillin-streptomycin (Gibco, 15140122), DMSO (Sigma, D2650), DPBS (Hyclone, SH30028.02).
  • C26 Mouse colon cancer cells (CT26) were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (1:1).
  • CT26 cells in logarithmic growth phase were collected, washed twice with pre-cooled DPBS, and the cell concentration was adjusted to 2 ⁇ 10 6 /mL.
  • Female hTIGIT mice were taken and CT26 cells were subcutaneously inoculated with an inoculation volume of 0.1 mL/mouse, that is, 2 ⁇ 10 5 /mouse.
  • Dosing frequency Isotype-hIgG1 10 10 i.p. Q3D*4 R0300-hIgG1 10 10 i.p. Q3D*4 15H9L2-hIgG1 10 10 i.p. Q3D*4 15H10L3-hIgG1 10 10 i.p. Q3D*4 316H1L1-hIgG1 10 10 i.p. Q3D*4
  • Isotype-hIgG1 represents the isotype control group (R0536).
  • Q3D*4 means 1 administration every 3 days, a total of 4 administrations.
  • the tumor volume was measured and the body weight of the mice was recorded. After that, the body weight was recorded twice a week and the long diameter and short diameter of the tumor were measured with a vernier caliper.
  • the tumor volume was calculated by the formula: (1/2) ⁇ long diameter ⁇ (short diameter) 2 .
  • the independent sample T test was used for comparison between two groups. More than 3 groups were compared using One-Way ANOVA. If the F value shows a significant difference, post hoc analysis among multiple groups can be performed. Data were processed using Prism GraphPad, when p ⁇ 0.05 indicated a statistically significant difference.
  • Tumor volume V 0.5a ⁇ b 2 , where a and b are the long and short diameters of the tumor, respectively.
  • TGI (%) [1-(Ti-T0)/(Vi-V0)] ⁇ 100
  • Ti is the average tumor volume of the treatment group on day i
  • T0 is the average tumor volume of the treatment group at the beginning of treatment
  • Vi is the average tumor volume of the vehicle control group on day i
  • V0 is the average tumor volume of the vehicle control group at the beginning of treatment.
  • Anti-TIGIT humanized antibody of hIgG1 subtype reduces the average tumor volume of CT26 colon cancer in hTIGIT knock in mice.
  • the results are shown in Table 17 and Figure 5.
  • the results show that: Humanized antibodies 15H9L2 and 15H10L3 had significant tumor-inhibitory effects on the tumor growth of hTIGIT knock in mouse CT26 model with TGIs of 56.38% and 62.83%, respectively; the anti-tumor effects of 316H1L1 were comparable to the positive control group R0300, with TGIs of 36.26% and 36.26%, respectively. 34.79%.
  • Isotype represents the isotype control group (R0536)
  • R0300 represents the positive control R0300-hIgG1
  • 15H9L2 represents 15H9L2-hIgG1
  • 15H10L3 represents 15H10L3-hIgG1
  • 316H1L1 represents 316H1L1-hIgG1.
  • mice Female, 6-8 weeks old (Balb/c background, source: Jiangsu Jicui Yaokang Biotechnology Co., Ltd., certificate number: 202102514); CT26 cells (Shanghai Cell Bank) RPMI 1640 medium (Gibco, 11875085), FBS (Gibco, 10091-148), 0.25% trypsin-EDTA (Gibco, 25200056), penicillin-streptomycin (Gibco, 15140122), DMSO (Sigma, D2650), DPBS (Hyclone, SH30028.02).
  • C26 Mouse colon cancer cells (CT26) were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (1:1).
  • CT26 cells in logarithmic growth phase were collected, washed twice with pre-cooled DPBS, and the cell concentration was adjusted to 2 ⁇ 10 6 /mL.
  • Female hTIGIT mice were taken and CT26 cells were subcutaneously inoculated with an inoculation volume of 0.1 mL/mouse, that is, 2 ⁇ 10 5 /mouse.
  • Dosing frequency Isotype-mIgG2a 10 10 i.p. Q3D*4 R0300-mIgG2a 10 10 i.p. Q3D*4 15H9L2-mIgG2a 10 10 i.p. Q3D*4 15H10L3-mIgG2a 10 10 i.p. Q3D*4 316H1L1-mIgG2a 10 10 i.p. Q3D*4
  • the administration volume is adjusted according to the animal's body weight (10L/g); if the body weight decreases by more than 15% during the administration period, the administration regimen will be adjusted.
  • Isotype represents the isotype control group (R0513).
  • Q3D*4 means 1 administration every 3 days, a total of 4 administrations.
  • the tumor volume was measured and the body weight of the mice was recorded. After that, the body weight was recorded twice a week and the long diameter and short diameter of the tumor were measured with a vernier caliper. The tumor volume was calculated by the formula: (1/2) ⁇ long diameter ⁇ (short diameter) 2 . When each mouse reached the end point of the experiment (the body weight decreased by 15% or the tumor volume exceeded 2000 mm 3 to reach the benevolent end point), the mice were killed by CO 2 asphyxiation, and the survival curve was recorded.
  • TGI (%) [1-(Ti-T0)/(Vi-V0)] ⁇ 100
  • Ti is the average tumor volume of the treatment group on day i
  • T0 is the average tumor volume of the treatment group at the beginning of treatment
  • Vi is the average tumor volume of the vehicle control group on day i
  • V0 is the average tumor volume of the vehicle control group at the beginning of treatment.
  • Anti-TIGIT humanized antibodies of mIgG2a subtype reduce the average tumor volume of CT26 colon cancer in hTIGIT knock in mice.
  • the results are shown in Table 19 and Figure 6.
  • the effect of survival time is shown in Figure 7.
  • the results show that: compared with the isotype control group, the anti-TIGIT humanized antibody 15H10L3 of the mIgG2a subtype has a significant tumor inhibitory effect on the tumor growth of the hTIGIT knock in mouse CT26 model, and TGI
  • the antitumor effect of 316H1L1 was equivalent to that of the positive control group R0300, and the TGI were 76.89% and 75.68%, respectively.
  • Isotype represents the isotype control group (R0513);
  • R0300 represents the positive control R0300-mIgG2a,
  • 15H9L2 represents 15H9L2-mIgG2a,
  • 15H10L3 represents 15H10L3-mIgG2a,
  • 316H1L1 represents 316H1L1-mIgG2a.
  • PBMCs were isolated from fresh blood, and the cell density was adjusted to 5E5 cells/mL with 10% FBS+x-vivo medium, and placed in an incubator overnight.
  • PBMC cell count donor-167 8.44E+05 cells/mL, 81.15%, take 20 mL of cells, donor-2345.53E+05 cells/mL, 78.38%, take 20 mL of cells;
  • Figure 8a is the result of single-tube staining; Figure 8b is the analysis steps; Figure 8c is the ratio diagram of CD4+/CD8+T cells; Figure 8d is the ratio diagram of Treg cells; Figure 8e is the target of human peripheral blood mononuclear cells ADCC results of the cells; the results show that: hIgG1 subtype anti-TIGIT humanized antibodies 15H9L2 and 15H10L3 can effectively reduce the proportion of Treg cells, while the hIgG1 subtype anti-TIGIT humanized antibody 316H1L1 is equivalent to the positive control group R0300.
  • the function of Treg cells was knocked out weakly, and the anti-TIGIT humanized antibody of hIgG1 subtype had no significant effect on the proportion of CD8+T cells.
  • hIgG1 subtype anti-TIGIT humanized antibodies 15H9L2 and 15H10L3 knockout Treg cells have stronger functions
  • hIgG1 subtype anti-TIGIT humanized antibody 316H1L Although there is no significant difference in the function of Treg cells compared with the positive control group R0300, the proportion of Treg cells in cells treated with 316H1L is lower; compared with the positive control group, anti-TIGIT humanized antibodies of the hIgG1 subtype can reduce cell ratio.
  • C26 Mouse colon cancer cells (CT26) were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (1:1).
  • CT26 cells in logarithmic growth phase were collected, washed twice with pre-cooled DPBS, and the cell concentration was adjusted to 2 ⁇ 10 6 /mL.
  • Female hTIGIT mice were taken and CT26 cells were subcutaneously inoculated with an inoculation volume of 0.1 mL/mouse, that is, 2 ⁇ 10 5 /mouse.
  • mice On the second day after the second administration, the mice were euthanized, and the tumor and spleen were dissected, and the lymphocytes in the spleen and tumor cells were analyzed after treatment.
  • Dosing frequency Isotype-mIgG2a 10 10 i.p. Q3D*2 R0300-mIgG2a 10 10 i.p. Q3D*2 15H9L2-mIgG2a 10 10 i.p. Q3D*2
  • the administration volume is adjusted according to the animal's body weight (10L/g); if the body weight decreases by more than 15% during the administration period, the administration regimen will be adjusted.
  • Isotype represents the isotype control group (R0513).
  • i.p. means intraperitoneal injection
  • Q3D*2 means administration once every 3 days, for a total of 2 administrations.
  • Washing resuspend cells with 5mL DPBS, count (using PBMC mode), transfer 3E6-4E6 cells to a 96-well plate, centrifuge at 300-400g for 5min.
  • Block non-specific Fc-mediated interactions Before staining mouse cells, pre-incubate the cells with 0.5-1 ⁇ g purified anti-mouse CD16/CD32/100 ⁇ L at 2-25°C for 10-20 minutes.
  • Figure 10a is a graph showing the effect of anti-TIGIT humanized antibody on T cells in tumor cell model mouse tissues
  • Figure 10b is the effect of anti-TIGIT humanized antibody tumor cell model mouse tissue on CD3+T cells and CD4+T cells
  • the results show that: compared with the isotype control group, the mIgG2a subtype anti-TIGIT humanized antibody 15H9L2 and the positive control group R0300 can significantly reduce the proportion of Treg cells in tumor infiltrating lymphocytes, and 15H9L2 has a more significant effect than R0300.
  • the evaluation of druggability can amplify potential risk sites, such as post-translational modification sites such as deamidation, isomerization, and oxidation, through early pressurization experiments and stability studies.
  • potential risk sites such as post-translational modification sites such as deamidation, isomerization, and oxidation
  • the link between stress conditions and changes in specific antibody risk sites can also be established, so that those environments that are not friendly to antibody molecules can be detected as early as possible.
  • antibody molecules will experience a variety of different environmental conditions, such as high temperature and oxidation in the cultivation process; acid and alkali conditions in the purification process; extreme acidic conditions in the virus fire extinguishing process; Conditions such as fusion and oscillation, and early detection of factors that are unstable to antibodies can provide corresponding reference strategies for later development.
  • Figure 11a and Figure 11b are the SEC-HPLC detection results of acid and alkali treatment of anti-TIGIT humanized antibody in turn;
  • Figure 11c and Figure 11d are the results of CE-SDS-NR detection of anti-TIGIT humanized antibody for acid and alkali treatment Figures;
  • Figure 11e, Figure 11f, and Figure 11g are the results of cIEF detection of anti-TIGIT humanized antibodies in turn, acid peak changes, main peak changes, and base peak changes after acid treatment;
  • Figure 11h, Figure 11i, and Figure 11j are anti-TIGIT humanized antibodies in turn cIEF detection results of acidic peak change, main peak change, and alkaline peak change after alkali treatment of the humanized antibody. , the purity results are stable.
  • Figure 12a is the result of SEC-HPLC detection of anti-TIGIT humanized antibody after repeated freeze-thaw treatment
  • Figure 12b is the result of repeated freeze-thaw treatment of CE-SDS-NR detection of anti-TIGIT humanized antibody
  • Figure 12c, Figure 12d, Figure 12e is the results of the cIEF detection of the anti-TIGIT humanized antibody after repeated freezing and thawing treatments for acidic peak changes, main peak changes, and basic peak changes. Stablize.
  • the three antibody samples were divided into two groups, the first group was controlled at 4°C, the second group was placed in a 37°C constant temperature incubator at high temperature, samples were taken in 1.5ml EP tubes at different time points, and sealed with parafilm, on the 0th, 7th, and 14th days , 28Day inspection and analysis, select some samples for mass spectrometry analysis according to the physical and chemical test results.
  • Figure 13a and Figure 13b are the SEC-HPLC detection results of high temperature treatment and 4°C control treatment of anti-TIGIT humanized antibody in sequence
  • Figure 13c and Figure 13d are the CE-SDS-NR detection high temperature treatment of anti-TIGIT humanized antibody in sequence , 4°C control treatment results
  • Figure 13e, Figure 13f, and Figure 13g are the results of cIEF detection of anti-TIGIT humanized antibody after high temperature treatment for acid peak changes, main peak changes, and base peak changes
  • Figure 13h, Figure 13i, Figure 13 13j is the results of cIEF detection of anti-TIGIT humanized antibody in turn after 4°C control treatment for acidic peak changes, main peak changes, and base peak changes. The results show that after 28 days of high temperature treatment for anti-TIGIT humanized antibodies, the detection results are stable.
  • the anti-TIGIT humanized antibody has undergone acid, alkali, repeated freeze-thaw, high temperature and other stress tests, and no significant changes have been found in its physical and chemical properties, indicating its good physical and chemical stability. Then we further tested the in vitro binding activity of the anti-TIGIT humanized antibody to prove that the anti-TIGIT humanized antibody can also maintain good activity under stress conditions.
  • Figure 14a, Figure 14b, and Figure 14c are the in vitro binding activity diagrams of hIgG1 subtype anti-TIGIT humanized antibodies 316H1L1, 15H9L2, and 15H10L3 after the stress test. The results show that the anti-TIGIT humanized antibodies have been tested under various stress conditions After that, the good antigen-binding activity was still maintained.

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Abstract

L'invention concerne un anticorps humanisé anti-TIGIT ou un fragment de liaison à l'antigène de celui-ci et une application de celui-ci. L'anticorps ou le fragment de liaison à l'antigène de celui-ci a une bonne activité de liaison et une bonne activité de blocage, a un effet antitumoral significatif, prolonge efficacement la période de survie des souris, réduit significativement la proportion de cellules Treg chez des des lymphocytes infiltrant les tumeurs et des PBMC humaines saines, présente une bonne stabilité et une bonne aptitude à la médication, et peut être largement utilisé pour prévenir ou traiter des maladies immunitaires ou des maladies associées à une tumeur.
PCT/CN2022/120984 2021-09-24 2022-09-23 Anticorps humanisé anti-tigit ou fragment de liaison à l'antigène de celui-ci et son application WO2023046097A1 (fr)

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