TW202321307A - Anti-tigit humanized antibody or antigen-binding fragment thereof and application thereof - Google Patents

Abstract

The present invention 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 significant anti-tumor effect, effectively prolongs the survival period of mice, and significantly reduces the proportion of Treg cells in healthy human PBMC and tumor-infiltrating lymphocytes. The antibody or the antigen-binding fragment thereof has good stability and good druggability, and can be widely used for preventing or treating immune diseases or tumor-related diseases.

Description

Anti-TIGIT humanized antibodies or antigen-binding fragments thereof and applications thereof

This application claims the priority of the Chinese patent application with the filing date of September 24, 2021 (Application Number: 202111123523.1, Invention Name: Anti-TIGIT humanized antibody or antigen-binding fragment thereof and its application). All the Chinese patent applications The contents are incorporated by reference into this application in their entirety.

The present invention relates to the field of biomedicine, specifically to anti-TIGIT humanized antibodies or antigen-binding fragments thereof and their applications.

TIGIT (T cell Ig and ITIM domain, also known as WUCAM, Vstm3, VSIG9) is a protein containing ITT domain (immunoglobulin tyrosine tail motif domain) and ITIM domain (immunoreceptor tyrosine inhibition motif). Domain) inhibitory receptor shared by T cells and NK cells, belongs to type I transmembrane protein, including IgV extracellular segment and immunoglobulin tyrosine tail-like phosphorylated fragment. The gene was discovered in 2008 by a research team from Genentech. The research team searched the genome for genes that met specific conditions (① expressed on immune cells; ② belongs to type I transmembrane protein; ③ has an immunoglobulin domain outside the cell; ④ has an immune regulatory domain inside the cell). Find this gene. Through sequence comparison, it was found that this protein belongs to a larger PVR protein family, whose members include activated receptors CD226 and CD96 that compete with TIGIT for ligands, as well as their ligands PVR, and so on. In 2012, the crystal structure of the TIGIT molecule was analyzed through X-ray diffraction. It was found that TIGIT expressed on immune cells first formed a cis homodimer on the same cell, and then the dimer passed through one TIGIT molecule on each side. Bind a PVR molecule and found that the preformed homodimer is necessary for the TIGIT-PVR interaction, because if the amino acids of the TIGIT-TIGIT binding interface are mutated in advance, the TIGIT-PVR interaction will be disrupted.

The TIGIT molecule is relatively conservative. The human TIGIT molecule has 88% and 58% sequence homology with monkey and mouse TIGIT respectively. TIGIT is expressed on the surface of immune cells such as T cells, NK cells and dendritic cells, and binds to PVR (polio virus receptor, CD155) on the surface of cancer cells to inhibit the activity of immune cells. The main ligands of TIGIT are CD155 (Necl-5, PVR poliovirus receptor) and CD112 (PVRL2, Nectin-2). Among them, the binding affinity to CD155 is the highest (3.15nM, Kd). Two TIGIT molecules and two CD155 molecules form a tetramer "lock-key structure" (TIGIT's Try113 and CD155's AX6G, and CD155's Phe128 and TIGIT's AX6G form "key-lock" respectively). These two ligands are also ligands of CD226 (DNAM-1). CD226 competes with TIGIT to stimulate T cell activity. The interaction between ligand and TIGIT defeats CD226, inhibiting immune activity, and tumor cells upregulate CD155 and CD122 to escape immune-mediated destruction.

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

The technical problem to be solved by the present invention is to develop anti-human TIGIT antibodies that bind with high affinity and specificity to TIGIT protein, and to carry out humanized transformation to reduce the risk of antibody immunogenicity while maintaining the functional activity of the antibody.

The invention provides anti-TIGIT humanized antibodies or antigen-binding fragments thereof. The antibodies include 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. , HCDR3, the amino acid sequences of LCDR1, LCDR2, and LCDR3 are selected from SEQ ID NO: 29 to 31 or SEQ ID NO: 32 to 34, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 are selected from SEQ ID NO: 35 to 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.

The invention also provides nucleic acids encoding said antibodies or antigen-binding fragments thereof.

The invention also provides vectors comprising the nucleic acids.

The invention also provides cells carrying said nucleic acid, containing said vector or expressing said antibody or antigen-binding fragment thereof.

The invention also provides a method of producing the antibody or antigen-binding fragment thereof, comprising: culturing the cell in a culture medium; and recovering the antibody or antigen-binding fragment thereof so produced from the culture medium or from the cultured cells.

The present invention also provides pharmaceutical compositions containing the antibody or antigen-binding fragment thereof, or the nucleic acid, or the vector or the cell.

The present invention also provides the use of the antibody or antigen-binding fragment thereof, the nucleic acid, the vector, the cell, and the pharmaceutical composition in the preparation of medicaments for preventing or treating immune diseases or tumor-related diseases. Application.

The present invention also provides any of the aforementioned antibodies or antigen-binding fragments thereof, nucleic acids, vectors, cells or pharmaceutical compositions used as medicines; in some technical solutions, the medicines are used to prevent or treat immune diseases, or tumor-related diseases.

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

The above-mentioned anti-TIGIT humanized antibodies have high affinity to TIGIT and have various functional properties, and can be used alone or in combination with other reagents to prevent or treat immune diseases or tumor-related diseases.

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used in another embodiment, to yield still further embodiments.

Thus, it is intended that the present invention cover such modifications and variations as come within the scope of the appended claims and their equivalents. Other objects, features, and aspects of the invention are disclosed in, or are apparent from, the following detailed description. Those of ordinary skill in the art will appreciate that this discussion is merely a description of exemplary embodiments and is not intended to limit the broader aspects of the invention.

The present invention relates to an anti-TIGIT humanized antibody or an antigen-binding fragment thereof. The antibody includes 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 consists of: the amino acid sequences of LCDR1, LCDR2 and LCDR3 are selected from SEQ ID NO:29～31 or SEQ ID NO:32～34; the amino acid sequences of HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO:35～37 or SEQ ID NO: 38 to 40. The amino acid sequence of the heavy chain variable region of the antibody is shown in any one of SEQ ID NO: 5 to 15 or SEQ ID NO: 21 to 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, and the amino acid sequences of HCDR1, HCDR2 and HCDR3 are respectively SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37, and the amino acid sequence of the antibody heavy chain variable region is as shown in any one of SEQ ID NO: 5 to 15; in some optional embodiments , the amino acid sequence of the antibody heavy chain variable region is 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, and the amino acid sequences of HCDR1, HCDR2 and HCDR3 are respectively are SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40, and the amino acid sequence of the antibody heavy chain variable region is as shown in any one of SEQ ID NO: 21 to 28; in some optional embodiments , the amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO: 21.

An important advantage of this antibody or its antigen-binding fragment is that it has high in vitro binding activity and species cross-binding activity with TIGIT.

An important advantage of this antibody or its antigen-binding fragment is that it has the activity to block the binding of PVR to TIGIT.

An important advantage of this antibody or its antigen-binding fragment is that it has significant anti-tumor effect.

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

Due to having one or more of the above properties, the antibody or its antigen-binding fragment can be used as an antibody drug.

In the present invention, the term "antibody or antigen-binding fragment thereof" refers to a protein that binds a specific antigen, which generally refers to all proteins and protein fragments including complementarity determining regions (CDR regions). "Antibody" refers specifically to a full-length antibody. The term "full-length antibody" includes both polyclonal and monoclonal antibodies, and the term "antigen-binding fragment" is a substance that contains part or all of the CDRs of an antibody that lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to 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 specifically recognizes and binds TIGIT.

"Humanized antibodies" are antibodies that retain the reactivity of non-human antibodies while having lower immunogenicity in humans. For example, it can be constructed by retaining the non-human antibody CDR regions and replacing the remainder of the antibody with its human antibody counterpart (i.e., the constant region and the framework portion of the variable region).

In the present invention, the term "specific binding" or similar expressions refers to the binding of an antibody or an antigen-binding fragment thereof to a predetermined epitope on an antigen. Typically, the antibody or antigen-binding fragment thereof binds with an affinity (KD) of approximately less than ^10-6 M, such as approximately less than ^10-7 M, ^10-8 M, ^10-9 M, or ^10-10 M or less. KD refers to the ratio of off-rate to on-rate (koff/kon), a quantity that can be measured by methods familiar to those skilled in the art.

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

In the present invention, the complementarity determining region of the heavy chain is represented by HCDR, and the complementarity determining region of the light chain is represented by LCDR. Commonly used CDR notation methods in this field include: Kabat numbering scheme, 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 immunoglobulin variable regions. In their compilation of "Sequences of Proteins of Immunological Interest", the amino acid sequences of the variable regions of the light chain (λ, κ) and the heavy chain of the antibody, as well as the variable region of the T cell receptor (α , β, γ, δ) aligned and numbered. Over the past few decades, the accumulation of sequences has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered the widely adopted standard for numbering antibody residues. The present invention uses the Kabat annotation standard to mark CDR regions, but CDR regions marked by other methods also belong to the protection scope of the present invention.

In the present invention, the terms "specific recognition", "selective binding", "selectively binding" and "specific binding" or similar expressions refer to the response of an antibody or an antigen-binding fragment thereof to a predetermined antigen. Epitope binding. Typically, the antibody or antigen-binding fragment thereof binds with an affinity (KD) of approximately less than ^10-6 M, such as approximately less than ^10-7 M, ^10-8 M, ^10-9 M, or ^10-10 M or less.

In the present invention, the objects specifically recognized by the provided antibodies or antigen-binding fragments thereof can be TIGITs derived from various genera, such as humans, mice, and monkeys (such as cynomolgus monkeys).

In some embodiments, the amino acid sequence of the light chain variable region of any of the aforementioned antibodies is as shown in any one of SEQ ID NO: 1 to 4 or SEQ ID NO: 16 to 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 as shown in any one of SEQ ID NO: 5 to 13, 15, and the light chain of the antibody The amino acid sequence of the variable region is as shown in SEQ ID NO:3, or as shown in any one of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:4; or the heavy chain variable region of the antibody The amino acid sequence is shown in any one of SEQ ID NO: 21 to 28, and the amino acid sequence of the light chain variable region of the antibody is shown in any one of SEQ ID NO: 16 to 20.

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

Table 1 No. light chain variable region heavy chain variable region 1 SEQ ID NO:3 SEQ ID NO:14 2 SEQ ID NO:16 SEQ ID NO:21 3 SEQ ID NO:1 SEQ ID NO:5 4 SEQ ID NO:1 SEQ ID NO:12 5 SEQ ID NO:4 SEQ ID NO:11 6 SEQ ID NO:4 SEQ ID NO:15 7 SEQ ID NO:2 SEQ ID NO:12 8 SEQ ID NO:2 SEQ ID NO:13 9 SEQ ID NO:2 SEQ ID NO:14 10 SEQ ID NO:2 SEQ ID NO:15 11 SEQ ID NO:4 SEQ ID NO:14 12 SEQ ID NO:16 SEQ ID NO:22 13 SEQ ID NO:20 SEQ ID NO:28 14 SEQ ID NO:19 SEQ ID NO:24 15 SEQ ID NO:20 SEQ ID NO:24 16 SEQ ID NO:18 SEQ ID NO:25 17 SEQ ID NO:18 SEQ ID NO:26 18 SEQ ID NO:18 SEQ ID NO:27

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

In some embodiments, the antibody contains a heavy chain constant region and a light chain constant region, and the heavy chain constant region sequence is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD. area sequence. In some embodiments, the heavy chain constant region sequence is selected from IgG1.

In some embodiments, the light chain constant region is a kappa or lambda chain. In some embodiments, the light chain constant region sequence is selected from kappa chains.

In some embodiments, the species source of the heavy chain constant region and light chain constant region is selected from the group consisting of cattle, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer , mink, chicken, duck, goose or any of the people. In some embodiments, the species origin of the heavy chain constant region and light chain constant region is human.

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. Show. In some embodiments, 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. In some embodiments, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:45, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:49.

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 set forth in SEQ ID NO: 53.

In some embodiments, the amino acid sequence of the heavy chain constant region of any of the preceding antibodies 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. In some embodiments, the amino acid sequence of the heavy chain constant region of the antibody described in any of the preceding items is shown in SEQ ID NO: 45, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 50. In some embodiments, the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO:3, and the amino acid sequence of the light chain constant region is set forth in SEQ ID NO:50; and the heavy region of the antibody is set forth in SEQ ID NO:3. The amino acid sequence of the chain variable region 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 set forth in SEQ ID NO: 16, and the amino acid sequence of the light chain constant region is set forth in SEQ ID NO: 50; and the amino acid sequence of the heavy chain region of the antibody is set forth in SEQ ID NO: 16. The amino acid sequence of the chain variable region is shown in SEQ ID NO:21, and the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:53 or SEQ ID NO:45. In some embodiments, the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO:2, and the amino acid sequence of the light chain constant region is set forth in SEQ ID NO:50; and the amino acid sequence of the heavy chain region of the antibody is set forth in SEQ ID NO:2. The amino acid sequence of the chain variable region is shown in SEQ ID NO:13, 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 heavy chain of the antibody is set forth in SEQ ID NO:51, and the amino acid sequence of the light chain is set forth in SEQ ID NO:52.

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

In the present invention, the term "F(ab') ₂ "includes two light chains and two heavy chains containing part of the constant region between the CH1 and CH2 domains such that an interchain is formed between the two heavy chains. Disulfide bonds. The F(ab') ₂ fragment thus consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

In the present 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, fusion of hybridomas or ligation of Fab' fragments. The two binding sites of a bispecific antigen-binding protein or antibody will bind two different epitopes, which may be present on the same or different protein targets.

The invention also relates to nucleic acids encoding said antibodies or antigen-binding fragments thereof.

In the present invention, the nucleic acid is usually RNA or DNA, and the nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. DNA is preferably used when ligated into a vector. In addition, since antibodies are membrane proteins, nucleic acids often carry signal peptide sequences.

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

The invention also relates to a vector comprising said nucleic acid.

In the present invention, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses , Papilloma vacuolating virus (such as SV40).

The invention also relates to cells carrying said nucleic acid, containing said vector or expressing said antibody or antigen-binding fragment thereof.

The invention also relates to a method of producing said antibody or antigen-binding fragment thereof, comprising: culturing said cells in a culture medium; and recovering the antibody or antigen-binding fragment thereof so produced from the culture medium or from the cultured cells.

The present invention also relates to a pharmaceutical composition containing the antibody or antigen-binding fragment thereof, or the nucleic acid, or the vector or the cell.

In the present invention, the term "pharmaceutical composition" is a form that is effective in allowing the biological activity of the active ingredient and does not contain additional ingredients that would have unacceptable toxicity to the subject to whom the composition is to be administered.

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

As used herein, the term "pharmaceutically acceptable carrier" may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible, Used to extend the shelf life or potency of antibodies.

The present invention also relates to the use of the antibody or antigen-binding fragment thereof, the nucleic acid, the vector, the cell, and the pharmaceutical composition in the preparation of drugs for preventing or treating immune diseases or tumor-related diseases. .

The present invention also provides any of the aforementioned antibodies or antigen-binding fragments thereof, nucleic acids, vectors, cells or pharmaceutical compositions used as medicines; in some technical solutions, the medicines are used to prevent or treat immune diseases, or tumor-related diseases.

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

In some embodiments, any of the aforementioned diseases is a disease related to human TIGIT, and more preferably is a T cell dysfunction disease.

In some embodiments, the present invention also provides reagents for detecting or determining human TIGIT, the reagents comprising the anti-TIGIT antibody or antigen-binding fragment thereof according to any one of the preceding items.

The invention has the following beneficial effects:

The humanized antibody against human TIGIT of the present invention has good binding activity and blocking activity, significant anti-tumor effect, effectively prolongs the survival period of mice, significantly reduces the proportion of Treg cells in healthy human PBMC and tumor-infiltrating lymphocytes, and has good stability. It has good medicinal properties and can be widely used to prevent or treat immune diseases or tumor-related diseases.

The embodiments of the present invention will be described in detail below with reference to examples. Example 1 Humanization of anti-human TIGIT antibody

The TIGIT-5 mouse monoclonal antibody (VL and VH sequences are shown in SEQ ID NO: 41 and SEQ ID NO: 42) and the 3TIGIT-16 mouse monoclonal antibody (VL and VH sequences are shown in SEQ ID NO: 42) in the Chinese patent application with patent application number 202011324100.1 SEQ ID NO:43, SEQ ID NO:44) has good binding affinity to CHO-hTIGIT, healthy human PBMC, and cynoTIGIT, blocks the binding of CHO-hTIGIT, CHO-hPVR, and hPVR-hFc, and blocks tumor cells, The binding kinetic constants of CHO-CD112 to TIGIT and the human TIGIT recombinant protein are all at the pM level, and it has a good ability to activate NK92-hTIGIT to kill U2-OS; therefore, this example tested the above-mentioned TIGIT-5 mouse single Anti- and 3TIGIT-16 mouse monoclonal antibodies were humanized. The specific methods are as follows:

Humanization transformation is carried out through complementarity-determining region transplantation (CDR-grafting). First, MOE (Molecular Operating Environment) software was used to conduct antibody homology modeling of the Fv regions of TIGIT-5 mouse monoclonal antibody and 3TIGIT-16 mouse monoclonal antibody. According to the model structure, the key amino acid residues that can affect the conformational stability of the antigen-binding region are analyzed. Subsequently, the human immunoglobulin database was searched, and human IGVH and IGVK sequences with high homology to TIGIT-5 and 3TIGIT-16 in the human germline antibody library were searched as templates for humanization transformation. Compare TIGIT-5 and 3TIGIT-16 with the matching human IGVH and IGVK sequences, focusing on analyzing the key amino acid residues on the original mouse monoclonal antibody that can affect the conformational stability of the antigen-binding region that are inconsistent with the human IGVH and IGVK sequences. point and observe whether humanized replacement can be performed in the model. Depending on the degree of humanization substitution, multiple humanized sequences can be generated simultaneously based on one parent sequence.

For TIGIT-5 mouse monoclonal antibody, the humanized VL sequence (VL1-VL4) is shown in Table 2-1 below, and the humanized VH sequence (VH1-VH11) is shown in Table 2-2 below:

table 2-1 No. TIGIT-5 humanized VL sequence TIGIT-5 (parent) LCDR1: RASENIYSNLA (SEQ ID NO:29) LCDR2: AASHLPD (SEQ ID NO:30) LCDR3: QHFWGTPRT (SEQ ID NO:31) DIQMIQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYAASHLPDGVPSRFSGSGSGTQYSLKINSLQSEDFGSYYCQHFWGTPRTFGGGTKLEIK (S EQ ID NO:41） VL1 DIQMTQSPSSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKSPKLLIYAASHLPDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHFWGTPRTFGQGTKLEIKR (SEQ ID NO: 1) VL2 DIQMTQSPSSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKSPKLLIYAASHLPDGVPSRFSGSGSGTDYSLTISSLQPEDFATYYCQHFWGTPRTFGQGTKLEIKR (SEQ ID NO: 2) VL3 DIQMTQSPSSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKSPKLLVYAASHLPDGVPSRFSGSGSGTDYSLTISSLQPEDFATYYCQHFWGTPRTFGQGTKLEIKR (SEQ ID NO: 3) VL4 DIQMIQSPSSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKSPKLLVYAASHLPDGVPSRFSGSGSGTDYSLTISSLQPEDFATYYCQHFWGTPRTFGQGTKLEIKR (SEQ ID NO: 4)

Table 2-2 No. TIGIT-5 humanized VH sequence TIGIT-5 (parent) HCDR1: SYWMN (SEQ ID NO: 35) HCDR2: MIRPSDSETRLNQMFKD (SEQ ID NO: 36) HCDR3: IHDYGHGAY (SEQ ID NO: 37) YGHGAYWGQGTLVTVSA (SEQ ID NO:42) VH1 QVQLVQSGAEVKKPGASVKVSCKASGYSFTSYWMNWVRQAPGQGLEWMGMIRPSDSETRLNQMFKDRVTMTTDTSSTAYMELRSLRSDDTAVYYCAGIHDYGHGAYWGQGTLVTVSS (SEQ ID NO: 5) VH2 QVQLVQSGAEVKKPGASVKVSCKASGYSFTSYWMNWVRQAPGQGLEWMGMIRPSDSETRLNQMFKDRVTMTTDTSSTAYMELSSLRSDDTAVYYCAGIHDYGHGAYWGQGTLVTVSS (SEQ ID NO: 6) VH3 QVQLVQSGAEVKKPGASVKVSCKASGYSFTSYWMNWVRQAPGQGLEWMGMIRPSDSETRLNQMFKDRVTMTVDTSTSTAYMELSSLRSDDTAVYYCAGIHDYGHGAYWGQGTLVTVSS (SEQ ID NO:7) VH4 QVQLVQSGAEVKKPGASVKVSCKASGYSFTSYWMNWVRQAPGQGLEWMGMIRPSDSETRLNQMFKDRVTMTTDKSTSTAYMELSSLRSDDTAVYYCAGIHDYGHGAYWGQGTLVTVSS (SEQ ID NO: 8) VH5 QVQLVQSGAEVKKPGASVKVSCKASGYSFTSYWMNWVRQAPGQGLEWIGMIRPSDSETRLNQMFKDRVTMTTDTSSTAYMELSSLRSDDTAVYYCAGIHDYGHGAYWGQGTLVTVSS (SEQ ID NO:9) VH6 QVQLVQSGAEVKKPGASVKVSCKASGYSFTSYWMNWVRQAPGQGLEWIGMIRPSDSETRLNQMFKDRVTMTVDKSTSTAYMELSSLRSDDTAVYYCAGIHDYGHGAYWGQGTLVTVSS (SEQ ID NO: 10) VH7 QVQLVQSGAEVKKPGASVKVSCKASGYSFTSYWMNWVRQAPGQGLEWIGMIRPSDSETRLNQMFKDRATLTVDKSTSTAYMELSSLRSDDTAVYYCAGIHDYGHGAYWGQGTLVTVSS (SEQ ID NO: 11) VH8 QVQLVQSGAEVKKPGSSVKVSCKASGYSFTSYWMNWVRQAPGQGLEWMGMIRPSDSETRLNQMFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCAGIHDYGHGAYWGQGTLVTVSS (SEQ ID NO: 12) VH9 QVQLVQSGAEVKKPGSSVKVSCKASGYSFTSYWMNWVRQAPGQGLEWMGMIRPSDSETRLNQMFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAGIHDYGHGAYWGQGTLVTVSS (SEQ ID NO: 13) VH10 QVQLVQSGAEVKKPGSSVKVSCKASGYSFTSYWMNWVRQAPGQGLEWIGMIRPSDSETRLNQMFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAGIHDYGHGAYWGQGTLVTVSS (SEQ ID NO: 14) VH11 QVQLVQSGAEVKKPGSSVKVSCKASGYSFTSYWMNWVRQAPGQGLEWIGMIRPSDSETRLNQMFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCAGIHDYGHGAYWGQGTLVTVSS (SEQ ID NO: 15)

For 3TIGIT-16 mouse monoclonal antibody, the humanized VL sequence (VL5-VL9) is shown in Table 3 below, and the humanized VH sequence (VH12-VH19) is shown in Table 4 below:

table 3 No. 3TIGIT-16 humanized VL sequence 3TIGIT-16 (parent) LCDR1: SASSSVSYMH (SEQ ID NO:32) LCDR2: DTSKLAS (SEQ ID NO:33) LCDR3: QQWSSNSLT (SEQ ID NO:34) QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPTRFSGSGSGTSYSLTISSMEAEEDAASYFCQQWSSNSLTFGAGTKLELKR (SEQ ID NO:43) VL5 DIQMTQSPSSSLSASVGDRVTITCSASSSVSYMHWYQQKPGKSPKRLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQWSSNSLTFGQGTKLEIKR (SEQ ID NO: 16) VL6 DIQMTQSPSSSLSASVGDRVTITCSASSSVSYMHWYQQKPGKSPKRWIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQWSSNSLTFGQGTKLEIKR (SEQ ID NO: 17) VL7 DIQMTQSPSSSLSASVGDRVTITCSASSSVSYMHWYQQKPGKSPKRWIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQWSSNSLTFGQGTKLEIKR (SEQ ID NO: 18) VL8 DIQMTQSPSSSLSASVGDRVTITCSASSSVSYMHWYQQKPGKSPKRWIYDTSKLASGVPSRFSGSGSGTEYSLTISSLQPEDFATYFCQQWSSNSLTFGQGTKLEIKR (SEQ ID NO: 19) VL9 DIVLTQSPSSSLSASVGDRVTITCSASSSVSYMHWYQQKPGKSPKRWIYDTSKLASGVPSRFSGSGSGTEYSLTISSLQPEDFATYFCQQWSSNSLTFGQGTKLEIKR (SEQ ID NO: 20)

Table 4 No. 3TIGIT-16 humanized VH sequence 3TIGIT-16 (parent) HCDR1: DYFMD (SEQ ID NO: 38) HCDR2: RLNPNNGRTSYNQKFKG (SEQ ID NO: 39) HCDR3: GDLGRWYFDV (SEQ ID NO: 40) EVQLQQSGPELVKPGASVKMSCKASGFTFTDYFMDWVKQSRGASFEWIGRLNPNNGRTSYNQKFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCAR GDLGRWYFDVWGAGTTVTVSS (SEQ ID NO:44) VH12 QVQLVQSGAEVKKPGASVKVSCKASGFTFTDYFMDWVRQAPGQRLEWMGRLNPNNGRTSYNQKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARGDLGRWYFDVWGQGTTVTVSS (SEQ ID NO: 21) VH13 QVQLVQSGAEVKKPGASVKVSCKASGFTFTDYFMDWVRQAPGQRFEWMGRLNPNNGRTSYNQKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARGDLGRWYFDVWGQGTTVTVSS (SEQ ID NO: 22) VH14 QVQLVQSGAEVKKPGASVKVSCKASGFTFTDYFMDWVRQAPGQRLEWMGRLNPNNGRTSYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARGDLGRWYFDVWGQGTTVTVSS (SEQ ID NO: 23) VH15 QVQLVQSGAEVKKPGASVKVSCKASGFTFTDYFMDWVRQAPGQRFEWMGRLNPNNGRTSYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARGDLGRWYFDVWGQGTTVTVSS (SEQ ID NO: 24) VH16 QVQLVQSGAEVKKPGASVKVSCKASGFTFTDYFMDWVRQAPGQRFEWMGRLNPNNGRTSYNQKFKGRVTITVDKSASTAYMELSSLRSEDTAVYYCARGDLGRWYFDVWGQGTTVTVSS (SEQ ID NO: 25) VH17 QVQLVQSGAEVKKPGASVKVSCKASGFTFTDYFMDWVRQAPGQRFEWIGRLNPNNGRTSYNQKFKGRVTITVDKSASTAYMELSSLRSEDTAVYYCARGDLGRWYFDVWGQGTTVTVSS (SEQ ID NO: 26) VH18 QVQLVQSGAEVKKPGSSVKVSCKASGFTFTDYFMDWVKQAPGQGFEWIGRLNPNNGRTSYNQKFKGKATLTVDKSTSTAYMELSSLRSEDTAVYYCARGDLGRWYFDVWGQGTTVTVSS (SEQ ID NO: 27) VH19 QVQLVQSGAEVKKPGASVKVSCKASGFTFTDYFMDWVRQAPGQRFEWIGRLNPNNGRTSYNQKFKGRATLTVDKSASTAYMELSSLRSEDTAVYYCARGDLGRWYFDVWGQGTTVTVSS (SEQ ID NO: 28)

Different humanized antibody molecules can be produced by combining different humanized VL and VH. The main consideration for different combination methods is the balance between the degree of humanization and the maintenance of antibody activity after humanization. Simply put, by pairing the most humanized VL and VH, you can get the most humanized antibody molecule, which means it may have lower immunogenicity, but a high degree of humanization may lead to changes in the modified The activity of the molecule is reduced (key amino acid residues that affect the activity of the parent antibody are replaced with humanized amino acids, resulting in changes in the antigen-binding region of the modified antibody). On the contrary, by pairing VL and VH with the lowest degree of humanization, you can get more consistent functional activity with the parent antibody (because most of the key amino acid residues are retained), but the degree of humanization is also lower, which may Will face a higher risk of immunogenicity. This is a balanced process. In the process of preparing humanized antibodies, it is necessary to express and prepare a variety of VL and VH combinations, and further verify them through in vivo and in vitro functional tests, and finally screen suitable humanized antibody molecules.

For TIGIT-5 mouse monoclonal antibody, the amino acid sequence of the anti-TIGIT humanized antibody obtained after humanization is shown in Table 5 below:

Table 5 Amino acid sequence of anti-TIGIT humanized antibodies obtained after humanizing TIGIT-5 mouse monoclonal antibody No. Anti-TIGIT humanized antibody Light chain variable region (VL) Heavy chain variable region (VH) R0766 H1L1 SEQ ID NO:1 SEQ ID NO:5 R0767 H8L1 SEQ ID NO:1 SEQ ID NO:12 R0768 H7L4 SEQ ID NO:4 SEQ ID NO:11 R0769 H11L4 SEQ ID NO:4 SEQ ID NO:15 R0770 H8L2 SEQ ID NO:2 SEQ ID NO:12 R0771 15H9L2 SEQ ID NO:2 SEQ ID NO:13 R0772 H10L2 SEQ ID NO:2 SEQ ID NO:14 R0773 H11L2 SEQ ID NO:2 SEQ ID NO:15 R0774 15H10L3 SEQ ID NO:3 SEQ ID NO:14 R0775 H10L4 SEQ ID NO:4 SEQ ID NO:14

For 3TIGIT-16 mouse monoclonal antibody, the amino acid sequence of the anti-TIGIT humanized antibody obtained after humanization is shown in Table 6 below:

Table 6 Amino acid sequence of anti-TIGIT humanized antibody obtained after humanization of 3TIGIT-16 mouse monoclonal antibody No. Anti-TIGIT humanized antibody Light chain variable region (VL) Heavy chain variable region (VH) R0750 316H1L1 SEQ ID NO:16 SEQ ID NO:21 R0751 H2L1 SEQ ID NO:16 SEQ ID NO:22 R0752 H3L1 SEQ ID NO:16 SEQ ID NO:23 R0753 H8L5 SEQ ID NO:20 SEQ ID NO:28 R0754 H4L1 SEQ ID NO:16 SEQ ID NO:24 R0755 H4L2 SEQ ID NO:17 SEQ ID NO:24 R0756 H4L3 SEQ ID NO:18 SEQ ID NO:24 R0757 H4L4 SEQ ID NO:19 SEQ ID NO:24 R0758 H4L5 SEQ ID NO:20 SEQ ID NO:24 R0759 H5L3 SEQ ID NO:18 SEQ ID NO:25 R0760 H6L3 SEQ ID NO:18 SEQ ID NO:26 R0761 H7L3 SEQ ID NO:18 SEQ ID NO:27

The amino-terminal sequence of the constant region used to construct the complete antibody from the variable region of the above anti-TIGIT humanized antibody is shown in Table 7 below:

Table 7. Antibody constant region sequences No. constant region sequence hIgG1-Fc Heavy chain constant region: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 45) hIgG4-Fc Heavy chain constant region: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:46) mIgG1-Fc Heavy chain constant region: AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSSELPIMHQDWLNGKEFKCRV NSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 47) mIgG2a-Fc Heavy chain constant region: AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWM SEQ ID NO:48 mouse-CL-κ Light chain constant region: AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKI (SEQ ID NO:49) Hu-CL-κ Light chain constant region: TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:50) hIgG1 mutation ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 53)

The specific numbered humanized antibodies listed in Table 5 and Table 6 above (such as R0774 (also identified as 15H10L3)), the heavy chain constant region of which is hIgG1 containing the K214R/D356E/L358M mutation (amino acid sequence such as SEQ ID NO. :53), the light chain constant region is Hu-CL-κ (the amino acid sequence is shown in SEQ ID NO:50). Exemplarily, the full-length amino acid sequence of humanized antibody: R0774 (also identified as 15H10L3) is:

R0774 heavy chain amino acid sequence (SEQ ID NO:51): QVQLVQSGAEVKKPGSSVKVSCKASGYSFTSYWMNWVRQAPGQGLEWIGMIRPSDSETRLNQMFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAGIHDYGHGAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

R0774 light chain amino acid sequence (SEQ ID NO:52): DIQMTQSPSSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKSPKLLVYAASHLPDGVPSRFSGSGSGTDYSLTISSLQPEDFATYYCQHFWGTPRTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Example 2 Preparation of anti- TIGIT humanized antibodies

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

Use AKTA avant 150 chromatography equipment, equilibrate the chromatography column (such as MabSelectSuRe LX, GE) with at least 5CV equilibration buffer (10mM PBS), load the sample to the chromatography column, so that the target protein is adsorbed on the chromatography column and other impurities pass through. Transparent separation. After loading, rinse the column again with at least 5CV of equilibration buffer (10mM PBS), and then use elution buffer (20mM NaAc, pH=3.4) to elute the target protein. Neutralization buffer (1M) is pre-added to the collection tube. Tris, pH8.0), the added volume of neutralizing buffer is determined based on the estimated content of the elution sample, generally 10% of the elution volume is added.

The concentration of the sample was measured using Biotek-Epoch-Take-3, and the antibody concentration was detected using the A280 method, that is, the extinction coefficient E.C.=1.37, the optical path = 0.05mm (the optical path length of different holes in the Take-3 plate has slight differences and will be automatically corrected). The equipment detects the absorbance value of the sample and calculates the concentration of the anti-TIGIT humanized antibody according to Lambert-Beer law. If the sample concentration is too low, ultrafiltration concentration is required. Use an ultrafiltration concentration tube (Amicon® Ultra-15 Centrifugal Filter Devices, 30kD) and follow the general operating methods provided in the instruction manual to concentrate the sample concentration to >0.5mg/ml; collect the concentration end Samples were sterilized and filtered through a 0.22um sterile syringe filter (Cobetter, PES, 0.22um, diameter 13mm), then aliquoted and frozen for later use.

The humanization degree and purity results of the anti-TIGIT humanized antibodies are shown in Table 8 and Table 9. The results show that the humanization degree, titer and purity results of the anti-TIGIT humanized antibodies prepared in this example are very good. good.

Table 8 No. Anti-TIGIT humanized antibody degree of humanization Titer (mg/L) Purity R0766 H1L1 99.12% 146.22 1.85%/98.15%/- R0767 H8L1 99.12% 175.74 2.56%/97.44%/- R0768 H7L4 94.69% 262.72 1.21%/98.8%/- R0769 H11L4 95.58% 196.94 0.95%/99.05%/- R0770 H8L2 98.23% 134.93 2.09%/97.91%/- R0771 15H9L2 97.79% 157.33 1.51%/98.49%/- R0772 H10L2 97.35% 176.06 2.7%/97.3%/- R0773 H11L2 96.46% 180.75 2.29%/97.71%/- R0774 15H10L3 96.90% 198.65 1.79%/98.21%/- R0775 H10L4 96.46% 238.73 1.44%/98.56%/-

Note: In Table 8, in the purity column, the leftmost value is the aggregate percentage, the rightmost value is the fragment percentage, and the middle value is the antibody monomer percentage. "-" means the value is 0%, such as "1.85% /98.15%/-", which indicates that in the antibody solution, the percentage of aggregates is 1.85%, the percentage of antibody monomer purity is 98.15%, and the percentage of fragments is 0%.

Table 9 No. Anti-TIGIT humanized antibody degree of humanization Titer(mg/L) Purity R0750 316H1L1 99.56% 197.55 4.54%/94.62%/0.84% R0751 H2L1 99.12% 109.14 3.56%/95.82%/0.62% R0752 H3L1 99.12% 138.22 3.67%/95.46%/0.87% R0753 H8L5 94.25% 142.30 2.5%/96.86%/0.63% R0754 H4L1 98.67% 140.67 3.97%/95.09%/0.94% R0755 H4L2 98.23% 181.83 2.55%/96.69%/0.76% R0756 H4L3 97.79% 77.17 1.47%/97.81%/0.71% R0757 H4L4 96.90% 134.37 1.55%/97.77%/0.67% R0758 H4L5 96.02% 170.05 1.19%/98.1%/0.71% R0759 H5L3 97.35% 197.55 2.8%/96.11%/1.09% R0760 H6L3 96.90% 109.14 2.6%/96.7%/0.7% R0761 H7L3 96.46% 138.22 2.76%/96.38%/0.86%

Note: In Table 9, in the purity column, the leftmost value is the aggregate percentage, the rightmost value is the fragment percentage, and the middle value is the antibody monomer percentage, such as "4.54%/94.62%/0.84%", where Indicates 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%. Example 3 Validation of anti -TIGIT humanized antibodies

The anti-TIGIT humanized antibodies obtained above were tested for in vitro binding activity, species cross-binding activity and blocking activity as follows:

(1) In vitro binding activity detection:

In order to verify the ability of anti-TIGIT humanized antibodies to bind to CHO-hTIGIT, the CHO-hTIGIT cells recombinantly expressed full-length human TIGIT to be tested were counted, centrifuged at 300g for 5 min, resuspended in FCM buffer, and the cell density was adjusted to 4E +06 pieces/mL for later use. Use FCM buffer to mix the samples to be tested (including positive control group, TIGIT-5 mouse monoclonal antibody (R0435), 3TIGIT-16 mouse monoclonal antibody (R0518), and anti-TIGIT humanized antibody; among which R0300-CH2a comes from patent WO2017053748 (A2 ) in tiragolumab, R0300-CH2a (the Fc region is replaced by the Fc region of mIgG1) was diluted to 20 μg/mL, and then FCM buffer was used for 3-fold serial dilution 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-type plate (the amount of each cell line added to the 96-well V-type plate is 2E+05 cells/well), and incubate on ice in the dark for 30 min. Centrifuge (300 g/5 min), discard the supernatant, add 200 μL FCM buffer to wash once, centrifuge (300 g/5 min), discard the supernatant. Add secondary antibody (PE Goat anti-mouse IgG, diluted 1:500) to a 96-well V-type plate (100 μL/well), and incubate on ice in the dark for 30 minutes. Centrifuge (300 g/5 min), discard the supernatant, add 200 μL FCM buffer to wash once, centrifuge (300 g/5 min), discard the supernatant. Add 100 μL 1×PBS to resuspend and run on the machine for detection.

(2) Species cross-binding activity detection:

In order to verify the binding ability of anti-TIGIT humanized antibodies to monkey TIGIT, the following experiments were specifically performed: CHO-cynoTIGIT cells, which are recombinantly expressed full-length cynoTIGIT to be tested, were counted, centrifuged at 300g for 5 min, resuspended in FCM buffer, and adjusted The cell density is set to 4E+06 cells/mL for later use. Use FCM buffer to dilute the sample to be tested (including the positive control group R0300-CH2a, R0435, R0518, and anti-TIGIT humanized antibody) to 20 μg/mL, and then use FCM buffer to conduct a 3-fold serial dilution 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-type plate (the amount of each cell line added to the 96-well V-type plate is 2E+05 cells/well), and incubate on ice in the dark for 30 min. Centrifuge (300 g/5 min), discard the supernatant, add 200 μL FCM buffer to wash once, centrifuge (300 g/5 min), discard the supernatant. Add secondary antibody (PE Goat anti-mouse IgG, diluted 1:500) to a 96-well V-type plate (100 μL/well), and incubate on ice in the dark for 30 minutes. Centrifuge (300 g/5 min), discard the supernatant, add 200 μL FCM buffer to wash once, centrifuge (300 g/5 min), discard the supernatant. Add 100 μL 1×PBS to resuspend and run on the machine for detection.

(3) Blocking activity detection:

In order to verify that anti-TIGIT humanized antibodies can block the ability of CHO-hTIGIT to bind to hPVR-hFc, the following experiments were performed: CHO-hTIGIT cells, which are recombinantly expressed full-length human TIGIT, were counted and centrifuged at 300g for 5 seconds. min, resuspend in FCM buffer, and adjust the cell density to 4E+06 cells/mL for later use. Use FCM buffer to dilute the sample to be tested (including the positive control group R0300-CH2a, R0435, R0518, and anti-TIGIT humanized antibody) to 20 μg/mL, and then use FCM buffer to conduct a 3-fold serial dilution to 12 concentration points; use FCM Buffer dilutes the ligand protein hPVR-hFc to 20 μg/mL. According to the experimental design, add 50 μL of antibody and 50 μL of cells to each well of a 96-well V-type plate (the amount of each cell line added to the 96-well V-type plate is 2E+05 cells/well), and incubate on ice in the dark for 30 min. Add the ligand protein to the 96-well V-shaped plate (50 μL/well), and incubate on ice in the dark for 30 minutes. Centrifuge (300 g/5 min), discard the supernatant, add 200 μL FCM buffer to wash once, centrifuge (300 g/5 min), discard the supernatant. Add secondary antibody (PE anti-human IgG Fc, diluted 1:500) to a 96-well V-type plate (100 μL/well), and incubate on ice in the dark for 30 minutes. Centrifuge (300 g/5 min), discard the supernatant, add 200 μL FCM buffer to wash once, centrifuge (300 g/5 min), discard the supernatant. Add 100 μL 1×PBS to resuspend and run on the machine for detection.

The in vitro binding activity, species cross-binding activity and blocking activity test results of anti-TIGIT humanized antibodies are shown in Table 10 and Table 11. The results show that most anti-TIGIT humanized antibodies can maintain similar activity to the parent In vitro binding activity, species cross-binding activity and blocking activity.

Table 10 No. combination hTIGIT EC50 cynoTIGIT EC50 hTIGIT-PVR IC50 R0300-CH2a BenchMarker 1.327 4.014 1.386 R0435 TIGIT-5 (mother parent) 0.7301 5.72 0.5582 R0766 H1L1 1.196 N/A N/A R0767 H8L1 0.8138 N/A N/A R0768 H7L4 1.154 3.018 0.6333 R0769 H11L4 0.9115 2.983 0.8835 R0770 H8L2 1.242 3.135 0.7169 R0771 15H9L2 0.6653 2.294 0.5179 R0772 H10L2 0.8769 2.578 0.6422 R0773 H11L2 1.062 3.512 0.6627 R0774 15H10L3 0.8043 1.922 0.5287 R0775 H10L4 1.145 2.585 0.6927

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

Table 11 No. combination hTIGIT EC50 cynoTIGIT EC50 hTIGIT-PVR IC50 R0300-CH2a BenchMarker 1.327 4.014 1.386 R0518 3TIGIT-16 (mother parent) 0.4844 1.211 0.3512 R0750 316H1L1 0.474 2.468 0.3431 R0751 H2L1 0.4293 1.861 0.4769 R0752 H3L1 N/A N/A N/A R0753 H8L5 0.4263 2.008 0.2512 R0754 H4L1 N/A N/A N/A R0755 H4L2 N/A N/A N/A R0756 H4L3 N/A N/A N/A R0757 H4L4 0.4963 2.371 0.4404 R0758 H4L5 0.5205 1.665 0.3629 R0759 H5L3 0.6349 2.481 0.4553 R0760 H6L3 0.495 1.374 0.4415 R0761 H7L3 0.3968 1.622 0.2941

Note: N/A: The activity is very weak and cannot be calculated. Example 4 In vivo functional evaluation of anti- TIGIT humanized antibodies 1. In vivo anti-tumor efficacy evaluation of different subtypes of anti-TIGIT antibodies against CT26 mouse colon cancer

(1) Experimental purpose: Compare different subtypes of anti-TIGIT antibodies R0226 (mIgG2a) [R0226 is derived from Oncomed, OMP-313R12, WO2016191643 (A2)], R0564 (mIgG1) in the CT26 mouse colon cancer subcutaneous transplant tumor model. [R0564 is the mIgG1 subtype of R0226], the anti-tumor effect of R0565 (mIgG2a DLE) [R0565 is the mIgG2a-DLE subtype of R0226], and an isotype control group R0513 (Isotype-mIgG2a) was also set.

(2) Experimental materials: Balb/c mice, female, 6-8 weeks (source: Beijing Vitong Lihua Experimental Technology Co., Ltd., certificate number: 1100112011045713); CT26 cells (Shanghai Cell Bank, catalog number 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).

(3) Instruments and equipment: electronic balance (Shanghai Sunny Hengping Scientific Instrument Co., Ltd., JA12002), vernier caliper (Shanghai Minate 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 (Price Machinery Co., Ltd., HH-S), carbon dioxide incubator (Japanese Panasonic Health Medical Instruments Co., Ltd., MCO-18AC ), biological safety cabinet (Guangzhou Qianjiang Experimental Technology Co., Ltd., ACZ-451), cell technology device (Shanghai Ruiyu Biotech, IC1000).

(4) Experimental methods

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).

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 /mL. Female Balb/c mice were taken and inoculated subcutaneously with CT26 cells. The inoculation volume was 0.1 mL/mouse, that is, 1×10 ⁵ /mouse.

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 can be divided into tumor groups. The volume was randomly divided into 4 groups, with 8 animals in each group, and administration was started (the dosage, method and frequency of CT26 tumor model administration are shown in Table 12).

Table 12 Group Dosage (mg/kg) Dosing volume (μL/g) Route of administration Dosing frequency R0513 (Isotype-mIgG2a) 10 10 ip Q3D*4 R0226 (mIgG2a) 10 10 ip Q3D*4 R0564(mIgG1) 10 10 ip Q3D*4 R0565 (mIgG2a DLE) 10 10 ip Q3D*4

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

4) Observation records:

The tumor volume was measured and the mice's weight was recorded on D13. After that, the body weight was recorded twice a week and the long and short diameters of the tumors were measured with vernier calipers. Calculate the tumor volume with the formula: (1/2) × long diameter × (short diameter) ² . When each mouse reaches the endpoint of the experiment (a 20% decrease in body weight or a mercy endpoint when the tumor volume exceeds 2000 ^mm3 ), the mice are killed by _CO2 asphyxiation, and the survival curve is recorded.

5) Data calculation, statistics and analysis:

Independent samples T test can be used to compare between two groups. One-Way ANOVA was used for comparisons of more than 3 groups. If the F value shows a significant difference, post hoc analysis between multiple groups can be performed. Data were processed using Prism GraphPad, and p<0.05 indicated statistically significant differences. 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, Ti is the average tumor volume of the treatment group on day i, T0 is the average tumor volume of the treatment group at the beginning of treatment, Vi is the average tumor volume of the solvent control group on day i, and V0 is the average tumor volume of the solvent control group at the beginning of treatment.

(5) Experimental results

The results of different subtypes of anti-TIGIT antibodies reducing the average tumor volume of CT26 colon cancer in Balb/c mice are shown in Table 13 and Figure 1. The results of the effects of different subtypes of anti-TIGIT antibodies on the survival of Balb/c mice are as follows As shown in Figure 2, the results show that the survival time and anti-tumor effect of anti-TIGIT antibodies mIgG2a subtype R0226 (TGI=88.5%) and mIgG2a DLE subtype R0565 (TGI=91.7%) are significantly better than those of mIgG1 subtype R0564 (TGI =44.6%).

Table 13 Anti-TIGIT antibodies of different subtypes reduce the mean tumor volume (mm ³ ) of CT26 colon cancer in Balb/c mice Days after implantation Isotype R0564 (10mg/kg) R0226 (10mg/kg) R0565 (10mg/kg) 13 45.99 43.60 40.70 45.65 17 142.98 85.62 105.95 121.60 twenty one 236.85 166.15 89.80 104.23 twenty four 541.09 279.63 102.69 129.81 28 1004.19 562.19 160.39 139.94 31 1526.54 863.21 210.29 168.67 35 2826.31 1652.90 344.84 227.59 38 \ 2115.96 508.97 325.59 42 \ 2466.77 816.10 482.39 45 \ 3171.10 1206.16 679.79 49 \ \ 1357.40 808.44 52 \ \ 1566.98 846.94

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). 2. Evaluation of the anti-tumor efficacy of TIGIT-5 mouse monoclonal antibody and 3TIGIT-16 mouse monoclonal antibody against colon cancer in CT26 mice in vivo

(1) Experimental purpose: To study the efficacy of mIgG2a subtype TIGIT-5 mouse monoclonal antibody (R0435) and 3TIGIT-16 mouse monoclonal antibody (R0518) in the CT26 mouse colon cancer subcutaneous transplant tumor model, and also set up an isotype control group (R0513) and positive control group (R0300-CH2a).

(2) Experimental materials: hTIGIT Knock in mice, female, 6-8 weeks (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).

(3) Instruments and equipment: electronic balance (Shanghai Sunny Hengping Scientific Instrument Co., Ltd., JA12002), vernier caliper (Shanghai Minate 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 (Price Machinery Co., Ltd., HH-S), carbon dioxide incubator (Japanese Panasonic Health Medical Instruments Co., Ltd., MCO-18AC ), biological safety cabinet (Guangzhou Qianjiang Experimental Technology Co., Ltd., ACZ-451), cell counter (Shanghai Ruiyu Biotechnology, IC1000).

(4) Experimental methods

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).

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 /mL. Female hTIGIT mice were taken and inoculated subcutaneously with CT26 cells. The inoculation volume was 0.1 mL/mouse, that is, 2×10 ⁵ /mouse.

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

Table 14 Group Dosage (mg/kg) Dosing volume (μL/g) Route of administration Dosing 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

Note: In Table 14, the dosing volume is adjusted according to the animal's weight (10 L/g); if the body weight decreases by more than 15% during the dosing 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.

4) Observation records:

The tumor volume was measured and the mice's weight was recorded on D8. After that, the body weight was recorded twice a week and the long and short diameters of the tumors were measured with vernier calipers. The tumor volume was calculated using the formula: (1/2) × long diameter × (short diameter) ² . When each mouse reaches the endpoint of the experiment (a 15% decrease in body weight or a mercy endpoint when the tumor volume exceeds 2000 ^mm3 ), the mice are killed by _CO2 asphyxiation, and the survival curve is recorded.

5) Data calculation, statistics and analysis:

Independent samples T test can be used to compare between two groups. One-Way ANOVA was used for comparisons of more than 3 groups. If the F value shows a significant difference, post hoc analysis between multiple groups can be performed. Data were processed using Prism GraphPad, and p<0.05 indicated statistically significant differences. 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, Ti is the average tumor volume of the treatment group on day i, T0 is the average tumor volume of the treatment group at the beginning of treatment, Vi is the average tumor volume of the solvent control group on day i, and V0 is the average tumor volume of the solvent control group at the beginning of treatment.

(5) Experimental results

TIGIT-5 mouse monoclonal antibody and 3TIGIT-16 mouse monoclonal antibody reduce the average tumor volume of CT26 colon cancer in hTIGIT knock in mice. The results are shown in Table 15 and Figure 3. TIGIT-5 mouse monoclonal antibody and 3TIGIT-16 mouse monoclonal antibody The results of the impact on the survival of hTIGIT knock in mice are shown in Figure 4. The results show that compared with the isotype control group, 3TIGIT-16 mouse monoclonal antibody (R0518) significantly inhibits the tumor growth of the hTIGIT knock in mouse CT26 model. The anti-tumor effect of TIGIT-5 mouse monoclonal antibody (R0435) is equivalent to that of the positive control group R0300-CH2a (TGI is 76.5% and 86.7% respectively).

Table 15 Anti-TIGIT antibody reduces mean tumor volume (mm ³ ) of CT26 colon cancer in hTIGIT knock in mice Days after implantation Isotype R0564 (10mg/kg) R0226 (10mg/kg) R0565 (10mg/kg) 8 64.71 78.74 77.53 74.60 11 96.87 735.42 88.72 106.42 14 171.24 179.30 100.63 147.89 17 293.07 198.26 82.22 163.82 20 684.85 318.11 115.47 192.07 twenty three 1374.13 449.21 155.76 297.70 26 2170.97 616.67 208.78 355.44 30 \ 1022.45 317.87 632.13 33 \ 1286.81 466.67 966.95 37 \ 1688.38 724.96 1484.91 39 \ 1994.65 952.17 1775.11 42 \ 2278.38 1168.35 2027.99 45 \ \ 1691.82 \

Note: In Table 15, Isotype represents the isotype control group (R0513). 3. Evaluation of the in vivo anti-tumor efficacy of anti-TIGIT humanized antibodies (hIgG1 subtype) against colon cancer in CT26 mice

(1) Experimental purpose: To study the efficacy of hIgG1 subtype anti-TIGIT humanized antibodies (15H9L2, 15H10L3, 316H1L1) in the CT26 mouse colon cancer subcutaneous transplant tumor model, and at the same time set up an isotype negative control group of hIgG1 subtype ( R0536) and the positive control group (R0300- hIgG1, a Tiragolumab mutant containing the K214R mutation in the heavy chain).

(2) Experimental materials: hTIGIT Knock in mice, female, 6-8 weeks (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).

(3) Instruments and equipment: electronic balance (Shanghai Sunny Hengping Scientific Instrument Co., Ltd., JA12002), vernier caliper (Shanghai Minate 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 (Price Machinery Co., Ltd., HH-S), carbon dioxide incubator (Japanese Panasonic Health Medical Instruments Co., Ltd., MCO-18AC ), biological safety cabinet (Guangzhou Qianjiang Experimental Technology Co., Ltd., ACZ-451), cell technology device (Shanghai Ruiyu Biotech, IC1000).

(4) Experimental methods

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).

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 /mL. Female hTIGIT mice were taken and inoculated subcutaneously with CT26 cells. The inoculation volume was 0.1 mL/mouse, that is, 2×10 ⁵ /mouse.

3) Dosing: The day of inoculation is recorded as day 0 (D0), and day 11 (D11). The mice are randomly divided into 5 groups according to tumor volume, with 8 mice in each group. Dosing begins (the dosing schedule is shown in Table 16 shown).

Table 16 Group Dosage (mg/kg) Dosing volume (μL/g) Route of administration Dosing 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

Note: In Table 16, the dosing volume is adjusted according to the animal's weight (10 L/g); if the body weight decreases by more than 15% during dosing, 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.

4) Observation records:

The tumor volume was measured and the mice's weight was recorded on D11. After that, the body weight was recorded twice a week and the long and short diameters of the tumors were measured with vernier calipers. The tumor volume was calculated using the formula: (1/2) × long diameter × (short diameter) ² .

5) Data calculation, statistics and analysis:

Independent samples T test can be used to compare between two groups. One-Way ANOVA was used for comparisons of more than 3 groups. If the F value shows a significant difference, post hoc analysis between multiple groups can be performed. Data were processed using Prism GraphPad, and p<0.05 indicated statistically significant differences. 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, Ti is the average tumor volume of the treatment group on day i, T0 is the average tumor volume of the treatment group at the beginning of treatment, Vi is the average tumor volume of the solvent control group on day i, and V0 is the average tumor volume of the solvent control group at the beginning of treatment.

(5) Experimental results

Anti-TIGIT humanized antibodies of hIgG1 subtype reduce the average tumor volume of CT26 colon cancer in hTIGIT knock in mice. The results are shown in Table 17 and Figure 5. The results show that compared with the isotype control group, anti-TIGIT of hIgG1 subtype Humanized antibodies 15H9L2 and 15H10L3 have significant inhibitory effects on the tumor growth of the hTIGIT knock in mouse CT26 model, with TGIs of 56.38% and 62.83% respectively; the anti-tumor effect of 316H1L1 is equivalent to that of the positive control group R0300, with TGIs of 36.26% and 36.26%, respectively. 34.79%.

Table 17 Study that Anti-TIGIT antibody reduces the mean tumor volume (mm ³ ) of CT26 colon cancer in hTIGIT knock in mice Days after implantation Isotype 15H9L2 (10mg/kg) 15H10L3 (10mg/kg) 316H1L1 (10mg/kg) R0300 (10mg/kg) 11 73.56 70.07 71.16 72.57 71.45 14 129.55 113.15 143.24 144.58 150.02 17 207.24 146.94 142.18 176.55 215.78 20 398.93 210.53 171.43 333.94 350.69 twenty four 910.40 340.72 294.64 517.77 559.18 27 1236.32 557.44 456.40 856.63 808.14 31 2161.64 980.86 847.27 1386.54 1433.18

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. 4. Evaluation of the in vivo anti-tumor efficacy of anti-TIGIT humanized antibodies (mIgG2a subtype) against colon cancer in CT26 mice

(1) Experimental purpose: To study the efficacy of mIgG2a subtype anti-TIGIT humanized antibodies (15H9L2, 15H10L3, 316H1L1) in the CT26 mouse colon cancer subcutaneous transplant tumor model, while setting up isotype negative control group (R0513) and positive Control group (R0300-mIgG2a, a Tiragolumab mutant in which the heavy chain constant region was replaced by mIgG2a).

(2) Experimental materials: hTIGIT Knock in mice, female, 6-8 weeks (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).

(3) Instruments and equipment: electronic balance (Shanghai Sunny Hengping Scientific Instrument Co., Ltd., JA12002), vernier caliper (Shanghai Minate 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 (Price Machinery Co., Ltd., HH-S), carbon dioxide incubator (Japanese Panasonic Health Medical Instruments Co., Ltd., MCO-18AC ), biological safety cabinet (Guangzhou Qianjiang Experimental Technology Co., Ltd., ACZ-451), cell counter (Shanghai Ruiyu Biotechnology, IC1000).

(4) Experimental methods

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).

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 /mL. Female hTIGIT mice were taken and inoculated subcutaneously with CT26 cells. The inoculation volume was 0.1 mL/mouse, that is, 2×10 ⁵ /mouse.

3) Dosing: The day of inoculation is recorded as day 0 (D0), and day 8 (D8). The mice are randomly divided into 5 groups according to tumor volume, with 8 mice in each group. Dosing begins (the dosing schedule is shown in Table 18 shown).

Table 18 Group Dosage (mg/kg) Dosing volume (μL/g) Route of administration Dosing 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 10 10 ip Q3D*4

Note: In Table 18, the dosing volume is adjusted according to the animal's weight (10 L/g); if the body weight decreases by more than 15% during the dosing period, the dosing regimen will be adjusted. Isotype stands for 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.

4) Observation records:

The tumor volume was measured and the mice's weight was recorded on D8. After that, the body weight was recorded twice a week and the long and short diameters of the tumors were measured with vernier calipers. The tumor volume was calculated using the formula: (1/2) × long diameter × (short diameter) ² . When each mouse reaches the endpoint of the experiment (a 15% decrease in body weight or a mercy endpoint when the tumor volume exceeds 2000 ^mm3 ), the mice are killed by _CO2 asphyxiation, and the survival curve is recorded.

5) Data calculation, statistics and analysis:

Independent samples T test can be used to compare between two groups. One-Way ANOVA was used for comparisons of more than 3 groups. If the F value shows a significant difference, post hoc analysis between multiple groups can be performed. Data were processed using Prism GraphPad, and p<0.05 indicated statistically significant differences. 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, Ti is the average tumor volume of the treatment group on day i, T0 is the average tumor volume of the treatment group at the beginning of treatment, Vi is the average tumor volume of the solvent control group on day i, and V0 is the average tumor volume of the solvent control group at the beginning of treatment.

Anti-TIGIT humanized antibodies of the mIgG2a subtype reduce the mean tumor volume of CT26 colon cancer in hTIGIT knock in mice. The results are shown in Table 19 and Figure 6. Anti-TIGIT humanized antibodies of the mIgG2a subtype reduce the mean tumor volume of CT26 colon cancer in hTIGIT knock in mice. The results of the impact on survival are shown in Figure 7. The results show that compared with the isotype control group, the anti-TIGIT humanized antibody 15H10L3 of the mIgG2a subtype has a significant inhibitory effect on tumor growth in the hTIGIT knock in mouse CT26 model. TGI The anti-tumor effect of 316H1L1 was equivalent to that of the positive control group R0300, with TGIs of 76.89% and 75.68% respectively.

Table 19 Study that Anti-TIGIT antibody reduces the mean tumor volume (mm ³ ) of CT26 colon cancer in hTIGIT knock in mice Days after implantation Isotype 15H9L2 (10mg/kg) 15H10L3 (10mg/kg) 316H1L1 (10mg/kg) R0300 (10mg/kg) 8 89.01 85.43 86.22 82.66 84.95 11 152.50 166.42 137.36 137.33 144.02 14 223.94 247.63 198.63 206.28 196.08 17 400.16 367.73 190.02 255.91 243.44 twenty one 867.42 547.86 278.70 358.66 353.78 twenty four 1382.88 757.28 341.39 482.46 423.82 28 2818.40 1319.52 524.63 717.06 744.31 31 \ 2024.05 696.22 1083.58 1220.31 37 \ 2989.35 1032.64 1855.84 2342.59

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. Example 5 Anti- TIGIT humanized antibody knocks out Treg cell function 1. Effect of anti-TIGIT humanized antibody on T cells in healthy human PBMC

Healthy human PBMC were used as target cells to study the impact of hIgG1 subtype anti-TIGIT humanized antibodies (15H9L2, 15H10L3, 316H1L1) on CD8+T cells and Treg cells in healthy human PBMC, and a positive control group (R0300- hIgG1 , a Tiragolumab mutant containing the K214R mutation in the heavy chain). The specific method is as follows:

Isolate PBMC from fresh blood, adjust the cell density to 5E5 cells/mL with 10% FBS+x-vivo medium, and place in an incubator overnight. PBMC cell count: donor-167 8.44E+05 cells/mL, 81.15%, take 20 mL of cells, donor-234 5.53E+05 cells/mL, 78.38%, take 20 mL of cells; NK92-CD16-5 cell count: P11 2.21E+05 cells/mL, 96.05%, take 33 mL of cells; centrifuge at 400g for 5 minutes, remove the supernatant, and wash the cells with 10% FBS+x-vivo medium (containing 1000 IU/mL IL-2) for 1 times; add 10% FBS+x-vivo medium (containing 1000 IU/mL IL-2) to resuspend, and count the cells (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+06 cells/mL, 97.08%, 1.5 mL), adjust the cell density to 2E+06 cells/mL respectively; antibody dilution ( All were 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 antibodies and SEB were added to 50 μL each. /well volume was added to the corresponding 96-well U-shaped plate, and the blank group was supplemented with the corresponding volume of 10% FBS+x-vivo culture medium (containing 1000 IU/mL IL-2). The total volume of each well was 200 μL. ;Continue to culture in the incubator for 40h.

After 40 hours, take out the cell plate, transfer the cells to a V-shaped plate, and wash once with 3% BSA buffer; mix the recommended amounts of various primary antibodies and add them to the cells, and mix gently (according to the recommended amount in the instruction manual). Incubate in the dark for 30 minutes at 2-8°C; add 100ul of 3% BSA buffer to wash the cells: centrifuge at 350g for 5 minutes and discard the supernatant. Add 200ul/well 3% BSA buffer, centrifuge at 350g for 5 minutes, and discard the supernatant. Add 200 µL Foxp3 fixation/membrane-breaking working solution to each well. Completely resuspend the cells in the added solution and incubate in the dark at 2-8°C for 60 minutes. Centrifuge at 400 g for 5 min. Discard supernatant. Add 200 µL 1× membrane-breaking buffer, centrifuge at 400 g for 5 min, discard the supernatant, and repeat once. Resuspend the pellet in the remaining volume and adjust the volume to approximately 100 μL with 1× membrane rupture buffer. Without washing, add the recommended amount of direct-coupled antibody (Foxp3) to detect intracellular antigens and incubate for 30 min at room temperature, protected from light. Add 200 µL of 1× membrane-breaking buffer to each well, and centrifuge at 400 g for 5 min at room temperature. Discard the supernatant and repeat. Resuspend stained cells in 300 µL of PBS. Pass through a 200 mesh gauze and transfer to a 1.5 mL centrifuge tube. On the machine, analyze by flow cytometry.

Figure 8a is a diagram of the staining results of a single tube; Figure 8b is a diagram of the analysis steps; Figure 8c is a ratio diagram of CD4+ /CD8+ T cells; Figure 8d is a ratio diagram of Treg cells; Figure 8e is using human peripheral blood mononuclear cells as target cells ADCC result chart; the results show that the anti-TIGIT humanized antibodies 15H9L2 and 15H10L3 of the hIgG1 subtype can effectively reduce the proportion of Treg cells, while the anti-TIGIT humanized antibody 316H1L1 of the hIgG1 subtype is equivalent to the positive control group R0300, with a higher The function of Treg cells was weakly knocked out, and the anti-TIGIT humanized antibody of hIgG1 subtype had no significant effect on the proportion of CD8+ T cells.

Then, more samples were tested according to the above method.

The test results of more samples are shown in Figure 9. The results show that the anti-TIGIT humanized antibodies 15H9L2 and 15H10L3 of hIgG1 subtype knockout Treg cells have stronger functions, while the anti-TIGIT humanized antibody 316H1L of hIgG1 subtype is more effective in knocking out Treg cells. Although there is no significant difference in function of Treg cells from the positive control group R0300, the proportion of Treg cells in cells treated with 316H1L is lower; compared with the positive control group, anti-TIGIT humanized antibodies of the hIgG1 subtype can reduce the number of Treg cells. cell ratio. 2. Effects of anti-TIGIT humanized antibodies on T cells in tumor cell model mouse tissues

(1) Experimental purpose: To study the mechanism of action of the anti-TIGIT humanized antibody 15H9L2-mIgG2a of mIgG2a subtype in the CT26 mouse colon cancer subcutaneous transplantation tumor model, and set up an isotype control group (R0513) and a positive control group (R0300- mIgG2a).

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

Table 20 Name brand Item number hTIGIT Knock in mouse female, 6-8 weeks Jiangsu Jicui Yaokang Biotechnology Co., Ltd. Certificate number: 202102514 CT26 cells Shanghai Cell Bank Catalog Number: TCM37 RPMI 1640 Gibco 11875085 FBS Gibco 10091-148 0.25% trypsin-EDTA Gibco 25200056 Penicillin-Streptomycin Gibco 15140122 DPBS Hyclone SH30028.02 Anti-mouse/Rat Foxp3 PE Invitrogen 12-5173-80A Rat IgG2a,k,iso Control PE Invitrogen 124321-81A Brilliant Violet 510TM anti-mouse CD45, Rat IgG2b Biolegend 103138 APC/Cyaninne7 anti-mouse CD3, Rat IgG2b Biolegend 100222 Anti-mouse CD25 APC, Rat/IgG2a Invitrogen 11-0042-81A Paeiific Blue ^TM anti-mouse CD8a, Rat IgG2a Biolegend 100725 Anti-mouse CD25 APC, Rat IgG1 Invitrogen 17-0251-81A Tumor Dissociation Kit, mouse Miltenyi 130-096-730 ACK Lysing Buffer Gibco A10492-01 DPBS Cytiva SH0028.02 CD45 MIcroBeads, mouse Miltenyi 130-052-301 eBioscience ^TM Fixation/Perm Diluent Invitrogen 00-5223-56 Permeabilization Buffer 10X Invitrogen 008333-56 Fixation/Permeabilization Concentrate Invitrogen 00-5123-43

(3) Experimental methods

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).

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 /mL. Female hTIGIT mice were taken and inoculated subcutaneously with CT26 cells. The inoculation volume was 0.1 mL/mouse, that is, 2×10 ⁵ /mouse.

3) Administration: The day of inoculation is recorded as day 0 (D0). On day 8 (D8), the tumor volume is 60-80 mm ^{. 3.} Select 40 animals for in vivo drug efficacy studies, and the remaining 15 animals will have tumor volumes of 300-500 mm. At ³ o'clock, the mice were randomly divided into 3 groups according to tumor volume, with 3 mice in each group, and drug administration was started (the drug administration schedule is shown in Table 21).

The day after the second administration, the mice were euthanized, and the tumors and spleens were dissected and dissected. After processing, the lymphocytes in the spleen and tumor cells were analyzed.

Table 21 Group Dosage (mg/kg) Dosing volume (μL/g) Route of administration Dosing frequency Isotype-mIgG2a 10 10 ip Q3D*2 R0300-mIgG2a 10 10 ip Q3D*2 15H9L2-mIgG2a 10 10 ip Q3D*2

Note: In Table 21, the dosing volume is adjusted according to the animal's weight (10 L/g); if the body weight decreases by more than 15% during the dosing period, the dosing regimen will be adjusted. Isotype stands for isotype control group (R0513). In addition, in the table, i.p. indicates intraperitoneal injection, and Q3D*2 indicates administration once every 3 days, for a total of 2 administrations.

4) Analysis of mouse spleen and tumor lymphocytes

a. Digestion of tumor tissue:

(1) Prepare 0.04g-1g of tumor tissue and cut it into small pieces (try to cut it into small pieces) into 2-4 mm.

(2) Digestive solution preparation: Add 2.35 mL of 1640 culture medium into a 5 mL centrifuge tube, then add 100 uL enzyme D, 50 uL enzyme R, and 12.5 uL enzyme A (if TILs analysis is performed, it is recommended to reduce the amount of enzyme R to 20% , for example: add 10 uL enzyme R to 2.5ml digestion solution).

(3) Add the chopped tumor tissue to the prepared digestive juice, digest at 37°C for 40 minutes, and blow evenly every 3-5 minutes.

(4) Place the cell strainer on a 50 mL centrifuge tube, remove the digested tissue supernatant, and add the cell strainer; add 10 mL of 1640 culture medium to the tissue fragments to rinse, and add the cell strainer to completely filter it.

(5) Centrifuge at 300g for 7 minutes, remove the supernatant, and the digested cells can be obtained.

(6) Experimental steps for CD45+ magnetic bead separation:

a) Prepare 250mL of separation buffer in advance: i. 50mL PBS+ 250μL FBS+ 125μL EDTA (0.8M) in ice bath for later use; b) Count the first part of digested tumor cells, take 5E7 cells and centrifuge at 300g for 10 minutes, discard the supernatant; c) Resuspend every 10e7 cells in 90μL separation buffer according to the number of tumor cells; d) Add 10μL of CD45 Microbeads per 10e7 cells and mix; e) Incubate at 4℃-8℃ for 15 minutes; f) Add 1-2mL of separation buffer to the test tube for each 1E7 cell, and centrifuge at 300g×4min for 10min; g) Discard the supernatant and add 500 μL of separation buffer to resuspend the cells (within 1E8 cells); h) Separation column preparation: add 500ul separation buffer to rinse the isolate (500ul/MS, 3ml/LS); i) Add the mixed cell suspension to the separation column (adsorbed to the magnet), and wait for all the liquid to naturally drip into the centrifuge tube; j) Add 500ul separation buffer again and wash 3 times (3×500ul/MS, 3×3ml/LS); k) Add 1mL of separation buffer to the separation column again, and remove the magnet at the same time. Add a piston to the separation column to push the cells out into a new centrifuge tube. You can also add 1mL of separation buffer to the separation column and repeat the above process. ; l) Centrifuge the collected cells, wash them once with PBS containing 1% FBS, resuspend them in flow cytometry staining solution, and count; m) Adjust the cell density to 2E7 cells/ml, and add an equal amount of 100 μL cell suspension to each well of the 96-well plate (can be adjusted according to the number of cells).

b. Spleen processing:

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

2) Centrifuge at 300g for 7 minutes, remove the supernatant, and the digested cells can be obtained.

3) Red blood lysis: After letting the blood sample stand for 30 minutes, add 1 mL of red blood cell lysis solution (depending on the volume of blood taken), pipet evenly, leave it for 5 minutes, centrifuge at 300-400g for 5 minutes, and discard the supernatant (if visible to the naked eye) Red blood cells are still visible, repeat the previous steps until red blood cells are completely broken).

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

c. Fluorescent staining

1) Block non-specific Fc-mediated interactions: Before staining mouse cells, pre-incubate the cells with 0.5-1 μg purified anti-mouse CD16/CD32/100 μL at 2-25°C for 10-20 minutes.

2) Mix the recommended amounts of various primary antibodies and add them to the cells, mix gently (according to the recommended amount in the instructions).

3) Incubate at 2-8℃ or on ice in the dark for more than 30 minutes.

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

5) 200ul/well, repeat step 4.

6) After the last wash, discard the supernatant and flick the sample to completely dissociate the pellet.

7) Add 200 µL Foxp3 fixation/membrane rupture 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).

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

9) Add 200 µL 1× membrane-breaking solution to each well, and centrifuge the sample at 400-600 g for 5 minutes at room temperature. Discard supernatant.

10) Repeat step 9.

11) Resuspend the pellet in the remaining volume and adjust the volume to approximately 100 μL with 1X membrane-breaking solution.

12) Without washing, add the recommended amount of direct-coupled antibody to detect intracellular antigens and incubate at room temperature for more than 30 minutes. Avoid light.

13) Add 200 µL 1× membrane-breaking solution to each well, and centrifuge the sample at 400-600 g for 5 minutes at room temperature. Discard supernatant.

14) Repeat step 14.

15) Resuspend the stained cells in an appropriate volume of flow cytometry staining solution. Analysis by flow cytometry.

Figure 10a is a graph showing the effect of anti-TIGIT humanized antibodies on T cells in tumor cell model mouse tissues. Figure 10b is a graph showing the effects of anti-TIGIT humanized antibodies on CD3+T cells and CD4+T cells in tumor cell model mouse tissues. The results show that compared with the isotype control group, the anti-TIGIT humanized antibody 15H9L2 of the mIgG2a subtype and the positive control group R0300 can significantly reduce the proportion of Treg cells in tumor-infiltrating lymphocytes, and 15H9L2 has a more significant effect than R0300. Example 6 Evaluation of Druggability of Anti- TIGIT Humanized Antibodies

Druggability assessment can use early pressurization experiments and stability studies to amplify potential risk sites, such as deamidation, isomerization, oxidation and other post-translational modification sites. The process can also establish links between stress conditions and changes in specific antibody risk sites, making it possible to detect environments that are unfriendly to antibody molecules as early as possible. Because during the entire preparation process, antibody molecules will experience a variety of different environmental conditions, such as high temperature and oxidation during the culture process; acid and alkali conditions during the purification process; extreme acidic conditions during virus extinguishing; freezing and thawing during long-term storage or transportation , oscillation and other conditions, early detection of factors that are unstable to antibodies can provide corresponding reference strategies for later development. Molecules with very poor stability can also be eliminated during the early druggability evaluation process to reduce the risk of late project failure and ensure the reliability of candidate molecules entering subsequent clinical stages. The druggability evaluation of anti-TIGIT humanized antibodies of hIgG1 subtype (15H9L2, 15H10L3, 316H1L1) is as follows: 1. Evaluation of the stability of anti-TIGIT humanized antibodies against acids and bases

First, replace all samples into 20 mmol (PH6.0) sodium acetate buffer, and adjust the sample concentration to 3 mg/ml. Take 2.5ml of each sample and adjust the pH to 3.5 with 0.5mol Citrate, or take 2.5ml of each sample and adjust the pH to 9.0 with 1M Tris-HCl. Aliquot it into 1.5ml EP centrifuge tubes and number them. Treat at room temperature for 0h, 4h, and 22h. After that, the pH was immediately adjusted back to around 6.0, samples were sent for physical, chemical and activity testing in a timely manner, and some samples were selected for mass spectrometry analysis based on the physical and chemical testing results.

Figures 11a and 11b are the acid and alkali treatment results of SEC-HPLC detection of anti-TIGIT humanized antibodies; Figure 11c and 11d are the acid and alkali treatment results of CE-SDS-NR detection of anti-TIGIT humanized antibodies. Figures; Figure 11e, Figure 11f, and Figure 11g are the cIEF detection results of the acidic peak change, main peak change, and alkali peak change of the anti-TIGIT humanized antibody after acid treatment; Figure 11h, Figure 11i, and Figure 11j are the anti-TIGIT humanized antibody, respectively. The cIEF test of humanized antibodies shows the changes in acidic peaks, main peaks, and alkaline peaks after alkali treatment. The results show that the anti-TIGIT humanized antibodies were treated with acid (pH3.5) and alkali (pH9.0) for 4 and 22 hours. , the purity results are stable. 2. Evaluation of the stability of anti-TIGIT humanized antibodies to repeated freezing and thawing

Replace all samples into 20 mmol (PH 6.0) sodium acetate buffer, and adjust the sample concentration to 3 mg/ml. After repeated freezing and thawing at -80°C and room temperature, samples were promptly sent for physical and chemical testing and activity testing, and some samples were selected for mass spectrometry analysis based on the physical and chemical testing results.

Figure 12a is a graph showing the results of SEC-HPLC detection of anti-TIGIT humanized antibodies and repeated freeze-thaw processing; Figure 12b is a graph showing the results of repeated freeze-thaw processing of CE-SDS-NR detection of anti-TIGIT humanized antibodies; Figure 12c, Figure 12d, Figure 12e shows the results of the cIEF detection of the anti-TIGIT humanized antibody after repeated freezing and thawing treatment of the acidic peak change, main peak change, and basic peak change. The results show that after 5 freeze-thaw cycles of the anti-TIGIT humanized antibody, the detection results stability. 3. Evaluation of the stability of anti-TIGIT humanized antibodies to high temperatures

Replace all samples into 20 mmol (PH 6.0) sodium acetate buffer, and adjust the sample concentration to 3 mg/ml. Divide the three antibody samples into two groups. The first group is controlled at 4°C, and the second group is placed in a constant temperature incubator at 37°C. Samples are taken into 1.5ml EP tubes at different time points and sealed with sealing film. On Day 0, Day 7, and Day 14 , 28 Day submission for inspection and analysis, and select some samples for mass spectrometry analysis based on the physical and chemical test results.

Figure 13a and Figure 13b are the results of SEC-HPLC detection of high-temperature treatment and 4°C control treatment of anti-TIGIT humanized antibodies respectively; Figures 13c and Figure 13d are respectively the results of CE-SDS-NR detection of anti-TIGIT humanized antibodies after high-temperature treatment. , 4°C control treatment results; Figure 13e, Figure 13f, and Figure 13g are the results of cIEF detection of anti-TIGIT humanized antibody in acidic peak changes, main peak changes, and alkali peak changes after high temperature treatment; Figure 13h, Figure 13i, Figure 13j are the results of cIEF detection of anti-TIGIT humanized antibody after 4°C control treatment of acidic peak changes, main peak changes, and alkaline peak changes. The results show that after high-temperature treatment of anti-TIGIT humanized antibodies for 28 days, the detection results are stable. 4. In vitro binding activity of anti-TIGIT humanized antibodies after stress test

The anti-TIGIT humanized antibody has been tested under acid, alkali, repeated freezing and thawing, high temperature and other stress tests, and no significant changes were found in the physical and chemical properties, indicating its good physical and chemical stability. We then further tested the in vitro binding activity of the anti-TIGIT humanized antibody to prove that the anti-TIGIT humanized antibody can maintain good activity under stress conditions.

Figure 14a, Figure 14b, and Figure 14c are the in vitro binding activity diagrams of the anti-TIGIT humanized antibodies 316H1L1, 15H9L2, and 15H10L3 of the hIgG1 subtype after stress testing. The results show that the anti-TIGIT humanized antibodies were tested under various stress conditions. , still maintains good antigen-binding activity.

The technical features of the above-described embodiments can be combined in any way. To simplify the description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, All should be considered to be within the scope of this manual.

The above-mentioned embodiments only express several implementation modes of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention. It should be noted that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the scope of protection of the patent of the present invention should be determined by the appended claims.

without

In order to more clearly explain the specific embodiments of the present invention or the technical solutions in the prior art, the drawings that need to be used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are: For some embodiments of the present invention, those of ordinary skill in the art can also obtain other drawings based on these drawings without exerting creative efforts. Figure 1 is a graph showing the results of different subtypes of anti-TIGIT antibodies reducing the average tumor volume of CT26 colon cancer in Balb/c mice. Figure 2 is a graph showing the effects of different subtypes of anti-TIGIT antibodies on the survival of Balb/c mice. Figure 3 is a graph showing the results of TIGIT-5 mouse monoclonal antibody and 3TIGIT-16 mouse monoclonal antibody reducing the average tumor volume of CT26 colon cancer in hTIGIT knock in mice. Figure 4 is a graph showing the effects of TIGIT-5 mouse monoclonal antibody and 3TIGIT-16 mouse monoclonal antibody on the survival of hTIGIT knock in mice. Figure 5 is a graph showing the results of anti-TIGIT humanized antibodies of hIgG1 subtype reducing the average tumor volume of CT26 colon cancer in hTIGIT knock in mice. Figure 6 is a graph showing the results of anti-TIGIT humanized antibodies of the mIgG2a subtype reducing the average tumor volume of CT26 colon cancer in hTIGIT knock in mice. Figure 7 is a graph showing the effect of anti-TIGIT humanized antibodies of mIgG2a subtype on the survival of hTIGIT knock-in mice. Figure 8a is a diagram of the staining results of a single tube; Figure 8b is a diagram of the analysis steps; Figure 8c is a ratio diagram of CD4+ /CD8+ T cells; Figure 8d is a ratio diagram of Treg cells; Figure 8e is using human peripheral blood mononuclear cells as target cells ADCC result graph. Figure 9 is a graph of the test results of more samples. Figure 10a is a graph showing the effect of anti-TIGIT humanized antibodies on T cells in tumor cell model mouse tissues; Figure 10b is a graph showing the effects of anti-TIGIT humanized antibodies on CD3+T cells and CD4+T cells in tumor cell model mouse tissues. Impact results graph. Figure 11a is the SEC-HPLC acid detection result of the anti-TIGIT humanized antibody; Figure 11b is the SEC-HPLC alkali treatment result of the anti-TIGIT humanized antibody; Figure 11c is the CE-SDS of the anti-TIGIT humanized antibody -NR acid detection result chart; Figure 11d is the CE-SDS-NR detection alkali treatment result chart of anti-TIGIT humanized antibody; Figure 11e is the cIEF detection acidic peak change result chart of anti-TIGIT humanized antibody after acid treatment; Figure 11f is the cIEF detection result of the anti-TIGIT humanized antibody showing the change of the main peak after acid treatment; Figure 11g is the cIEF detection result of the anti-TIGIT humanized antibody showing the change of the base peak after acid treatment; Figure 11h is the result of the anti-TIGIT humanized antibody Figure 11i is the cIEF detection result of the main peak change after alkali treatment of the anti-TIGIT humanized antibody; Figure 11j is the cIEF detection result of the main peak change of the anti-TIGIT humanized antibody after alkali treatment. Figure. Figure 12a is a graph showing the results of SEC-HPLC detection of anti-TIGIT humanized antibodies and repeated freeze-thaw processing; Figure 12b is a graph showing the results of repeated freeze-thaw processing of CE-SDS-NR detection of anti-TIGIT humanized antibodies; Figure 12c is a graph showing the results of anti-TIGIT humanized antibodies Figure 12d is the cIEF detection result of the anti-TIGIT humanized antibody and the main peak change after repeated freezing and thawing treatment; Figure 12e is the cIEF of the anti-TIGIT humanized antibody. Detection of changes in alkaline peaks after repeated freezing and thawing. Figure 13a is a graph showing the results of SEC-HPLC detection of anti-TIGIT humanized antibodies after high temperature treatment; Figure 13b is a graph showing the results of SEC-HPLC detection of anti-TIGIT humanized antibodies subjected to 4°C control treatment; Figure 13c is a graph showing the results of SEC-HPLC detection of anti-TIGIT humanized antibodies The CE-SDS-NR detection results of high-temperature treatment; Figure 13d is the CE-SDS-NR detection of anti-TIGIT humanized antibody after 4°C control treatment; Figure 13e is the cIEF detection of anti-TIGIT humanized antibody acidity after high-temperature treatment Peak change results; Figure 13f is the cIEF detection result of the anti-TIGIT humanized antibody after high-temperature treatment; Figure 13g is the cIEF detection of the anti-TIGIT humanized antibody alkali peak change results after high-temperature treatment; Figure 13h is the anti- Figure 13i is the cIEF detection result of the anti-TIGIT humanized antibody and the change of the main peak after the control treatment at 4°C. Figure 13i is the result of the cIEF detection of the anti-TIGIT humanized antibody and the main peak change after the control treatment at 4°C. Figure 13j is the result of the anti-TIGIT humanized antibody. cIEF detection results chart of alkali peak changes after control treatment at 4°C. Figure 14a is a graph of the in vitro binding activity of the anti-TIGIT humanized antibody 316H1L1 of the hIgG1 subtype after the stress test; Figure 14b is a graph of the in vitro binding activity of the anti-TIGIT humanized antibody 15H9L2 of the hIgG1 subtype after the stress test; Figure 14c It is the in vitro binding activity diagram of anti-TIGIT humanized antibody 15H10L3 of hIgG1 subtype after stress test; among them, R0750 represents 316H1L1, R0771 represents 15H9L2, and R0774 represents 15H10L3.

TW202321307A_111136259_SEQL.xml

Claims (13)

An anti-TIGIT humanized antibody or an antigen-binding fragment thereof, the antibody comprising 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 selected from SEQ ID NO: 29 to 31 or SEQ ID NO: 32 to 34, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 are selected from SEQ ID NO: 35 to 37 or SEQ ID NO. :38～40, wherein the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO:14, or as SEQ ID NO:5～13, SEQ ID NO:15 or SEQ ID NO:21～ 28 either shown. The anti-TIGIT humanized antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO: 3, or as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 16 to 20. The anti-TIGIT humanized antibody or antigen-binding fragment thereof as described in claim 1 or 2, wherein the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO: 14, or as shown in SEQ ID NO: 5 ~13 and 15 are any one shown, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:3, or as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:4 or the amino acid sequence of the heavy chain variable region of the antibody is as shown in any one of SEQ ID NO: 21 to 28, and the amino acid sequence of the light chain variable region of the antibody is as SEQ ID NO: 16 Any one of ~20 is shown; Optionally, the amino acid sequences of the light chain variable region and heavy chain variable region of the antibody are as follows: No. light chain variable region heavy chain variable region 1 SEQ ID NO:3 SEQ ID NO:14 2 SEQ ID NO:16 SEQ ID NO:21 3 SEQ ID NO:1 SEQ ID NO:5 4 SEQ ID NO:1 SEQ ID NO:12 5 SEQ ID NO:4 SEQ ID NO:11 6 SEQ ID NO:4 SEQ ID NO:15 7 SEQ ID NO:2 SEQ ID NO:12 8 SEQ ID NO:2 SEQ ID NO:13 9 SEQ ID NO:2 SEQ ID NO:14 10 SEQ ID NO:2 SEQ ID NO:15 11 SEQ ID NO:4 SEQ ID NO:14 12 SEQ ID NO:16 SEQ ID NO:22 13 SEQ ID NO:20 SEQ ID NO:28 14 SEQ ID NO:19 SEQ ID NO:24 15 SEQ ID NO:20 SEQ ID NO:24 16 SEQ ID NO:18 SEQ ID NO:25 17 SEQ ID NO:18 SEQ ID NO:26 18 SEQ ID NO:18 SEQ ID NO:27 . The anti-TIGIT humanized antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 3, and the heavy chain The amino acid sequence of the 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 SEQ ID NO :21; or the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:13. The anti-TIGIT humanized antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody contains a heavy chain constant region and a light chain constant region, and 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; optionally, the heavy chain constant region sequence is selected from IgG1; The light chain constant region is a kappa or lambda chain; optionally, the light chain constant region sequence is selected from a kappa chain; The species source of the heavy chain constant region and the light chain constant region is selected from cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, and ducks , goose or human being. The anti-TIGIT humanized antibody or antigen-binding fragment thereof as described in claim 5, wherein the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 53 or as shown in any one of SEQ ID NO: 45 to 48 , the amino acid sequence of the light chain constant region is shown in SEQ ID NO:50 or SEQ ID NO:49; optionally, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:53, and the light chain The amino acid sequence of the constant region is shown in SEQ ID NO:50; optionally, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:51, and the amino acid sequence of the light chain is shown in SEQ ID NO:52 . The anti-TIGIT humanized antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antigen-binding fragment is any one of F(ab')2, Fab, scFv and bispecific antibodies . A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7; Preferably, 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. . A vector comprising the nucleic acid according to claim 8. A cell that carries the nucleic acid as described in claim 8, contains the vector as described in claim 9, or expresses the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 7. A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, comprising: culturing the cells according to claim 10 in a culture medium; and recovering the antibody from the culture medium or from the cultured cells. of antibodies or antigen-binding fragments thereof. A pharmaceutical composition, characterized in that the composition contains the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 7, or the nucleic acid as described in claim 8, or the vector as described in claim 9 Or a cell as described in claim 10; optionally, the pharmaceutical composition includes a pharmaceutically acceptable carrier and/or excipient. An antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 7, a nucleic acid as described in claim 8, a vector as described in claim 9, a cell as described in claim 10, as described in claim 12 Application of pharmaceutical compositions in the preparation of medicaments for preventing or treating immune diseases or tumor-related diseases.

Images