Anti-tigit humanized antibodies or antigen binding fragments thereof and uses thereof

Abstract

The application discloses an anti-TIGIT humanized antibody or an antigen binding fragment thereof and application thereof. The antibody or the antigen binding fragment thereof has good binding activity and blocking activity, has a remarkable anti-tumor effect, effectively prolongs the survival period of a mouse, remarkably reduces the proportion of Treg cells in healthy human PBMCs and tumor infiltrating lymphocytes, has good stability and good drug property, and can be widely used for preventing or treating immune diseases or tumor related diseases.

Description

[0001] This application claims priority to Chinese patent application filed on September 24, 2021 (application number: 202111123523.1, invention title: Anti-TIGIT humanized antibody or antigen-binding fragment thereof and its application), the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of biomedicine, and more specifically, to anti-TIGIT humanized antibodies or their antigen-binding fragments and their applications. Background Technology

[0003] TIGIT (T cell Ig and ITIM domain, also known as WUCAM, Vstm3, VSIG9) is an inhibitory receptor shared by T cells and NK cells, containing both the ITT domain (immunoglobulin tyrosine tail motif domain) and the ITIM domain (immunoreceptor tyrosine inhibitory motif domain). It belongs to the type I transmembrane protein family and includes the extracellular segment of IgV and a phosphorylated segment resembling the immunoglobulin tyrosine tail. This gene was discovered in 2008 by a research group at Genentech. This group found the gene by searching the genome for genes that met specific criteria (① expressed on immune cells; ② belonging to the type I transmembrane protein family; ③ containing an extracellular immunoglobulin domain; ④ containing an intracellular immunoregulatory domain). Sequence alignment revealed that this protein belongs to a large PVR protein family, whose members include the activating receptors CD226 and CD96, which compete with TIGIT for ligands, as well as their ligands PVR, etc. In 2012, the crystal structure of the TIGIT molecule was resolved by X-ray diffraction. The study found that TIGIT expressed on immune cells first forms cis-homodimers on the same cell. Then, each of these dimers binds to a PVR molecule through a TIGIT molecule on one side. It was also found that the pre-formed homodimers are necessary for the TIGIT-PVR interaction because if the amino acids at the TIGIT-TIGIT binding interface are mutated beforehand, the TIGIT-PVR interaction will be disrupted.

[0004] TIGIT molecules are relatively conserved, with human TIGIT molecules sharing 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 (poliovirus receptor, CD155) on the surface of cancer cells, inhibiting immune cell activity. The main ligands for TIGIT are CD155 (Necl-5, PVR poliovirus receptor) and CD112 (PVRL2, Nectin-2). CD155 exhibits the highest binding affinity (3.15 nM, Kd), and two TIGIT molecules form a tetrameric "lock-and-key" structure with two CD155 molecules (TIGIT's Try113 and CD155's AX6G, and CD155's Phe128 and TIGIT's AX6G form "key-lock" pairs, respectively). These two ligands are also ligands of CD226 (DNAM-1). CD226 competes with TIGIT to stimulate T cell activity. The interaction between the ligands and TIGIT defeats CD226, suppressing immune activity. Tumor cells upregulate CD155 and CD122 to evade immune-mediated destruction.

[0005] Therefore, TIGIT-specific antagonistic antibodies can inhibit CD155 and CD112-induced T cell responses and enhance anti-tumor immunity. Developing antibodies that can specifically bind to TIGIT is of great significance for the prevention or treatment of immune diseases or tumor-related diseases. Summary of the Invention

[0006] The technical problem to be solved by this invention is to develop anti-human TIGIT antibodies that bind with high affinity and specificity to the TIGIT protein, and to humanize them to reduce the risk of antibody immunogenicity while maintaining the functional activity of the antibody.

[0007] This invention provides a humanized anti-TIGIT antibody or its antigen-binding fragment, wherein the antibody comprises a light chain CDR region and a heavy chain CDR region. The light chain CDR region is composed of LCDR1, LCDR2, and LCDR3, and the heavy chain CDR region is composed of HCDR1, HCDR2, and 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.

[0008] The present invention also provides a nucleic acid that encodes the antibody or an antigen-binding fragment thereof.

[0009] The present invention also provides a vector containing the nucleic acid.

[0010] The present invention also provides cells that carry the nucleic acid, contain the vector, or express the antibody or its antigen-binding fragment.

[0011] The present invention also provides a method for producing the antibody or an antigen-binding fragment thereof, comprising: culturing the cells in a culture medium; and recovering the antibody or an antigen-binding fragment thereof thus produced from the culture medium or from the cultured cells.

[0012] The present invention also provides pharmaceutical compositions comprising the antibody or its antigen-binding fragment, or the nucleic acid, or the carrier or the cell.

[0013] 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 a medicament for the prevention or treatment of immune diseases or tumor-related diseases.

[0014] The present invention also provides any of the aforementioned antibodies or antigen-binding fragments thereof, nucleic acids, carriers, cells or pharmaceutical compositions for use as medicaments; in some embodiments, the medicaments are used to prevent or treat immune diseases or tumor-related diseases.

[0015] The present invention also provides a method for preventing or treating a disease, the method comprising administering to an individual in need a therapeutically effective amount of any of the aforementioned antibodies or their antigen-binding fragments, nucleic acids, vectors, cells or pharmaceutical compositions; in some embodiments, the disease is an immune disease or a tumor-related disease.

[0016] The aforementioned anti-TIGIT humanized antibody has a high affinity for TIGIT and possesses a variety of functional properties. It can be used alone or in combination with other reagents for the prevention or treatment of immune diseases or tumor-related diseases. Attached Figure Description

[0017] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0018] Figure 1This figure shows the results of different subtypes of anti-TIGIT antibodies reducing the average tumor volume of CT26 colon cancer in Balb / c mice.

[0019] Figure 2 This is a graph showing the effects of different subtypes of anti-TIGIT antibodies on the survival of Balb / c mice.

[0020] Figure 3 This is a graph showing the results of reducing the average tumor volume of CT26 colon cancer in mice with TIGIT-5 mouse monoclonal antibody and 3TIGIT-16 mouse monoclonal antibody.

[0021] Figure 4 The figure shows the effect of TIGIT-5 mouse monoclonal antibody and 3TIGIT-16 mouse monoclonal antibody on the survival of hTIGIT knock in mice.

[0022] Figure 5 The figure shows the results of reducing the mean tumor volume of CT26 colon cancer in hTIGIT knock-in mice with the humanized anti-TIGIT antibody of the hIgG1 subtype.

[0023] Figure 6 This is a graph showing the results of reducing the mean tumor volume of CT26 colon cancer in mice with the mIgG2a subtype of anti-TIGIT humanized antibody.

[0024] Figure 7 This is a graph showing the effect of mIgG2a subtype anti-TIGIT humanized antibody on the survival of hTIGIT knock-in mice.

[0025] Figure 8a This is a single-tube staining result image; Figure 8b This is a diagram of the analysis steps; Figure 8c This is a graph showing the ratio of CD4+ to CD8+ T cells; Figure 8d This is a ratio plot of Treg cells; Figure 8e This is an ADCC result diagram using human peripheral blood mononuclear cells as target cells.

[0026] Figure 9 This is a graph showing the test results for more samples.

[0027] Figure 10a This is a graph showing the effect of anti-TIGIT humanized antibody on T cells in mouse tumor cell model tissues; Figure 10b This is a graph showing the effects of the humanized TIGIT antibody on CD3+ T cells and CD4+ T cells in mouse tumor cell model tissues.

[0028] Figure 11aThis is a graph showing the SEC-HPLC detection results of the anti-TIGIT humanized antibody. Figure 11b This is a graph showing the SEC-HPLC detection results of anti-TIGIT humanized antibody after alkaline treatment. Figure 11c This is a graph showing the acid results of CE-SDS-NR detection for anti-TIGIT humanized antibody; Figure 11d This is a graph showing the alkaline treatment results of CE-SDS-NR detection of anti-TIGIT humanized antibody; Figure 11e This is a graph showing the changes in the acid peak after acid treatment in the cIEF detection of anti-TIGIT humanized antibody; Figure 11f This is a graph showing the changes in the main peak of cIEF detection after acid treatment for anti-TIGIT humanized antibody. Figure 11g This is a graph showing the changes in the alkaline peak after acid treatment in the cIEF detection of anti-TIGIT humanized antibody. Figure 11h This is a graph showing the changes in the acidic peak after alkaline treatment in the cIEF detection of anti-TIGIT humanized antibody. Figure 11i This is a graph showing the changes in the main peak of cIEF detection after alkaline treatment with anti-TIGIT humanized antibody. Figure 11j This is a graph showing the changes in the alkaline peak after alkaline treatment in the cIEF detection of anti-TIGIT humanized antibody.

[0029] Figure 12a This is a graph showing the results of repeated freeze-thaw cycles in SEC-HPLC detection of anti-TIGIT humanized antibody; Figure 12b This is a graph showing the results of repeated freeze-thaw cycles in the CE-SDS-NR assay for anti-TIGIT humanized antibody; Figure 12c This is a graph showing the changes in the acid peak after repeated freeze-thaw cycles in the cIEF assay of anti-TIGIT humanized antibody. Figure 12d This is a graph showing the changes in the main peak after repeated freeze-thaw cycles in the cIEF assay of the anti-TIGIT humanized antibody. Figure 12e This is a graph showing the changes in the basic peak after repeated freeze-thaw cycles in the cIEF assay of anti-TIGIT humanized antibody.

[0030] Figure 13a This is a graph showing the results of high-temperature processing for SEC-HPLC detection of anti-TIGIT humanized antibodies; Figure 13b This is a graph showing the SEC-HPLC detection results of anti-TIGIT humanized antibody at 4℃ control treatment. Figure 13c This is a graph showing the high-temperature processing results of CE-SDS-NR detection of anti-TIGIT humanized antibody; Figure 13d This is a graph showing the CE-SDS-NR detection results of anti-TIGIT humanized antibody at 4℃ control treatment. Figure 13e This is a graph showing the changes in the acid peak after high-temperature treatment in the cIEF detection of anti-TIGIT humanized antibody; Figure 13f This is a graph showing the changes in the main peak of cIEF detection after high-temperature treatment of anti-TIGIT humanized antibody; Figure 13gThis is a graph showing the changes in the alkaline peak after high-temperature treatment in the cIEF detection of anti-TIGIT humanized antibody. Figure 13h This is a graph showing the changes in the acid peak after 4℃ control treatment in the cIEF detection of anti-TIGIT humanized antibody; Figure 13i This is a graph showing the changes in the main peak of cIEF detection after 4℃ control treatment with anti-TIGIT humanized antibody. Figure 13j This is a graph showing the changes in the alkaline peak after 4℃ control treatment in the cIEF detection of anti-TIGIT humanized antibody.

[0031] Figure 14a This is an in vitro binding activity diagram of the hIgG1 subtype anti-TIGIT humanized antibody 316H1L1 after a stress test; Figure 14b This is a graph showing the in vitro binding activity of the hIgG1 subtype anti-TIGIT humanized antibody 15H9L2 after a stress test. Figure 14c This is an in vitro binding activity diagram of the hIgG1 subtype anti-TIGIT humanized antibody 15H10L3 after a stress test; where R0750 represents 316H1L1, R0771 represents 15H9L2, and R0774 represents 15H10L3. Detailed Implementation

[0032] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0033] Therefore, this invention is intended to cover such modifications and variations falling within the scope of the appended claims and their equivalents. Other objects, features, and aspects of the invention are disclosed in or will be apparent from the following detailed description. It will be understood by those skilled in the art that this discussion is merely a description of exemplary embodiments and is not intended to limit the broader aspects of the invention.

[0034] This invention relates to a humanized anti-TIGIT antibody or its antigen-binding fragment, wherein the antibody comprises a light chain CDR region and a heavy chain CDR region, the light chain CDR region being composed of LCDR1, LCDR2, and LCDR3, and the heavy chain CDR region being composed of HCDR1, HCDR2, and HCDR3, wherein 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, and 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. In some optional embodiments, the amino acid sequences of LCDR1, LCDR2, and LCDR3 are SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31, respectively; the amino acid sequences of HCDR1, HCDR2, and HCDR3 are SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37, respectively; and the amino acid sequence of the variable region of the antibody heavy chain is as shown in any one of SEQ ID NO:5 to 15. In some optional embodiments, the amino acid sequence of the variable region of the antibody heavy chain is as shown in SEQ ID NO:14. In other optional embodiments, the amino acid sequences of LCDR1, LCDR2, and LCDR3 are SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34, respectively; the amino acid sequences of HCDR1, HCDR2, and HCDR3 are SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, respectively; and the amino acid sequence of the variable region of the antibody heavy chain is as shown in any one of SEQ ID NO:21 to 28. In some optional embodiments, the amino acid sequence of the variable region of the antibody heavy chain is as shown in SEQ ID NO:21.

[0035] One important advantage of this antibody or its antigen-binding fragment is its high in vitro binding activity and species cross-binding activity with TIGIT.

[0036] One important advantage of this antibody or its antigen-binding fragment is that it has the activity of blocking the binding of PVR to TIGIT.

[0037] One important advantage of this antibody or its antigen-binding fragment is its significant anti-tumor activity.

[0038] One important advantage of this antibody or its antigen-binding fragment is its ability to reduce the proportion of Treg cells.

[0039] Because it possesses one or more of the above-mentioned characteristics, the antibody or its antigen-binding fragment can be used as an antibody drug.

[0040] In this invention, the term "antibody or antigen-binding fragment thereof" refers to a protein that binds to a specific antigen, and broadly refers to all proteins and protein fragments containing a complementarity-determining region (CDR). "Antibody" specifically refers to a full-length antibody. The term "full-length antibody" includes both polyclonal and monoclonal antibodies. The term "antigen-binding fragment" is a substance containing part or all of the antibody's CDR, lacking at least some amino acids present in the full-length chain but still capable of specifically binding to an antigen. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. In some embodiments, the antigen-binding fragment has the function of specifically recognizing and binding TIGIT.

[0041] "Humanized antibodies" are antibodies that retain the reactivity of non-human antibodies while exhibiting lower immunogenicity in humans. For example, they can be constructed by retaining the CDR region of a non-human antibody and replacing the rest of the antibody with a human antibody counterpart (i.e., the framework portion of the constant region and the variable region).

[0042] In this invention, the term "specific binding" or similar expressions refer to the binding of an antibody or its antigen-binding fragment to a pre-defined epitope on an antigen. Typically, the antibody or its antigen-binding fragment binds at a rate of approximately less than 10... -6 M, for example, approximately less than 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less affinity (KD) binding. KD refers to the ratio of dissociation rate to binding rate (koff / kon), which can be measured by methods familiar to those skilled in the art.

[0043] In this invention, the term "complementarity-determining region" or "CDR" refers to a highly variable region of the heavy and light chains of an immunoglobulin. There are three heavy chain CDRs and three light chain CDRs. Here, depending on the context, the terms "CDR" and "CDRs" are used to refer to regions containing one or more, or even all, of the major amino acid residues that contribute to the binding affinity of an antibody or its antigen-binding fragment to the antigen or epitope it recognizes.

[0044] In this invention, the complementarity-determining region (CDR) of the heavy chain is represented by HCDR, and the complementarity-determining region of the light chain is represented by LCDR. Commonly used CDR labeling methods in the art include the Kabat numbering scheme, the Chothia and Lesk numbering scheme, and the 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 the variable regions of immunoglobulins. In their compilation of "Sequences of Proteins of Immunological Interest," the amino acid sequences of the light chain (λ, κ) variable regions and antibody heavy chains, as well as the variable regions (α, β, γ, δ) of T cell receptors, are aligned and numbered. Over the past few decades, the accumulation of sequences has led to the creation of the Kabat database, and the Kabat numbering scheme is generally considered the widely adopted standard for numbering antibody residues. This invention uses the Kabat annotation standard to label CDR regions, but CDR regions labeled by other methods are also within the scope of this invention.

[0045] In this invention, the terms "specific recognition," "selective binding," "selective binding," and "specific binding," or similar expressions, refer to the binding of an antibody or its antigen-binding fragment to a predetermined epitope on an antigen. Typically, the antibody or its antigen-binding fragment binds at a rate of approximately less than 10... -6 M, for example, approximately less than 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or lower affinity (K) D (Combined)

[0046] In this invention, the provided antibody or its antigen-binding fragment can specifically recognize TIGIT from various genera, such as humans, mice, and monkeys (e.g., cynomolgus monkeys).

[0047] In some embodiments, the amino acid sequence of the light chain variable region of any of the preceding antibodies is as shown in SEQ ID NO:1-4 or any of SEQ ID NO:16-20. In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO:14, or any of SEQ ID NO:5-13, 15, and the amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO:3, or any of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:4; or the amino acid sequence of the heavy chain variable region of the antibody 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.

[0048] In some embodiments, the amino acid sequences of the light chain variable region and the heavy chain variable region of the antibody are shown in Table 1 below:

[0049] Table 1

[0050]

[0051]

[0052] In some embodiments, the amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO:2 and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:13, or the amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO:3 and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:14, or the amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO:16 and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:21.

[0053] In some embodiments, the antibody comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region sequence is selected from the constant region sequences of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD. In some embodiments, the heavy chain constant region sequence is selected from IgG1.

[0054] In some embodiments, the light chain constant region is a κ or λ chain. In some embodiments, the light chain constant region sequence is selected from κ chains.

[0055] In some embodiments, the species source of the heavy chain constant region and the light chain constant region is selected from any one of cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, geese, or humans. In some embodiments, the species source of the heavy chain constant region and the light chain constant region is humans.

[0056] In some embodiments, 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. In some embodiments, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO:53, and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO:50. In some embodiments, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO:45, and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO:49.

[0057] In some embodiments, the amino acid sequence of the heavy chain constant region is as 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 as shown in SEQ ID NO:53.

[0058] In some embodiments, the antibody described in any of the preceding claims has an amino acid sequence of the heavy chain constant region as shown in SEQ ID NO:53 and an amino acid sequence of the light chain constant region as shown in SEQ ID NO:50. In some embodiments, the antibody described in any of the preceding claims has an amino acid sequence of the heavy chain constant region as shown in SEQ ID NO:45 and an amino acid sequence of the light chain constant region as shown in SEQ ID NO:50. In some embodiments, 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 amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:14, and the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:53 or SEQ ID NO:45. In some embodiments, 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 amino acid sequence of the heavy chain variable region of the antibody 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. In some embodiments, the amino acid sequence of the variable region of the light chain of the antibody is as shown in SEQ ID NO:2, and the amino acid sequence of the constant region of the light chain is as shown in SEQ ID NO:50; and the amino acid sequence of the variable region of the heavy chain of the antibody is as shown in SEQ ID NO:13, and the amino acid sequence of the constant region of the heavy chain is as shown in SEQ ID NO:53 or SEQ ID NO:45. In some embodiments, the amino acid sequence of the heavy chain of the antibody is as shown in SEQ ID NO:51, and the amino acid sequence of the light chain is as shown in SEQ ID NO:52.

[0059] In some embodiments, the antigen-binding fragment is any one of F(ab')2, Fab, scFv, and bispecific antibodies.

[0060] In this invention, the term "F(ab')2" comprises two light chains and two heavy chains containing portions of a constant region between CH1 and CH2 structural domains, so as to form interchain disulfide bonds between the two heavy chains. The F(ab')2 segment thus consists of two Fab' segments held together by disulfide bonds between the two heavy chains.

[0061] In this invention, the term "bispecific antibody" is a multispecific antigen-binding protein or multispecific antibody, and can be produced by a variety of methods, including, but not limited to, hybridoma fusion or Fab' fragment linkage. The two binding sites of a bispecific antigen-binding protein or antibody will bind two different epitopes, which are located on the same or different protein targets.

[0062] The present invention also relates to nucleic acids that encode the antibody or an antigen-binding fragment thereof.

[0063] In this invention, nucleic acids are typically RNA or DNA, and the nucleic acid molecule can be single-stranded or double-stranded, but double-stranded DNA is preferred. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "effectively linked." For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence. DNA is preferred when it is linked into a vector. Furthermore, since antibodies are membrane proteins, nucleic acids typically carry a signal peptide sequence.

[0064] In some embodiments, the nucleic acid includes: a first nucleic acid encoding a heavy chain variable region of the antibody or an antigen-binding fragment thereof, and / or a second nucleic acid encoding a light chain variable region of the antibody or an antigen-binding fragment thereof.

[0065] The present invention also relates to a vector comprising the nucleic acid.

[0066] In this invention, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ 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, and papillomaviruses (such as SV40).

[0067] The present invention also relates to cells that carry the nucleic acid, contain the vector, or express the antibody or its antigen-binding fragment.

[0068] The present invention also relates to a method for producing the antibody or an antigen-binding fragment thereof, comprising: culturing the cells in a culture medium; and recovering the antibody or an antigen-binding fragment thereof thus produced from the culture medium or from the cultured cells.

[0069] The present invention also relates to pharmaceutical compositions comprising the antibody or its antigen-binding fragment, or the nucleic acid, or the carrier or the cell.

[0070] In this invention, the term "pharmaceutical composition" refers to a form in which the biological activity of the active ingredient is permitted and which does not contain any additional ingredients that would have unacceptable toxicity to the subject to which the composition is applied.

[0071] In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipient.

[0072] In this invention, the term "pharmaceuticalally acceptable carrier" may include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and delayed absorption agents, etc., used to extend the shelf life or efficacy of antibodies.

[0073] The present invention also relates to the use of the antibody or its antigen-binding fragment, the nucleic acid, the carrier, the cell, or the pharmaceutical composition in the preparation of a medicament for the prevention or treatment of immune diseases or tumor-related diseases.

[0074] The present invention also provides any of the aforementioned antibodies or antigen-binding fragments thereof, nucleic acids, carriers, cells or pharmaceutical compositions for use as medicaments; in some embodiments, the medicaments are used to prevent or treat immune diseases or tumor-related diseases.

[0075] The present invention also provides a method for preventing or treating a disease, the method comprising administering to an individual in need a therapeutically effective amount of any of the aforementioned antibodies or their antigen-binding fragments, nucleic acids, vectors, cells or pharmaceutical compositions; in some technical solutions, the disease is an immune disease or a tumor-related disease;

[0076] In some embodiments, the disease described in any of the preceding claims is a disease related to human TIGIT, more preferably a T-cell dysfunction disease.

[0077] In some embodiments, the present invention also provides reagents for detecting or measuring human TIGIT, said reagents comprising the anti-TIGIT antibody or its antigen-binding fragment as described in any of the preceding claims.

[0078] The present invention has the following beneficial effects:

[0079] The humanized antibody against human TIGIT of this invention exhibits good binding and blocking activity, significant anti-tumor effects, effectively prolongs the survival of mice, significantly reduces the proportion of Treg cells in healthy human PBMCs and tumor-infiltrating lymphocytes, has good stability and drug-like properties, and can be widely used for the prevention or treatment of immune diseases or tumor-related diseases.

[0080] The embodiments of the present invention will now be described in detail with reference to examples.

[0081] Example 1: Humanization of Anti-human TIGIT Antibody

[0082] The TIGIT-5 mouse monoclonal antibody (VL, VH sequences as shown in SEQ ID NO:41, SEQ ID NO:42) and 3TIGIT-16 mouse monoclonal antibody (VL, VH sequences as shown in SEQ ID NO:43, SEQ ID NO:44) in Chinese patent application No. 202011324100.1 exhibit good binding affinity to CHO-hTIGIT, healthy human PBMCs, and cynoTIGIT. They also block the binding of CHO-hTIGIT, CHO-hPVR, and hPVR-hFc, as well as the binding of tumor cells and CHO-CD112 to TIGIT. The binding kinetics of the human TIGIT recombinant protein are all in the pM range, and they demonstrate good ability to activate NK92-hTIGIT and kill U2-OS. Therefore, this embodiment humanizes the above-mentioned TIGIT-5 mouse monoclonal antibody and 3TIGIT-16 mouse monoclonal antibody using the following specific method:

[0083] Humanization was performed using complementarity-determining region (CDR) grafting. First, antibody homology models were created for the Fv regions of TIGIT-5 and 3TIGIT-16 mouse monoclonal antibodies using MOE (Molecular Operating Environment) software. Based on the model structure, key amino acid residues affecting the conformational stability of the antigen-binding region were analyzed. Subsequently, human immunoglobulin databases were searched, and human IGVH and IGVK sequences with high homology to TIGIT-5 and 3TIGIT-16 in human germline antibody libraries were used as templates for humanization. TIGIT-5 and 3TIGIT-16 were compared with matching human IGVH and IGVK sequences, focusing on analyzing sites on the original mouse monoclonal antibodies where key amino acid residues affecting the conformational stability of the antigen-binding region were inconsistent with the human IGVH and IGVK sequences. The feasibility of humanized replacement was observed in the model. Depending on the degree of humanization replacement, multiple humanized sequences could be generated simultaneously from a single parent sequence.

[0084] For the TIGIT-5 mouse monoclonal antibody, the humanized VL sequences (VL1-VL4) are shown in Table 2-1 below, and the humanized VH sequences (VH1-VH11) are shown in Table 2-2 below.

[0085] Table 2-1

[0086]

[0087]

[0088] Table 2-2

[0089]

[0090]

[0091] For the 3TIGIT-16 mouse monoclonal antibody, the humanized VL sequences (VL5-VL9) are shown in Table 3 below, and the humanized VH sequences (VH12-VH19) are shown in Table 4 below.

[0092] Table 3

[0093]

[0094] Table 4

[0095]

[0096]

[0097] Combining different humanized VL and VH pairs can produce different humanized antibody molecules. The main consideration in different combinations is balancing the degree of humanization with the maintenance of antibody activity after humanization. Simply put, pairing the VL and VH with the highest degree of humanization yields the antibody molecule with the highest degree of humanization. This implies potentially lower immunogenicity, but a high degree of humanization may lead to reduced activity of the modified molecule (key amino acid residues affecting the activity of the parent antibody are replaced with humanized amino acids, causing changes in the antigen-binding region of the modified antibody). Conversely, pairing the VL and VH with the lowest degree of humanization yields functional activity more consistent with the parent antibody (because most key amino acid residues are retained), but the degree of humanization is also lower, potentially leading to a higher risk of immunogenicity. This is a balancing act; the preparation of humanized antibodies requires expression of various VL and VH combinations, followed by validation through in vivo and in vitro functional experiments, ultimately screening for suitable humanized antibody molecules.

[0098] The amino acid sequences of the humanized anti-TIGIT humanized antibody obtained after humanization of TIGIT-5 mouse monoclonal antibody are shown in Table 5 below:

[0099] Table 5. Amino acid sequences of anti-TIGIT humanized antibodies obtained after humanization of TIGIT-5 mouse monoclonal antibody.

[0100]

[0101]

[0102] The amino acid sequences of the anti-TIGIT humanized antibodies obtained after humanization of the 3TIGIT-16 mouse monoclonal antibody are shown in Table 6 below:

[0103] Table 6. Amino acid sequences of anti-TIGIT humanized antibodies obtained after humanization of 3TIGIT-16 mouse monoclonal antibody.

[0104]

[0105] The amino-terminal sequences of the constant regions used to construct complete antibodies from the variable regions of the above anti-TIGIT humanized antibodies are shown in Table 7 below:

[0106] Table 7. Antibody constant region sequence

[0107]

[0108]

[0109] The humanized antibodies with specific numbers listed in Tables 5 and 6 above (e.g., R0774 (also identified as 15H10L3)) have a heavy chain constant region containing hIgG1 with the K214R / D356E / L358M mutation (amino acid sequence as shown in SEQ ID NO:53) and a light chain constant region containing Hu-CL-κ (amino acid sequence as shown in SEQ ID NO:50). For example, the full-length amino acid sequence of the humanized antibody R0774 (also identified as 15H10L3) is as follows:

[0110] R0774 heavy chain amino acid sequence (SEQ ID NO:51):

[0111] QVQLVQSGAEVKKPGSSVKVSCKASGYSFTSYWMNWVRQAPGQGLEWIGMIRPSDSETRLNQMFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAGIHDYGHGAYWGQGT LVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0112] R0774 light chain amino acid sequence (SEQ ID NO:52):

[0113] DIQMTQSPSSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKSPKLLVYAASHLPDGVPSRFSGSGSGTDYSLTISSLQPEDFATYYCQHFWGTPRTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0114] Example 2: Preparation of anti-TIGIT humanized antibody

[0115] Antibodies were prepared using conventional methods, and the expression supernatant was purified by ProA affinity chromatography. The procedure is as follows:

[0116] Using an AKTA Avant 150 chromatography instrument, equilibrate the column (e.g., MabSelect SuRe LX, GE) with at least 5 CV equilibration buffer (10 mM PBS). Load the sample onto the column, allowing the target protein to adsorb onto the column while other impurities pass through and separate. After loading, wash the column again with at least 5 CV equilibration buffer (10 mM PBS), then elute the target protein with elution buffer (20 mM NaAc, pH 3.4). Add neutralization buffer (1 M Tris, pH 8.0) to the collection tube beforehand; the volume of neutralization buffer added depends on the estimated concentration of the eluted sample, generally 10% of the elution volume.

[0117] Sample concentration was determined using the Biotek-Epoch-Take-3 assay. Antibody concentration was detected using the A280 method, with an extinction coefficient EC = 1.37 and a path length of 0.05 mm (slight differences in path length may occur between wells in the Take-3 plate, which is automatically corrected). The absorbance of the sample was measured using the instrument, and the concentration of the anti-TIGIT humanized antibody was calculated according to the Lambert-Beer law. If the sample concentration was too low, ultrafiltration concentration was required using an ultrafiltration concentrator (…). Using the Ultra-15 Centrifugal Filter Devices (30kD), concentrate the sample to >0.5mg / ml according to the general operating procedure provided in the instruction manual; collect the concentrated sample, sterilize it with a 0.22um sterile syringe filter (Cobbat, PES, 0.22um, 13mm diameter), and then aliquot and freeze for later use.

[0118] The results of the humanization degree and purity of the anti-TIGIT humanized antibody are shown in Tables 8 and 9. The results show that the humanization degree, titer and purity of the anti-TIGIT humanized antibody prepared in this example are very good.

[0119] Table 8

[0120]

[0121] Note: In Table 8, the leftmost value in the purity column is the percentage of aggregates, the rightmost value is the percentage of fragments, and the middle value is the percentage of antibody monomers. "-" indicates a value of 0%. For example, "1.85% / 98.15% / -" indicates that the percentage of aggregates in the antibody solution is 1.85%, the percentage of antibody monomer purity is 98.15%, and the percentage of fragments is 0%.

[0122] Table 9

[0123]

[0124]

[0125] Note: In Table 9, the leftmost value in the purity column is the percentage of aggregates, the rightmost value is the percentage of fragments, and the middle value is the percentage of antibody monomers. For example, "4.54% / 94.62% / 0.84%" means that in the antibody solution, the percentage of aggregates is 4.54%, the percentage of antibody monomer purity is 94.62%, and the percentage of fragments is 0.84%.

[0126] Example 3: Validation of anti-TIGIT humanized antibody

[0127] The in vitro binding activity, species cross-binding activity, and blocking activity of the anti-TIGIT humanized antibodies obtained above were detected by the following methods:

[0128] (1) In vitro binding activity assay:

[0129] To verify the binding ability of the anti-TIGIT humanized antibody to CHO-hTIGIT, the recombinant CHO engineered cells (CHO-hTIGIT cells) expressing full-length human TIGIT were counted, centrifuged at 300g for 5 min, resuspended in FCM buffer, and the cell density was adjusted to 4E+06 cells / mL. The test samples (including the positive control group, TIGIT-5 mouse monoclonal antibody (R0435), 3TIGIT-16 mouse monoclonal antibody (R0518), and the anti-TIGIT humanized antibody; where R0300-CH2a is derived from tiragolumab in patent WO2017053748(A2), and R0300-CH2a has its Fc region replaced with the Fc region of mIgG1) were diluted to 20 μg / mL using FCM buffer, and then serially diluted 3-fold to 12 concentration points. According to the experimental design, add 50 μL of antibody and 50 μL of cells to each well of a 96-well V-plate (2E+05 cells / well per cell line) and incubate on ice in the dark for 30 min. Centrifuge (300 g / 5 min), discard the supernatant, wash once with 200 μL of FCM buffer, centrifuge again (300 g / 5 min), and discard the supernatant. Add secondary antibody (PE Goat anti-mouse IgG, 1:500 dilution) to the 96-well V-plate (100 μL / well) and incubate on ice in the dark for 30 min. Centrifuge (300 g / 5 min), discard the supernatant, wash once with 200 μL of FCM buffer, centrifuge again (300 g / 5 min), and discard the supernatant. Resuspend in 100 μL of 1×PBS and analyze.

[0130] (2) Species cross-binding activity detection:

[0131] To verify the binding ability of the anti-TIGIT humanized antibody to monkey TIGIT, the following experiment was conducted: CHO engineered cells (CHO-cynoTIGIT cells) expressing full-length cynoTIGIT were counted, centrifuged at 300g for 5 min, resuspended in FCM buffer, and the cell density was adjusted to 4E+06 cells / mL. The test samples (including positive control groups R0300-CH2a, R0435, R0518, and the anti-TIGIT humanized antibody) were diluted to 20 μg / mL using FCM buffer, and then serially diluted 3-fold to 12 concentration points using FCM buffer. According to the experimental design, 50 μL of antibody and 50 μL of cells were added to each well of a 96-well V-plate (2E+05 cells / well), and incubated on ice in the dark for 30 min. Centrifuge (300g / 5min), discard supernatant, wash once with 200μL FCM buffer, centrifuge (300g / 5min), discard supernatant. Add secondary antibody (PE Goat anti-mouse IgG, 1:500 dilution) to 96-well V-plates (100μL / well), incubate on ice in the dark for 30min. Centrifuge (300g / 5min), discard supernatant, wash once with 200μL FCM buffer, centrifuge (300g / 5min), discard supernatant. Resuspend in 100μL 1×PBS, and analyze.

[0132] (3) Detection of blocking activity:

[0133] To verify the ability of the anti-TIGIT humanized antibody to block the binding of CHO-hTIGIT to hPVR-hFc, the following experiments were conducted: Recombinant CHO-hTIGIT engineered cells expressing full-length human TIGIT were counted, centrifuged at 300g for 5 min, resuspended in FCM buffer, and the cell density was adjusted to 4E+06 cells / mL. The test samples (including positive control groups R0300-CH2a, R0435, R0518, and the anti-TIGIT humanized antibody) were diluted to 20 μg / mL using FCM buffer, and then serially diluted 3-fold to 12 concentration points using FCM buffer. The ligand protein hPVR-hFc was diluted to 20 μg / mL using FCM buffer. According to the experimental design, 50 μL of antibody and 50 μL of cells were added to each well of a 96-well V-plate (2E+05 cells / well), and incubated on ice in the dark for 30 min. Add ligand protein to 96-well V-type plates (50 μL / well) and incubate on ice in the dark for 30 min. Centrifuge (300 g / 5 min), discard the supernatant, wash once with 200 μL FCM buffer, centrifuge (300 g / 5 min), and discard the supernatant. Add secondary antibody (PE anti-human IgG Fc, 1:500 dilution) to 96-well V-type plates (100 μL / well) and incubate on ice in the dark for 30 min. Centrifuge (300 g / 5 min), discard the supernatant, wash once with 200 μL FCM buffer, centrifuge (300 g / 5 min), and discard the supernatant. Resuspend in 100 μL 1×PBS and analyze.

[0134] The results of in vitro binding activity, species cross-binding activity and blocking activity of anti-TIGIT humanized antibodies are shown in Tables 10 and 11. The results show that most anti-TIGIT humanized antibodies can maintain similar in vitro binding activity, species cross-binding activity and blocking activity as the parent antibody.

[0135] Table 10

[0136]

[0137] Note: N / A: The activity is very weak and cannot be calculated.

[0138] Table 11

[0139]

[0140]

[0141] Note: N / A: The activity is very weak and cannot be calculated.

[0142] Example 4: In vivo functional evaluation of anti-TIGIT humanized antibody

[0143] 1. Evaluation of the in vivo antitumor efficacy of different subtypes of anti-TIGIT antibodies against colon cancer in CT26 mice.

[0144] (1) Experimental objective: To compare the anti-tumor effects of different subtypes of anti-TIGIT antibodies R0226 (mIgG2a) [R0226 is derived from Oncomed, OMP-313R12, WO2016191643(A2)], R0564 (mIgG1) [R0564 is the mIgG1 subtype of R0226], and R0565 (mIgG2a DLE) [R0565 is the mIgG2a-DLE subtype of R0226] in a CT26 mouse subcutaneous xenograft model of colon cancer. An isotype control group R0513 (Isotype-mIgG2a) was also set up.

[0145] (2) Experimental materials: Balb / c mice, female, 6-8 weeks old (Source: Beijing Vital River Laboratory Technology Co., Ltd., Certificate No.: 1100112011045713); CT26 cells (Shanghai Cell Bank, Catalog No. TCM37); 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).

[0146] (3) Instruments and equipment: Electronic balance (Shanghai Shunyu Hengping Scientific Instruments Co., Ltd., JA12002), Vernier caliper (Shanghai Meinate Industrial Co., Ltd., MNT-150T), Microscope (Chongqing Aote Optical Instrument Co., Ltd., BDS200), Medical centrifuge (Hunan Xiangyi Laboratory Development Co., Ltd., L530R), Digital display constant temperature water bath (Pruis Machinery Co., Ltd., HH-S), Carbon dioxide incubator (Panasonic Health Medical Devices Co., Ltd., MCO-18AC), Biosafety cabinet (Guangzhou Qianjiang Experimental Technology Co., Ltd., ACZ-451), Cell technology instrument (Shanghai Ruiyu Biotechnology, IC1000).

[0147] (4) Experimental methods

[0148] 1) Cell culture: Mouse colon cancer cells (CT26) were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (1:1).

[0149] 2) Inoculation: Collect CT26 cells in the logarithmic growth phase, wash twice with pre-cooled DPBS, and adjust the cell concentration to 1×10⁻⁶. 6 / mL. Female Balb / c mice were subcutaneously inoculated with CT26 cells at a volume of 0.1 mL / mouse, i.e., 1 × 10⁹ / mL. 5 / mouse.

[0150] 3) Administration: The day of inoculation is recorded as day 0 (D0). When the average tumor volume reaches 60-80 mm... 3 Mice can be randomly divided into groups according to tumor volume. On day 13 (D13), mice are randomly divided into 4 groups of 8 mice each, and drug administration begins (the dosage, method and frequency of drug administration in the CT26 tumor model are shown in Table 12).

[0151] Table 12

[0152]

[0153] Note: Dosage volume is adjusted according to animal body weight (10L / g); if the animal's body weight decreases by more than 15% during the dosing period, the dosing regimen will be adjusted. Additionally, in the table, ip indicates intraperitoneal injection, and Q3D*4 indicates dosing once every 3 days for a total of 4 doses.

[0154] 4) Observation Records:

[0155] Tumor volume was measured and mouse weight was recorded starting on day 13. Weight was then recorded twice weekly, and the major and minor diameters of the tumor were measured using calipers. The formula was: (1 / 2) × major diameter × (minor diameter). 2 Tumor volume was calculated. Each mouse reached the experimental endpoint (a 20% decrease in body weight or a tumor volume exceeding 2000 mm²). 3 (To reach the merciful endpoint), mice were euthanized by CO2 asphyxiation, and survival curves were recorded.

[0156] 5) Data calculation, statistics and analysis:

[0157] Independent samples t-test can be used to compare two groups. One-way aNOVA should be used for comparisons of three or more groups. If the F-value shows a significant difference, a post-hoc analysis of multiple groups can be performed. Data were processed using Prism GraphPad, and p < 0.05 was considered statistically significant. Tumor volume V = 0.5a × b 2 a and b are the long and short diameters of the tumor, respectively. Tumor growth inhibition TGI (%) = [1 - (Ti - T0) / (Vi - V0)] × 100, where 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 start of treatment, Vi is the average tumor volume of the solvent control group on day i, and V0 is the average tumor volume of the solvent control group at the start of treatment.

[0158] (5) Experimental Results

[0159] The results of different subtypes of anti-TIGIT antibodies reducing the mean tumor volume of CT26 colon cancer in Balb / c mice are shown in Table 13 and 2014. Figure 1 As shown, the effects of different subtypes of anti-TIGIT antibodies on the survival of Balb / c mice are as follows: Figure 2 As shown, the results indicate that the anti-TIGIT antibodies mIgG2a subtype R0226 (TGI = 88.5%) and mIgG2a DLE subtype R0565 (TGI = 91.7%) have significantly better survival and antitumor activity than mIgG1 subtype R0564 (TGI = 44.6%).

[0160] Table 13

[0161]

[0162] Note: In Table 13, Isotype represents the isotype control group R0513 (Isotype-mIgG2a), R0226 represents R0226 (mIgG2a), R0564 represents R0564 (mIgG1), and R0565 represents R0565 (mIgG2a DLE).

[0163] 2. Evaluation of the in vivo antitumor efficacy of TIGIT-5 mouse monoclonal antibody and 3TIGIT-16 mouse monoclonal antibody against colon cancer in CT26 mice.

[0164] (1) Experimental objective: To study the efficacy of TIGIT-5 mouse monoclonal antibody (R0435) and 3TIGIT-16 mouse monoclonal antibody (R0518) of mIgG2a subtype in a CT26 mouse subcutaneous xenograft model of colon cancer, and to set up an isotype control group (R0513) and a positive control group (R0300-CH2a).

[0165] (2) Experimental materials: female hTIGIT Knock in mice, 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).

[0166] (3) Instruments and equipment: Electronic balance (Shanghai Shunyu Hengping Scientific Instruments Co., Ltd., JA12002), Vernier caliper (Shanghai Meinate Industrial Co., Ltd., MNT-150T), Microscope (Chongqing Aote Optical Instrument Co., Ltd., BDS200), Medical centrifuge (Hunan Xiangyi Laboratory Development Co., Ltd., L530R), Digital display constant temperature water bath (Pruis Machinery Co., Ltd., HH-S), Carbon dioxide incubator (Panasonic Health Medical Devices Co., Ltd., MCO-18AC), Biosafety cabinet (Guangzhou Qianjiang Experimental Technology Co., Ltd., ACZ-451), Cell counter (Shanghai Ruiyu Biotechnology, IC1000).

[0167] (4) Experimental methods

[0168] 1) Cell culture: Mouse colon cancer cells (CT26) were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (1:1).

[0169] 2) Inoculation: Collect CT26 cells in the logarithmic growth phase, wash twice with pre-cooled DPBS, and adjust the cell concentration to 2×10⁻⁶. 6 / mL. Female hTIGIT mice were subcutaneously inoculated with CT26 cells at a volume of 0.1 mL / mouse, i.e., 2 × 10⁹ / mL. 5 / mouse.

[0170] 3) Administration: The day of inoculation is recorded as day 0 (D0). When the average tumor volume reaches 60-80 mm... 3 Mice can be randomly divided into groups according to tumor volume. On day 8 (D8), mice are randomly divided into 4 groups of 9 mice each, and drug administration begins (the dosage, method and frequency of drug administration in the CT26 tumor model are shown in Table 14).

[0171] Table 14

[0172] Group Dosage (mg / kg) Dosage volume (μL / g) route of administration Dosage frequency Isotype-m 10 10 IP Q3D*4 R0300-CH2a 10 10 IP Q3D*4 R0435 10 10 IP Q3D*4 R0518 10 10 IP Q3D*4

[0173] Note: In Table 14, the dosage 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 dosing regimen will be adjusted. Isotype-m represents the isotype control group (R0513). In addition, in the table, ip indicates intraperitoneal injection, and Q3D*4 indicates administration once every 3 days for a total of 4 administrations.

[0174] 4) Observation Records:

[0175] Tumor volume was measured and mouse weight was recorded starting on day 8. Weight was then recorded twice weekly, and the major and minor diameters of the tumor were measured using calipers. The formula was: (1 / 2) × major diameter × (minor diameter). 2Tumor volume was calculated. Each mouse reached the experimental endpoint (a 15% decrease in body weight or a tumor volume exceeding 2000 mm²). 3 (To reach the merciful endpoint), mice were euthanized by CO2 asphyxiation, and survival curves were recorded.

[0176] 5) Data calculation, statistics and analysis:

[0177] Independent samples t-test can be used to compare two groups. One-way aNOVA should be used for comparisons of three or more groups. If the F-value shows a significant difference, a post-hoc analysis of multiple groups can be performed. Data were processed using Prism GraphPad, and p < 0.05 was considered statistically significant. Tumor volume V = 0.5a × b 2 a and b are the long and short diameters of the tumor, respectively. Tumor growth inhibition TGI (%) = [1 - (Ti - T0) / (Vi - V0)] × 100, where 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 start of treatment, Vi is the average tumor volume of the solvent control group on day i, and V0 is the average tumor volume of the solvent control group at the start of treatment.

[0178] (5) Experimental Results

[0179] The results of TIGIT-5 mouse monoclonal antibody and 3TIGIT-16 mouse monoclonal antibody reducing the mean tumor volume of CT26 colon cancer in hTIGIT knock-in mice are shown in Table 15. Figure 3 As shown, the effects of TIGIT-5 mouse monoclonal antibody and 3TIGIT-16 mouse monoclonal antibody on the survival of hTIGIT knock-in mice are as follows: Figure 4 As shown, the results indicated that, compared with the isotype control group, 3TIGIT-16 mouse monoclonal antibody (R0518) had a significant inhibitory effect on tumor growth in the hTIGIT knock in mouse CT26 model (TGI = 93.8%), and R0518 significantly prolonged the survival of mice; the antitumor effect of TIGIT-5 mouse monoclonal antibody (R0435) was comparable to that of the positive control group R0300-CH2a (TGI = 76.5% and 86.7%, respectively).

[0180] Table 15

[0181]

[0182] Note: In Table 15, Isotype represents the isotype control group (R0513).

[0183] 3. Evaluation of the in vivo antitumor efficacy of anti-TIGIT humanized antibody (hIgG1 subtype) against colon cancer in CT26 mice.

[0184] (1) Experimental objective: To study the efficacy of anti-TIGIT humanized antibodies (15H9L2, 15H10L3, 316H1L1) of hIgG1 subcutaneous xenografts in CT26 mice with colon cancer. At the same time, a negative control group (R0536) and a positive control group (R0300-hIgG1, which is a Tiragolumab mutant with K214R mutation in the heavy chain) of hIgG1 were set up.

[0185] (2) Experimental materials: female hTIGIT Knock in mice, 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).

[0186] (3) Instruments and equipment: Electronic balance (Shanghai Shunyu Hengping Scientific Instruments Co., Ltd., JA12002), Vernier caliper (Shanghai Meinate Industrial Co., Ltd., MNT-150T), Microscope (Chongqing Aote Optical Instrument Co., Ltd., BDS200), Medical centrifuge (Hunan Xiangyi Laboratory Development Co., Ltd., L530R), Digital display constant temperature water bath (Pruis Machinery Co., Ltd., HH-S), Carbon dioxide incubator (Panasonic Health Medical Devices Co., Ltd., MCO-18AC), Biosafety cabinet (Guangzhou Qianjiang Experimental Technology Co., Ltd., ACZ-451), Cell technology instrument (Shanghai Ruiyu Biotechnology, IC1000).

[0187] (4) Experimental methods

[0188] 1) Cell culture: Mouse colon cancer cells (CT26) were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (1:1).

[0189] 2) Inoculation: Collect CT26 cells in the logarithmic growth phase, wash twice with pre-cooled DPBS, and adjust the cell concentration to 2×10⁻⁶. 6 / mL. Female hTIGIT mice were subcutaneously inoculated with CT26 cells at a volume of 0.1 mL / mouse, i.e., 2 × 10⁹ / mL. 5 / mouse.

[0190] 3) Administration: The day of inoculation is recorded as day 0 (D0) and day 11 (D11). Mice are randomly divided into 5 groups of 8 mice each according to tumor volume and administration begins (administration regimen is shown in Table 16).

[0191] Table 16

[0192] Group Dosage (mg / kg) Dosage volume (μL / g) route of administration Dosage frequency Isotype-hIgG1 10 10 IP Q3D*4 R0300-hIgG1 10 10 IP Q3D*4 15H9L2-hIgG1 10 10 IP Q3D*4 15H10L3-hIgG1 10 10 IP Q3D*4 316H1L1-hIgG1 10 10 IP Q3D*4

[0193] Note: In Table 16, the dosage 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 dosing regimen will be adjusted. Isotype-hIgG1 represents the isotype control group (R0536). In addition, in the table, ip indicates intraperitoneal injection, and Q3D*4 indicates administration once every 3 days for a total of 4 administrations.

[0194] 4) Observation Records:

[0195] Tumor volume was measured and mouse weight was recorded starting on day 11. Weight was then recorded twice weekly, and the major and minor diameters of the tumor were measured using calipers. The formula was: (1 / 2) × major diameter × (minor diameter). 2 Calculate the tumor volume.

[0196] 5) Data calculation, statistics and analysis:

[0197] Independent samples t-test can be used to compare two groups. One-way aNOVA should be used for comparisons of three or more groups. If the F-value shows a significant difference, a post-hoc analysis of multiple groups can be performed. Data were processed using Prism GraphPad, and p < 0.05 was considered statistically significant. Tumor volume V = 0.5a × b 2 a and b are the long and short diameters of the tumor, respectively. Tumor growth inhibition TGI (%) = [1 - (Ti - T0) / (Vi - V0)] × 100, where 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 start of treatment, Vi is the average tumor volume of the solvent control group on day i, and V0 is the average tumor volume of the solvent control group at the start of treatment.

[0198] (5) Experimental Results

[0199] Humanized anti-TIGIT antibodies of the hIgG1 subtype reduced the mean tumor volume of CT26 colon cancer in hTIGIT knock-in mice. Results are shown in Table 17. Figure 5As shown, the results indicated that, compared with the isotype control group, the humanized anti-TIGIT antibodies 15H9L2 and 15H10L3 of the hIgG1 subtype had significant inhibitory effects on tumor growth in the hTIGIT knock-in mouse CT26 model, with TGIs of 56.38% and 62.83%, respectively; the anti-tumor effect of 316H1L1 was comparable to that of the positive control group R0300, with TGIs of 36.26% and 34.79%, respectively.

[0200] Table 17

[0201]

[0202] Note: In Table 17, Isotype represents the isotype control group (R0536), R0300 represents the positive control R0300-hIgG1, 15H9L2 represents 15H9L2-hIgG1, 15H10L3 represents 15H10L3-hIgG1, and 316H1L1 represents 316H1L1-hIgG1.

[0203] 4. Evaluation of the in vivo antitumor efficacy of anti-TIGIT humanized antibody (mIgG2a subtype) against colon cancer in CT26 mice.

[0204] (1) Experimental objective: To study the efficacy of anti-TIGIT humanized antibodies (15H9L2, 15H10L3, 316H1L1) of mIgG2a subcutaneous xenografts of colon cancer in CT26 mice. At the same time, a negative control group (R0513) and a positive control group (R0300-mIgG2a, which is a Tiragolumab mutant in which the heavy chain constant region is replaced by mIgG2a) were set up.

[0205] (2) Experimental materials: female hTIGIT Knock in mice, 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).

[0206] (3) Instruments and equipment: Electronic balance (Shanghai Shunyu Hengping Scientific Instruments Co., Ltd., JA12002), Vernier caliper (Shanghai Meinate Industrial Co., Ltd., MNT-150T), Microscope (Chongqing Aote Optical Instrument Co., Ltd., BDS200), Medical centrifuge (Hunan Xiangyi Laboratory Development Co., Ltd., L530R), Digital display constant temperature water bath (Pruis Machinery Co., Ltd., HH-S), Carbon dioxide incubator (Panasonic Health Medical Devices Co., Ltd., MCO-18AC), Biosafety cabinet (Guangzhou Qianjiang Experimental Technology Co., Ltd., ACZ-451), Cell counter (Shanghai Ruiyu Biotechnology, IC1000).

[0207] (4) Experimental methods

[0208] 1) Cell culture: Mouse colon cancer cells (CT26) were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (1:1).

[0209] 2) Inoculation: Collect CT26 cells in the logarithmic growth phase, wash twice with pre-cooled DPBS, and adjust the cell concentration to 2×10⁻⁶. 6 / mL. Female hTIGIT mice were subcutaneously inoculated with CT26 cells at a volume of 0.1 mL / mouse, i.e., 2 × 10⁹ / mL. 5 / mouse.

[0210] 3) Administration: The day of vaccination is recorded as day 0 (D0) and day 8 (D8). Mice are randomly divided into 5 groups of 8 mice each according to tumor volume and administration begins (administration regimen is shown in Table 18).

[0211] Table 18

[0212] Group Dosage (mg / kg) Dosage volume (μL / g) route of administration Dosage frequency Isotype-mIgG2a 10 10 IP Q3D*4 R0300-mIgG2a 10 10 IP Q3D*4 15H9L2-mIgG2a 10 10 IP Q3D*4 15H10L3-mIgG2a 10 10 IP Q3D*4 316H1L1-mIgG2a 110 10 IP Q3D*4

[0213] Note: In Table 18, the dosage 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 dosing regimen will be adjusted. Isotype represents the isotype control group (R0513). In addition, in the table, ip indicates intraperitoneal injection, and Q3D*4 indicates administration once every 3 days for a total of 4 administrations.

[0214] 4) Observation Records:

[0215] Tumor volume was measured and mouse weight was recorded starting on day 8. Weight was then recorded twice weekly, and the major and minor diameters of the tumor were measured using calipers. The formula was: (1 / 2) × major diameter × (minor diameter). 2 Tumor volume was calculated. Each mouse reached the experimental endpoint (a 15% decrease in body weight or a tumor volume exceeding 2000 mm²). 3 (To reach the merciful endpoint), mice were euthanized by CO2 asphyxiation, and survival curves were recorded.

[0216] 5) Data calculation, statistics and analysis:

[0217] Independent samples t-test can be used to compare two groups. One-way aNOVA should be used for comparisons of three or more groups. If the F-value shows a significant difference, a post-hoc analysis of multiple groups can be performed. Data were processed using Prism GraphPad, and p < 0.05 was considered statistically significant. Tumor volume V = 0.5a × b 2 a and b are the long and short diameters of the tumor, respectively. Tumor growth inhibition TGI (%) = [1 - (Ti - T0) / (Vi - V0)] × 100, where 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 start of treatment, Vi is the average tumor volume of the solvent control group on day i, and V0 is the average tumor volume of the solvent control group at the start of treatment.

[0218] The mIgG2a subtype of anti-TIGIT humanized antibody reduced the mean tumor volume of CT26 colon cancer in hTIGIT knock-in mice. Results are shown in Table 19. Figure 6 As shown, the effect of mIgG2a subtype anti-TIGIT humanized antibody on the survival of hTIGIT knock-in mice is as follows: Figure 7 As shown, the results indicated that, compared with the isotype control group, the mIgG2a subtype anti-TIGIT humanized antibody 15H10L3 had a significant inhibitory effect on tumor growth in the hTIGIT knock in mouse CT26 model, with a TGI of 83.95%; the anti-tumor effect of 316H1L1 was comparable to that of the positive control group R0300, with TGIs of 76.89% and 75.68%, respectively.

[0219] Table 19

[0220]

[0221] Note: In Table 19, Isotype represents the isotype control group (R0513); R0300 represents the positive control R0300-mIgG2a; 15H9L2 represents 15H9L2-mIgG2a; 15H10L3 represents 15H10L3-mIgG2a; and 316H1L1 represents 316H1L1-mIgG2a.

[0222] Example 5: Anti-TIGIT humanized antibody knocks out Treg cell function

[0223] 1. Effects of anti-TIGIT humanized antibody on T cells in PBMCs of healthy individuals

[0224] Using healthy human PBMCs as target cells, this study investigated the effects of humanized anti-TIGIT antibodies of hIgG1 subtypes (15H9L2, 15H10L3, 316H1L1) on CD8+ T cells and Treg cells in healthy human PBMCs. A positive control group (R0300-hIgG1, a Tiragolumab mutant with a K214R mutation in the heavy chain) was also included. The specific methods are as follows:

[0225] PBMCs were isolated from fresh blood and adjusted to a density of 5E5 cells / mL using 10% FBS + x-vivo medium, then incubated overnight. PBMC cell counts: donor-167 8.44E+05 cells / mL, 81.15%, 20mL of cells were collected; donor-23 45.53E+05 cells / mL, 78.38%, 20mL of cells were collected. NK92-CD16-5 cell count: P11 2.21E+05 cells / mL, 96.05%, 33 mL of cells were collected; after centrifugation at 400g for 5 min, the supernatant was discarded, and the cells were washed once with 10% FBS + x-vivo medium (containing 1000 IU / mL IL-2); resuspended in 10% FBS + x-vivo medium (containing 1000 IU / mL IL-2), and the cells were counted (PBMC-donor167: 2.27E+06 cells / mL, 90.84%, 5 mL; PBMC-donor234: 2.06E+06 cells / mL, 92.19%, 4 mL; NK92-CD16-5: 3.93E+0). 6 cells / mL, 97.08%, 1.5 mL), and the cell density was adjusted to 2E+06 cells / mL respectively; antibody was diluted (all diluted with 10% FBS + x-vivo medium containing 1000 IU / mL IL-2); according to the experimental design, NK92-CD16-5 cells, PBMC cells, anti-TIGIT humanized antibody and SEB were added to the corresponding 96-well U-shaped plates at a volume of 50 μL / well, and the blank group was supplemented with the corresponding volume of 10% FBS + x-vivo medium (containing 1000 IU / mL IL-2), with a total volume of 200 μL per well; and the cells were incubated in the incubator for 40 h.

[0226] After 40 hours, remove the cell culture plate and transfer the cells to a V-plate. Wash once with 3% BSA buffer. Mix the recommended amounts of various primary antibodies and add them to the cells, mixing gently (according to the manufacturer's instructions). Incubate at 2-8°C in the dark for 30 minutes. Add 100 μL of 3% BSA buffer to wash the cells. Centrifuge at 350g for 5 minutes and discard the supernatant. Add 200 μL of 3% BSA buffer per well, centrifuge at 350g for 5 minutes, and discard the supernatant. Add 200 μL of Foxp3 fixation / permeabilization working solution to each well. Completely resuspend the cells in the added solution and incubate at 2-8°C in the dark for 60 minutes. Centrifuge at 400g for 5 minutes. Discard the supernatant. Add 200 μL of 1× permeabilization buffer, centrifuge at 400g for 5 minutes, and discard the supernatant. Repeat once. Resuspend the pellet in the remaining volume and adjust the volume to approximately 100 μL with 1× permeabilization buffer. Without washing, add the recommended amount of direct-conjugated antibody (Foxp3) to detect intracellular antigens and incubate at room temperature for 30 min, protected from light. Add 200 μL of 1× cell permeabilization buffer to each well and centrifuge at 400 g for 5 min at room temperature. Discard the supernatant and repeat once. Resuspend the stained cells in 300 μL of PBS. Pass through a 200-mesh screen and transfer to a 1.5 mL centrifuge tube. Analyze by flow cytometry.

[0227] Figure 8a This is a single-tube staining result image; Figure 8b This is a diagram of the analysis steps; Figure 8c This is a graph showing the ratio of CD4+ to CD8+ T cells; Figure 8d This is a ratio plot of Treg cells; Figure 8e This is an ADCC result diagram using human peripheral blood mononuclear cells as target cells. The results show that the humanized anti-TIGIT antibodies 15H9L2 and 15H10L3 of the hIgG1 subtype can effectively reduce the proportion of Treg cells, while the humanized anti-TIGIT antibody 316H1L1 of the hIgG1 subtype is comparable to the positive control group R0300, and has a weak function of knocking out Treg cells. At the same time, the humanized anti-TIGIT antibody of the hIgG1 subtype has no significant effect on the proportion of CD8+ T cells.

[0228] Then, following the same method, more samples were tested.

[0229] More sample test results are as follows Figure 9As shown, the results indicate that the humanized anti-TIGIT antibodies 15H9L2 and 15H10L3 of the hIgG1 subtype have a strong knockout function of Treg cells, while the humanized anti-TIGIT antibody 316H1L of the hIgG1 subtype, although not significantly different from the positive control group R0300 in knocking out Treg cells, has a lower proportion of Treg cells in cells treated with 316H1L. Compared with the positive control group, all humanized anti-TIGIT antibodies of the hIgG1 subtype can reduce the proportion of Treg cells.

[0230] 2. Effects of anti-TIGIT humanized antibody on T cells in mouse tumor cell model tissues

[0231] (1) Experimental objective: To investigate the mechanism of action of anti-TIGIT humanized antibody 15H9L2-mIgG2a of mIgG2a subtype in a CT26 mouse subcutaneous xenograft model of colon cancer, and to set up an isotype control group (R0513) and a positive control group (R0300-mIgG2a).

[0232] (2) Experimental materials: as shown in Table 20.

[0233] Table 20

[0234]

[0235] (3) Experimental methods

[0236] 1) Cell culture: Mouse colon cancer cells (CT26) were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (1:1).

[0237] 2) Inoculation: Collect CT26 cells in the logarithmic growth phase, wash twice with pre-cooled DPBS, and adjust the cell concentration to 2×10⁻⁶. 6 / mL. Female hTIGIT mice were subcutaneously inoculated with CT26 cells at a volume of 0.1 mL / mouse, i.e., 2 × 10⁹ / mL. 5 / mouse.

[0238] 3) Administration: The day of vaccination is recorded as day 0 (D0), and the tumor volume is 60-80 mm on day 8 (D8). 3 Forty mice were selected for in vivo efficacy studies; the remaining 15 mice had tumors that had grown to 300-500 mm in size. 3 At that time, the mice were randomly divided into 3 groups according to the tumor volume, with 3 mice in each group, and the drug was started (the drug administration regimen is shown in Table 21).

[0239] The day after the second administration, the mice were euthanized, and the tumors and spleens were dissected and processed for analysis of lymphocytes in the spleen and tumor cells.

[0240] Table 21

[0241] Group Dosage (mg / kg) Dosage volume (μL / g) route of administration Dosage frequency Isotype-mIgG2a 10 10 IP Q3D*2 R0300-mIgG2a 10 10 IP Q3D*2 15H9L2-mIgG2a 10 10 IP Q3D*2

[0242] Note: In Table 21, the dosage 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 dosing regimen will be adjusted. Isotype represents the isotype control group (R0513). In addition, in the table, ip indicates intraperitoneal injection, and Q3D*2 indicates administration once every 3 days for a total of 2 administrations.

[0243] 4) Analysis of mouse spleen and tumor lymphocytes

[0244] a. Digestion of tumor tissue:

[0245] (1) Prepare 0.04g-1g of tumor tissue and cut it into small pieces (as small as possible) to 2-4mm.

[0246] (2) Preparation of digestion solution: Add 2.35 mL of 1640 medium to a 5 mL centrifuge tube, and then add 100 μL of enzyme D, 50 μL of enzyme R, and 12.5 μL of enzyme A respectively (if performing TILs analysis, it is recommended to reduce the amount of enzyme R by up to 20%, for example: add 10 μL of enzyme R to 2.5 mL of digestion solution).

[0247] (3) Add the chopped tumor tissue to the prepared digestion solution and digest at 37°C for 40 minutes, blowing it evenly every 3-5 minutes.

[0248] (4) Place the cell strainer on a 50mL centrifuge tube, remove the precipitated tissue supernatant, and add a cell strainer; add 10mL of 1640 culture medium to the tissue fragments to rinse them, and then add the cell strainer to filter them completely.

[0249] (5) Centrifuge at 300g for 7 minutes, remove the supernatant, and you will get the digested cells.

[0250] (6) Experimental steps for separating CD45+ magnetic beads:

[0251] a) Prepare 250 mL of separation buffer in advance:

[0252] i. 50mL PBS + 250μL FBS + 125μL EDTA (0.8M) in an ice bath for later use;

[0253] b) Count the digested tumor cells from the first part, take 5E7 cells, centrifuge at 300g for 10min, and discard the supernatant;

[0254] c) Resuspend each 10e7 cells in 90 μL of separation buffer according to the number of tumor cells;

[0255] d) Add 10 μL of CD45 Microbeads per 10e7 cells and mix well;

[0256] e) Incubate at 4℃-8℃ for 15 minutes;

[0257] f) Add 1-2 mL of separation buffer to each 1E7 cell in a test tube, and centrifuge at 300g for 4 min for 10 min;

[0258] g) After discarding the supernatant, add 500 μL of separation buffer to resuspend the cells (up to 1E8 cells);

[0259] h) Preparation of the separation column: Add 500ul of separation buffer to wash the isolate (500ul / MS, 3ml / LS);

[0260] i) Add the mixed cell suspension to the separation column (adsorbed onto the magnet) and wait for all the liquid to drip naturally into the centrifuge tube;

[0261] j) Add 500ul of separation buffer again and wash 3 times (3×500ul / MS, 3×3ml / LS);

[0262] k) Add 1 mL of separation buffer to the separation column again, remove the magnet, attach a stopcock to the separation column and push the cells into a new centrifuge tube. You can add another 1 mL of separation buffer to the separation column and repeat the above process.

[0263] l) Centrifuge the collected cells, wash them once with PBS containing 1% FBS, resuspend them with flow cytometry staining solution, and count them;

[0264] m) Adjust the cell density to 2E7 cells / ml, and add an equal amount of 100μL of cell suspension to each well of a 96-well plate (adjust according to the number of cells).

[0265] b. Spleen treatment:

[0266] 1) Grind the spleen with the flat end of a syringe, place the filter membrane on a 50mL centrifuge tube, take the spleen suspension and add it to the cell sieve; then add 10mL of 1640 culture medium to rinse it, add it to the cell sieve and filter it completely.

[0267] 2) Centrifuge at 300g for 7 minutes, remove the supernatant, and you will get the digested cells.

[0268] 3) Red blood cell lysis: After the blood sample has stood for 30 minutes, add 1 mL of red blood cell lysis buffer (depending on the volume of blood collected), mix well, let stand for 5 minutes, centrifuge at 300-400g for 5 minutes, and discard the supernatant (if red blood cells are still visible to the naked eye, repeat the previous steps until red blood cell lysis is complete).

[0269] 4) Washing: Resuspend cells in 5 mL of DPBS, count (using PBMC mode), take 3E6-4E6 cells and transfer to a 96-well plate, centrifuge at 300-400g for 5 min.

[0270] c. Fluorescent staining

[0271] 1) Blocking nonspecific Fc-mediated interactions: Before staining mouse cells, pre-incubate the cells with 0.5-1 μg of purified anti-mouse CD16 / CD32 / 100 μL at 2-25℃ for 10-20 minutes.

[0272] 2) Mix the recommended amounts of various primary antibodies and add them to the cells, mixing gently (according to the recommended amounts in the instructions).

[0273] 3) Incubate at 2-8℃ or on ice in the dark for at least 30 minutes.

[0274] 4) Add 100 μL of flow cytometry staining solution to wash the cells; centrifuge at 350 g for 5 minutes at room temperature and discard the supernatant.

[0275] 5) 200ul / well, repeat step 4.

[0276] 6) After the final wash, discard the supernatant and gently tap the sample to completely dissociate the precipitate.

[0277] 7) Add 200 μL of Foxp3 fixation / permeabilization working solution to each well. It is best to completely resuspend the cells in the added solution; incubate at 2-8°C or room temperature in the dark for 30-60 minutes (mouse samples can be incubated at 2-8°C in the dark for up to 18 hours).

[0278] 8) Centrifuge the sample at 400-600g for 5 minutes at room temperature. Discard the supernatant.

[0279] 9) Add 200 μL of 1× membrane rupture buffer to each well and centrifuge the sample at 400-600g for 5 minutes at room temperature. Discard the supernatant.

[0280] 10) Repeat step 9.

[0281] 11) Resuspend the precipitate in the remaining volume and adjust the volume to approximately 100 μL with 1X membrane rupture buffer.

[0282] 12) Without washing, add the recommended amount of direct-conjugated antibody to detect intracellular antigens and incubate at room temperature for at least 30 minutes. Protect from light.

[0283] 13) Add 200 μL of 1× membrane rupture buffer to each well and centrifuge the sample at 400-600g for 5 minutes at room temperature. Discard the supernatant.

[0284] 14) Repeat step 14.

[0285] 15) Resuspend the stained cells in an appropriate volume of flow cytometry staining solution. Analyze by flow cytometry.

[0286] Figure 10a This is a graph showing the effect of anti-TIGIT humanized antibody on T cells in a mouse tumor cell model. Figure 10b The figure shows the effects of anti-TIGIT humanized antibody on CD3+ T cells and CD4+ T cells in mouse tumor cell model tissues. The results show that, compared with the isotype control group, both 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, with 15H9L2 showing a more significant effect than R0300.

[0287] Example 6: Drugability Assessment of Anti-TIGIT Humanized Antibody

[0288] Drugability assessment can amplify potential risk sites, such as post-translational modification sites like deamidation, isomerization, and oxidation, through early stress experiments and stability studies. This process can also establish a link between stress conditions and changes in specific antibody risk sites, allowing for the early detection of environments unfavorable to antibody molecules. Because antibody molecules undergo various environmental conditions throughout the preparation process—such as high temperatures and oxidation during culture; acidic and alkaline conditions during purification; extreme acidic conditions during virus inactivation; and freeze-thaw cycles and shaking during long-term storage or transportation—early identification of factors causing antibody instability can provide corresponding reference strategies for later development. Molecules with very poor stability can also be excluded during early drugability assessment, reducing the risk of later project failure and ensuring the reliability of candidate molecules entering subsequent clinical stages. The following is a drugability assessment of humanized anti-TIGIT antibodies against hIgG1 subtypes (15H9L2, 15H10L3, 316H1L1):

[0289] 1. Stability assessment of anti-TIGIT humanized antibody against acid and alkali

[0290] First, all samples were replaced with 20 mmol (pH 6.0) sodium acetate buffer, and the sample concentration was adjusted to 3 mg / ml. 2.5 ml of each sample was taken and the pH was adjusted to 3.5 with 0.5 mol Citrate, or 2.5 ml of each sample was taken and the pH was adjusted to 9.0 with 1 M Tris-HCl. The samples were then aliquoted into 1.5 ml EP centrifuge tubes and labeled. After incubation at room temperature for 0 h, 4 h, and 22 h, the pH was immediately adjusted back to around 6.0. The samples were then promptly sent for physicochemical and activity assays. Based on the physicochemical assay results, a selection of samples was chosen for mass spectrometry analysis.

[0291] Figure 11a , Figure 11bThe following are the SEC-HPLC detection results of anti-TIGIT humanized antibodies after acid and alkali treatment. Figure 11c , Figure 11d The images shown are, in order, the acid and alkali treatment results of CE-SDS-NR detection of anti-TIGIT humanized antibody; Figure 11e , Figure 11f , Figure 11g The images, in order, show the changes in the acid peak, the main peak, and the alkali peak after acid treatment in the cIEF detection of anti-TIGIT humanized antibody. Figure 11h , Figure 11i , Figure 11j The images show the changes in the acid peak, main peak, and alkaline peak after alkaline treatment in cIEF assay of anti-TIGIT humanized antibody, in that order. The results show that the purity of anti-TIGIT humanized antibody remained stable after treatment with acid (pH 3.5) and alkaline (pH 9.0) for 4 and 22 hours, respectively.

[0292] 2. Stability assessment of anti-TIGIT humanized antibody against repeated freeze-thaw cycles

[0293] All samples were replaced with 20 mmol (pH 6.0) sodium acetate buffer solution, and the sample concentration was adjusted to 3 mg / ml. After repeated freeze-thaw cycles at -80℃ and room temperature, the samples were promptly sent for physicochemical and activity testing. Based on the physicochemical test results, a selection of samples were chosen for mass spectrometry analysis.

[0294] Figure 12a This is a graph showing the results of repeated freeze-thaw cycles in SEC-HPLC detection of anti-TIGIT humanized antibody; Figure 12b This is a graph showing the results of repeated freeze-thaw cycles in the CE-SDS-NR assay for anti-TIGIT humanized antibody; Figure 12c , Figure 12d , Figure 12e The images show the changes in the acid peak, main peak, and basic peak of the anti-TIGIT humanized antibody after repeated freeze-thaw cycles in cIEF detection. The results show that the anti-TIGIT humanized antibody remained stable after 5 freeze-thaw cycles.

[0295] 3. Stability assessment of anti-TIGIT humanized antibody at high temperatures

[0296] All samples were replaced with 20 mmol (pH 6.0) sodium acetate buffer, and the sample concentration was adjusted to 3 mg / ml. The three antibody samples were divided into two groups: the first group was a 4℃ control, and the second group was incubated at 37℃. Samples were taken at different time points into 1.5 ml EP tubes, sealed with sealing film, and sent for analysis on days 0, 7, 14, and 28. Based on the physicochemical test results, a portion of the samples were selected for mass spectrometry analysis.

[0297] Figure 13a , Figure 13b The images are, in order, the SEC-HPLC detection results of anti-TIGIT humanized antibody after high-temperature treatment and 4℃ control treatment; Figure 13c , Figure 13d The images, in order, show the results of CE-SDS-NR detection of anti-TIGIT humanized antibody after high-temperature treatment and 4℃ control treatment. Figure 13e , Figure 13f , Figure 13g The images, in order, show the changes in the acid peak, the main peak, and the alkali peak after high-temperature treatment in the cIEF detection of anti-TIGIT humanized antibodies. Figure 13h , Figure 13i , Figure 13j The images show the changes in the acid peak, main peak, and alkali peak of the anti-TIGIT humanized antibody after 4℃ control treatment for cIEF detection. The results show that the anti-TIGIT humanized antibody was stable after 28 days of high-temperature treatment.

[0298] 4. In vitro binding activity of anti-TIGIT humanized antibody after stress test

[0299] The anti-TIGIT humanized antibody underwent stress tests including acid, alkali, repeated freeze-thaw cycles, and high temperatures, and no significant changes in its physicochemical properties were observed, indicating its good physicochemical stability. We then further tested the in vitro binding activity of the anti-TIGIT humanized antibody to demonstrate that it maintains good activity even under stress conditions.

[0300] Figure 14a , Figure 14b , Figure 14c The images show the in vitro binding activity of the hIgG1 subtype anti-TIGIT humanized antibodies 316H1L1, 15H9L2, and 15H10L3 after stress testing. The results show that the anti-TIGIT humanized antibodies maintained good antigen-binding activity after various stress conditions.

[0301] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0302] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A humanized anti-TIGIT antibody or its antigen-binding fragment, characterized in that, The amino acid sequences of the light chain variable region and heavy chain variable region of the antibody or its antigen-binding fragment are as follows: 。 2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO:3, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:14; or the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO:16, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:21; or the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO:2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

13.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment contains a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region sequence is selected from the constant region sequence of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD. The constant region of the light chain is a κ or λ chain.

4. The antibody or its antigen-binding fragment according to claim 3, characterized in that, The heavy chain constant region sequence is selected from IgG1; The light chain constant region sequence is selected from the κ chain; The species source of the heavy chain constant region and the light chain constant region is selected from any one of cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans.

5. The antibody or its antigen-binding fragment according to claim 3, characterized in that, The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:53 or any of SEQ ID NO:45-48, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:50 or SEQ ID NO:

49.

6. The antibody or its antigen-binding fragment according to claim 3, characterized in that, 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.

7. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the heavy chain of the antibody or its antigen-binding fragment is shown in SEQ ID NO:51, and the amino acid sequence of the light chain is shown in SEQ ID NO:

52.

8. Nucleic acid, characterized in that, The nucleic acid encodes the antibody or its antigen-binding fragment as described in any one of claims 1-7.

9. The nucleic acid according to claim 8, characterized in that, The nucleic acid comprises: a first nucleic acid encoding a heavy chain variable region of the antibody or an antigen-binding fragment thereof, and a second nucleic acid encoding a light chain variable region of the antibody or an antigen-binding fragment thereof.

10. A carrier, characterized in that, The vector comprises the nucleic acid as described in claim 8 or 9.

11. A cell, characterized in that, The cell carries the nucleic acid of claim 8 or 9, contains the vector of claim 10, or expresses the antibody or antigen-binding fragment of any one of claims 1-7.

12. A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1-7, comprising: The cells of claim 11 are cultured in a culture medium; And antibodies or antigen-binding fragments thereof recovered from the culture medium or from the cultured cells.

13. A pharmaceutical composition, characterized in that, The composition contains the antibody or antigen-binding fragment thereof as described in any one of claims 1-7, or the nucleic acid as described in claim 8 or 9, or the vector as described in claim 10, or the cell as described in claim 11.

14. The pharmaceutical composition according to claim 13, characterized in that, The pharmaceutical composition includes a pharmaceutically acceptable carrier and / or excipient.

15. The use of the antibody or antigen-binding fragment thereof of any one of claims 1-7, the nucleic acid of claim 8 or 9, the vector of claim 10, the cell of claim 11, or the pharmaceutical composition of claim 13 or 14 in the preparation of a medicament for the prevention or treatment of colon cancer.

Images