WO2021093849A1

WO2021093849A1 - A novel antibody against tigit

Info

Publication number: WO2021093849A1
Application number: PCT/CN2020/128659
Authority: WO
Inventors: Yong Zheng; Jie Luo; Jing Li
Original assignee: Wuxi Biologics (Shanghai) Co., Ltd.; WuXi Biologics Ireland Limited
Priority date: 2019-11-14
Filing date: 2020-11-13
Publication date: 2021-05-20
Also published as: TW202124452A; CN114641502A; CN114641502B

Abstract

The present invention provides TIGIT monoclonal antibodies, particularly humanized monoclonal antibodies specifically binding to TIGIT with high affinity. The present invention also provides functional monoclonal antibodies cross-reactive to TIGIT of human, cynomolgus monkey and mouse. The present invention further provides amino acid sequences of the antibodies of the invention, cloning or expression vectors, host cells and methods for expressing or isolating the antibodies. The epitopes of the antibodies are identified. Therapeutic compositions comprising the antibodies of the invention are also provided. The invention also provides methods for treating cancers and other diseases with anti-TIGIT antibodies.

Description

A novel antibody against TIGIT

Technical Field

The present invention relates generally to antibodies against TIGIT and compositions thereof, and therapy in the treatment of tumor or inflammatory diseases using anti-TIGIT antibodies.

Background of the Invention

TIGIT (T cell immune receptor with Ig and ITIM domains) also known as Vstm3 and WUCAM is an inhibitory receptor that is expressed on NK and CD8+T cells, as well as a subset CD4+ T cells including immunosuppressive Tregs (X. Yu, K. Harden, L.C. Gonzalez et al., Nature Immunology, vol. 10, no. 1, pp. 48–57, 2009.; K.S. Boles, W. Vermi, F. Facchetti et al., European Journal of Immunology, vol. 39, no. 3, pp. 695–703, 2009.; N. Stanietsky, H. Simic, J. Arapovic et al., Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 42, pp. 17858–17863, 2009.; R.J. Johnston, L. Comps-Agrar, J. Hackney et al., Cancer Cell, vol. 26, no. 6, pp. 923–937, 2014.; N. Joller, E. Lozano, P.R. Burkett et al., Immunity, vol. 40, no. 4, pp. 569–581, 2014. ) . It has been identified as a member of CD28 family based on gene structure (S.D. Levin, D.W. Taft, C.S. Brandt et al., European Journal of Immunology, vol. 41, no. 4, pp. 902–915, 2011) . TIGIT binds to CD155 and CD112, both of which are expressed by tumors and antigen-presenting cells, resulting in immune suppression (Stanietsky, N. et al. Proc. Natl. Acad. Sci. U.S.A. 106, 17858–17863, 2019.; Stengel, K.F. et al. Proc. Natl. Acad. Sci. U.S.A. 109, 5399–5404, 2012.; Stanietsky, N. et al. Eur. J. Immunol. 43, 2138–2150, 2013. ) . These ligands also bind the co-stimulatory molecule CD226 resulting in NK and T cell activation. Blockade antibodies of TIGIT disrupt binding of TIGIT to its ligands and block its inhibitory signals, shifting the balance in favor of CD226-mediated activating signals, which induces a strong anti-tumor immune response (E. Lozano, M. Dominguez-Villar, V. Kuchroo, and D.A. Hafler, Journal of Immunology, vol. 188, no. 8, pp. 3869–3875, 2012.; K.E. Pauken and E.J. Wherry, et al., Cancer Cell, vol. 26, no. 6, pp. 785–787, 2014.; Stamm H, Wellbrock J, Fiedler W. Mamm Genome. 29 (11-12) : 694-702, 2018. ) . TIGIT is up regulated and identified as a exhaustion marker in cancer and inflammatory diseases (Chauvin JM, Pagliano O, Fourcade J, Sun Z, Wang H, Sander C, et al. J Clin Invest 2015; 125: 2046–58.; Y. Kong, L. Zhu, T.D. Schell et al., Clinical Cancer Research, vol. 22, no. 12, pp. 3057–3066, 2016.; Johnston RJ, Comps-Agrar L, Hackney J, Yu X, HuseniM, Yang Y, et al. Cancer Cell 2014; 26: 923–37. ) as other exhaustion markers like PD-1, LAG3 and TIM3 (Fleury M, Belkina AC, Proctor EA et al., Arthritis Rheumatol. 2018 Apr; 70 (4) : 566-577.; Fromentin R, Bakeman W, Lawani MB, et al., PLoS Pathog. 2016 Jul 14; 12 (7) ) . Recently, more and more studies demonstrate that TIGIT is expressed with PD-1 in parallel (Josefsson SE, Beiske K, Blaker YN, et al., Cancer Immunol Res. 2019 Mar; 7 (3) : 355-362; Niclas C. Blessin, Ronald Simon, et al., Dis Markers. 2019; 2019: 5160565) in tumor tissues and blockade TIGIT with PD-1 could reverse immune suppression (Hung AL, Maxwell R, Theodros D, Belcaid Z, et al., Oncoimmunology. 2018 May 24; 7 (8) ) . Over all, TIGIT could be a promising therapeutic target for tumor immunotherapy as single agent or in combination with other immune modulators.

Disclosure of the Invention

The present invention provides isolated antibodies, in particular monoclonal antibodies or fully human antagonist antibody against TIGIT.

In one aspect, the present invention provides an antibody or an antigen binding-fragment thereof, wherein the antibody or the antigen binding fragment binds to human, Cynomolgus and mouse TIGIT.

The present invention provides an antibody, or an antigen-binding fragment thereof, comprising:

a) a variable region of a heavy chain having an amino acid sequence that is at least 70%, 80%, 90%, 95%or 99%homologous to a sequence of SEQ ID NO: 7; and

b) a variable region of a light chain having an amino acid sequence that is at least 70%, 80%, 90%, 95%or 99%homologous to a sequence SEQ ID NO: 8,

wherein the antibody or the antigen binding fragment specifically binds to TIGIT.

In various embodiments, the antibody or an antigen binding fragment thereof comprises:

a) a variable region of a heavy chain having an amino acid sequence of SEQ ID NO: 7; and

b) a variable region of a light chain having an amino acid sequence of SEQ ID NO: 8,

In another aspect, the invention provides an antibody, or an antigen binding fragment thereof, comprising: a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences; and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences,

wherein the heavy chain variable region CDR3 sequence comprises an amino acid sequence of SEQ ID NO: 5, and conservative modifications thereof,

Preferably, wherein the light chain variable region CDR3 sequence comprises an amino acid sequence of SEQ ID NO: 6, and conservative modifications thereof.

Preferably, wherein the heavy chain variable region CDR2 sequence comprises an amino acid sequence of SEQ ID NO: 3, and conservative modifications thereof.

Preferably, wherein the light chain variable region CDR2 sequence comprises an amino acid sequence of SEQ ID NO: 4, and conservative modifications thereof.

Preferably, wherein the heavy chain variable region CDR1 sequence comprises an amino acid sequence of SEQ ID NO: 1, and conservative modifications thereof.

Preferably, wherein the light chain variable region CDR1 sequence comprises an amino acid sequence of SEQ ID NO: 2, and conservative modifications thereof.

A preferred antibody or an antigen binding fragment thereof comprises:

a) a heavy chain variable region CDR1 comprising SEQ ID NO: 1;

b) a heavy chain variable region CDR2 comprising SEQ ID NO: 3;

c) a heavy chain variable region CDR3 comprising SEQ ID NO: 5;

d) a light chain variable region CDR1 comprising SEQ ID NOs: 2;

e) a light chain variable region CDR2 comprising SEQ ID NOs: 4;

f) a light chain variable region CDR3 comprising SEQ ID NOs: 6;

wherein the antibody or the antigen binding-fragment specifically binds to TIGIT.

The antibodies of the invention can be chimeric antibody.

The antibodies of the invention can be humanized antibody.

The antibodies of the invention can be fully human antibody.

The antibodies of the invention can be rat antibody.

In a further aspect, the invention provides a nucleic acid molecule encoding the antibody, or antigen binding fragment thereof.

The invention provides a cloning or expression vector comprising the nucleic acid molecule encoding the antibody, or antigen binding fragment thereof.

The invention also provides a host cell comprising one or more cloning or expression vectors.

In yet another aspect, the invention provides a process, comprising culturing the foresaid host cell and isolating the antibody;

wherein, the antibody is prepared through immunization in a rat with human TIGIT protein.

In a further aspect, the invention provides pharmaceutical composition comprising the antibody, or the antigen binding fragment of said antibody in the invention, and one or more of a pharmaceutically acceptable excipient, a diluent or a carrier.

The invention also provides a method for preparing an anti-TIGIT antibody or an antigen-binding fragment thereof comprising:

(a) providing:

(i) a heavy chain variable region antibody sequence comprising a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO: 3; and a CDR3 sequence of SEQ ID NO: 5; and/or

(ii) a light chain variable region antibody sequence comprising a CDR1 sequence of SEQ ID NO: 2, a CDR2 sequence of SEQ ID NO: 4, and a CDR3 of SEQ ID NO: 6; and

(b) expressing the altered antibody sequence as a protein.

The invention also provides a method of preventing or treating a disease associated with bone loss in a subject comprising administering to a subject in need a therapeutically effective amount of foresaid antibody or antigen-binding fragment in this invention.

The invention also provides a combined method of preventing or treating tumor or inflammatory disease comprising administering to a subject in need a therapeutically effective amount of foresaid antibody or antigen-binding fragment in the invention and administering to the subject a therapeutically effective amount of immune checkpoint antibody.

The invention also provides the use of said antibody or the antigen binding fragment thereof in the manufacture of a medicament for the prevention or treatment of tumor or inflammatory disease.

Said cancer is selected from a group consisting of melanoma, renal cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, liver cancer, gastrointestinal cancer, glioblastoma, cervical cancer, bladder cancer, and rectal cancer.

These and other objectives are provided for by the present disclosure which, in a broad sense, is directed to compounds, methods, compositions and articles of manufacture that provided antibodies with improved efficacy. The benefits provided by the present disclosure are broadly applicable in the field of antibody therapeutics and diagnostics and may be used in conjunction with antibodies that react with a variety of targets.

In one aspect, the present invention provides an isolated antibody or an antigen binding fragment thereof, wherein the antibody or the antigen binding fragment binds to human, Cynomolgus and/or mouse TIGIT with a high affinity.

In some embodiments, the isolated antibody or the antigen binding fragment thereof as described herein comprises:

A) one or more heavy chain CDRs (HCDRs) selected from the group consisting of:

(i) a HCDR1 with at least 90%sequence identity to a HCDR1 as set forth in SEQ ID NO: 1;

(ii) a HCDR2 with at least 90%sequence identity to a HCDR2 as set forth in SEQ ID NO: 3; and

(iii) a HCDR3 with at least 90%sequence identity to a HCDR3 as set forth in SEQ ID NO: 5;

B) one or more light chain CDRs (LCDRs) selected from the group consisting of:

(i) a LCDR1 with at least 90%sequence identity to a LCDR1 as set forth in SEQ ID NO: 2;

(ii) a LCDR2 with at least 90%sequence identity to a LCDR2 as set forth in SEQ ID NO: 4; and

(iii) a LCDR3 with at least 90%sequence identity to a LCDR3 as set forth in SEQ ID NO: 6;

or

C) one or more HCDRs of A) and one or more LCDRs of B) .

A) one or more heavy chain CDRs (HCDRs) selected from the group consisting of:

(i) a HCDR1 comprising SEQ ID NO: 1 or a HCDR1 that differs in amino acid sequence from the HCDR1 by an amino acid addition, deletion or substitution of not more than 2 amino acids;

(ii) a HCDR2 comprising SEQ ID NO: 3 or a HCDR2 that differs in amino acid sequence from the HCDR2 by an amino acid addition, deletion or substitution of not more than 2 amino acids; and

(iii) a HCDR3 comprising SEQ ID NO: 5 or a HCDR3 that differs in amino acid sequence from the HCDR3 by an amino acid addition, deletion or substitution of not more than 2 amino acids;

B) one or more light chain CDRs (LCDRs) selected from the group consisting of:

(i) a LCDR1 comprising SEQ ID NO: 2 or a LCDR1 that differs in amino acid sequence from the LCDR1 by an amino acid addition, deletion or substitution of not more than 2 amino acids;

(ii) a LCDR2 comprising SEQ ID NO: 4 or a LCDR2 that differs in amino acid sequence from the LCDR2 by an amino acid addition, deletion or substitution of not more than 2 amino acids; and

(iii) a LCDR3 comprising SEQ ID NO: 6 or a LCDR3 that differs in amino acid sequence from the LCDR3 by an amino acid addition, deletion or substitution of not more than 2 amino acids; or

C) one or more HCDRs of A) and one or more LCDRs of B) .

In some embodiments, the isolated antibody or the antigen binding fragment thereof as described herein comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein

(A) the VH comprises:

(i) a HCDR1 comprising SEQ ID NO: 1;

(ii) a HCDR2 comprising SEQ ID NO: 3; and

(iii) a HCDR3 comprising SEQ ID NO: 5; and/or

(B) the VL comprises:

(i) a LCDR1 comprising SEQ ID NO: 2;

(ii) a LCDR2 comprising SEQ ID NO: 4; and

(iii) a LCDR3 comprising SEQ ID NO: 6.

(A) a heavy chain variable region comprising:

(i) the amino acid sequence of SEQ ID NO: 7;

(ii) an amino acid sequence at least 85%, 90%, or 95%identical to SEQ ID NO: 7; or

(iii) an amino acid sequence with addition, deletion and/or substitution of one or more amino acids compared with SEQ ID NO: 7; and/or

(B) a light chain variable region comprising:

(i) the amino acid sequence of SEQ ID NO: 8;

(ii) an amino acid sequence at least 85%, at least 90%, or at least 95%identical to SEQ ID NO: 8; or

(iii) an amino acid sequence with addition, deletion and/or substitution of one or more amino acids compared with SEQ ID NO: 8.

In some embodiments, the isolated antibody or the antigen binding fragment thereof as described herein comprises a heavy chain variable region as set forth in SEQ ID NO: 7 and a light chain variable region as set forth in SEQ ID NO: 8.

In some embodiments, the isolated antibody or the antigen binding fragment thereof as disclosed herein is a chimeric antibody, a humanized antibody or a fully human antibody or a rat antibody. Preferably, the antibody is a fully human monoclonal antibody.

In some embodiments, the isolated antibody or the antigen binding fragment thereof as disclosed herein comprises a human IgG constant domain, wherein the human IgG constant domain is a human IgG1 or IgG4 constant domain, preferably a human IgG1 constant domain.

(A) a heavy chain comprising:

(i) the amino acid sequence of SEQ ID NO: 11;

(ii) an amino acid sequence at least 85%, 90%, or 95%identical to SEQ ID NO: 11; or

(iii) an amino acid sequence with addition, deletion and/or substitution of one or more amino acids compared with SEQ ID NO: 11; and/or

(B) a light chain comprising:

(i) the amino acid sequence of SEQ ID NO: 12;

(ii) an amino acid sequence at least 85%, at least 90%, or at least 95%identical to SEQ ID NO: 12; or

(iii) an amino acid sequence with addition, deletion and/or substitution of one or more amino acids compared with SEQ ID NO: 12.

In some embodiments, the isolated antibody or the antigen binding fragment thereof as described herein comprises a heavy chain comprising SEQ ID NO: 11 and a light chain comprising SEQ ID NO: 12.

In some aspects, the present disclosure is directed to an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding the heavy chain variable region and/or the light chain variable region of the isolated antibody as disclosed herein.

In some aspects, the present disclosure is directed to a vector comprising the nucleic acid molecule encoding the antibody or antigen binding fragment thereof as disclosed herein.

In some aspects, the present disclosure is directed to a host cell comprising the expression vector as disclosed herein.

In some aspects, the present disclosure is directed to a pharmaceutical composition comprising at least one antibody or antigen binding fragment thereof as disclosed herein and a pharmaceutically acceptable carrier.

In some aspects, the present disclosure is directed to a method for preparing an anti-TIGIT antibody or antigen binding fragment thereof which comprises expressing the antibody or antigen binding fragment thereof in the host cell as disclosed herein and isolating the antibody or antigen binding fragment thereof from the host cell.

In some aspects, the present disclosure is directed to a method of modulating an immune response in a subject, comprising administering the antibody or antigen binding fragment thereof as disclosed herein to the subject such that an immune response in the subject is modulated, optionally the immune response is TIGIT related.

In some aspects, the present disclosure is directed to a method for inhibiting growth of tumor cells in a subject, comprising administering an effective amount of the antibody or antigen binding fragment thereof or the pharmaceutical composition as disclosed herein to the subject.

In some aspects, the present disclosure is directed to a method for treating or preventing diseases comprising proliferative disorders (such as cancers) in a subject comprising administering an effective amount of the antibody or antigen binding fragment thereof or the pharmaceutical composition as disclosed herein to the subject.

In some aspects, the present disclosure is directed to the use of the antibody or antigen binding fragment thereof as disclosed herein in the manufacture of a medicament for treating or preventing diseases comprising proliferative disorders (such as cancers) .

In some embodiments, said cancer is selected from a group consisting of melanoma, renal cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, liver cancer, gastrointestinal cancer, glioblastoma, cervical cancer, bladder cancer, and rectal cancer.

In some aspects, the present disclosure is directed to kits or devices and associated methods that employ the antibody or antigen-binding portion thereof as disclosed herein, and pharmaceutical compositions as disclosed herein, which are useful for the treatment of diseases comprising cancers. The foregoing is a summary and thus contains, by necessity, simplifications, generalizations, and omissions of detail; consequently, those skilled in the art will appreciate that the summary is illustrative only and is not intended to be in any way limiting. Other aspects, features, and advantages of the methods, compositions and/or devices and/or other subject matter described herein will become apparent in the teachings set forth herein. The summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

Brief Description of the Drawings

Figure 1 shows human TIGIT binding by FACS.

Figure 2a shows ELISA binding to human TIGIT; 2b shows ELISA binding to human CD28; 2c shows ELISA binding to human PD-1; 2d shows ELISA binding to human ICOS; 2e shows ELISA binding to human CTLA4; 2f shows ELISA binding to human PVRIG.

Figure 3 shows cynomolgus TIGIT binding assay by FACS.

Figure 4 shows mouse TIGIT binding.

Figure 5 shows human TIGIT ligand competition assay by FACS.

Figure 6 shows report gene assay of W3645-2.131.4-hIgG1L3.

Figure 7 shows human TIGIT-expressing Jurkat cell activation assay.

Figure 8 shows CD8+T cell activation assay.

Figure 9 shows NK killing assay.

Figure 10 shows ADCC effect of W3645-2.131.4-hIgG1L3.

Figure 11 shows melting curve of W3645-2.131.4-hIgG1L3

Figure 12 shows serum stability of W3645-2.131.4-hIgG1L3.

Figure 13 shows CT26 syngeneic model.

Figure 14 shows C57 (hTIGIT) /MC38 model.

Figure 15 shows frequency of CD4+T and CD8+ in spleen.

Figure 16 shows frequency of CD4+T and CD8+ in TIL

Figure 17 shows frequency of Tregs in spleen and TIL.

Figure 18 shows M1 population in TIL.

Figure 19 shows Intracellular IFNg upon stimulation in TILs.

Detailed description

In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

The term “TIGIT” , “T cell immune receptor with Ig and ITIM domains” as referred to herein are also known as Vstm3 and WUCAM, is an inhibitory receptor that is expressed on NK and CD8+T cells, as well as a subset CD4+T cells including immunosuppressive Tregs.

The term “antibody” as referred to herein includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion" ) or single chains thereof. An "antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR) , interspersed with regions that are more conserved, termed framework regions (FR) . Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. CDR boundaries for antibodies are defined or identified by Kabat numbering system.

The term "antibody, " as used in this disclosure, refers to an immunoglobulin or a fragment or a derivative thereof, and encompasses any polypeptide comprising an antigen-binding site, regardless whether it is produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, non-specific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies. The term "antibody" also includes antibody fragments such as Fab, F (ab') 2, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function. Typically, such fragments would comprise an antigen-binding fragment.

The terms "antigen-binding fragment, " "antigen-binding domain, " and "binding fragment" refer to a part of an antibody molecule that comprises amino acids responsible for the specific binding between the antibody and the antigen. In instances, where an antigen is large, the antigen-binding fragment may only bind to a part of the antigen. A portion of the antigen molecule that is responsible for specific interactions with the antigen-binding fragment is referred to as "epitope" or "antigenic determinant. "

An antigen-binding fragment typically comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH) , however, it does not necessarily have to comprise both. For example, a so-called Fd antibody fragment consists only of a VH domain, but still retains some antigen-binding function of the intact antibody.

In line with the above the term "epitope" defines an antigenic determinant, which is specifically bound/identified by a binding fragment as defined above. The binding fragment may specifically bind to/interact with conformational or continuous epitopes, which are unique for the target structure. A conformational or discontinuous epitope is characterized for polypeptide antigens by the presence of two or more discrete amino acid residues which are separated in the primary sequence, but come together on the surface of the molecule when the polypeptide folds into the native protein/antigen. The two or more discrete amino acid residues contributing to the epitope are present on separate sections of one or more polypeptide chain (s) . These residues come together on the surface of the molecule when the polypeptide chain (s) fold (s) into a three-dimensional structure to constitute the epitope. In contrast, a continuous or linear epitope consists of two or more discrete amino acid residues, which are present in a single linear segment of a polypeptide chain.

The term "cross-reactivity" refers to binding of an antigen fragment described herein to the same target molecule in human, monkey, and/or murine (mouse or rat) . Thus, "cross-reactivity" is to be understood as an interspecies reactivity to the same molecule X expressed in different species, but not to a molecule other than X. Cross-species specificity of a monoclonal antibody recognizing, to monkey, and/or to a murine (mouse or rat) TIGIT, can be determined, for instance, by FACS analysis.

As used herein, the term "subject" includes any human or nonhuman animal. The term "nonhuman animal" includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Except when noted, the terms "patient" or "subject" are used interchangeably.

The terms "treatment" and "therapeutic method" refer to both therapeutic treatment and prophylactic/preventative measures. Those in need of treatment may include individuals already having a particular medical disorder as well as those who may ultimately acquire the disorder.

The terms "conservative modifications" i.e., nucleotide and amino acid sequence modifications which do not significantly affect or alter the binding characteristics of the antibody encoded by the nucleotide sequence or containing the amino acid sequence. Such conservative sequence modifications include nucleotide and amino acid substitutions, additions and deletions. Modifications can be introduced into the sequence by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine) , acidic side chains (e.g., aspartic acid, glutamic acid) , uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan) , nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine) , beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine) .

The experimental methods in the following examples are conventional methods, unless otherwise specified.

Examples

Example 1: Materials and Antibody Generation

1. General Materials

Antigens were purchased from vendors or prepared in house (see the details in Table 1) W364-hPro1. ECD. His is the extracellular domain of human TIGIT (NP_776160.2) with a C-terminal polyhistidine tag; W364-hPro1. ECD. hFc is the extracellular domain of human TIGIT (NP_776160.2) with the Fc region of human IgG1 at the C-terminus; W364-mPro1. ECD. His is the extracellular domain of mouse TIGIT (NP_001139797.1) with a C-terminal polyhistidine tag; W364-mPro1. ECD. hFc is the extracellular domain of mouse TIGIT (NP_001139797.1) with the Fc region of human IgG1 at the C-terminus; W364-hPro1L1. ECD. hFc is the extracellular domain of human CD155 (NP_006496.3) with the Fc region of human IgG1 at the C-terminus. W364-hPro1L1. ECD. mFc is the extracellular domain of human CD155 (NP_006496.3) with the Fc region of mouse IgG1 at the C-terminus.

Table. 1. Antigens and ligands

General research materials and their sources are listed in Table 2 below.

Table 2. General cell lines and reagents

2. Construction of expression vector of BMK antibodies

The amino acid sequences encoding the variable domain of the anti-TIGIT antibody (WBP364-BMK1; Tiragolumab US patent US20170088613A1/4.1D3) , anti-TIGIT antibody (WBP364-BMK4; US patent US20160176963A1, 22G2) and anti-TIGIT antibody (WBP364-BMK6; WO2017059095A1, MAB10) were first codon optimized for mammalian expression and then synthesized by GENEWIZ (SuZhou, CHINA) . The DNA segments were then sub-cloned into pcDNA expression vectors with constant region of human IgG1 or IgG4.

Table 3. Information of benchmark antibodies

3. Hybridoma sequencing and construction of in-house discovered antibodies The anti-TIGIT antibodies were discovered in-house after immunizing (Omni rat) with human TIGIT. The total RNA of the hybridoma cell sample is first extracted, following the instruction of the TaKaRa MiniBEST Universal RNA Extraction Kit. The SMART RACE cDNA Amplication Kit from Clonetech is then used to convert RNA to cDNA. The heavy and light chain DNA sequences were amplified from cDNA with 30 cycles of PCR, each with denaturation at 94℃ for 30 sec, anneal at 60℃ for 30 sec, then elongation at 72℃ for 30 sec. The PCR product is then sub-cloned to TA-cloning vector, then sent for GENEWIZ for sequencing. Once sequencing data confirms monoclonality of the hybridoma cell sample, the amino acid sequences of the VH and VL domains were codon optimized for mammalian expression then synthesized by GENEWIZ (SuZhou, CHINA) . The DNA segments were then sub-cloned into pcDNA expression vectors with constant region of human IgG1 or IgG4.

4. Expression and Purification of Antibodies

The plasmids containing VH and VL gene were co-transfected into Expi293 cells (Thermofisher, A14635) . The cells were cultured for 5 days following the manufacturer suggested protocol. After the supernatants were confirmed by SDS-PAGE, the antibodies were purified with Protein a column (GE Healthcare, Cat. 175438) . The concentration of purified Fc-tagged proteins was determined by absorbance at 280 nm. The size and purity were confirmed by SDS-PAGE and SEC-HPLC, respectively; and then stored at -80℃. The purity of antibodies was tested by SEC-HPLC using Agilent 1260 Infinity HPLC. 50 μL of antibody solution was injected on a TSKgel SuperSW3000 column using 50 mM sodium phosphate, 0.15 M NaCl, pH 7.0 buffer. The running time was 20 min. Peak retention times on the column were monitored at 280 nm. Data was analyzed using ChemStation software (V2.99.2.0) . The purity of all antibodies is required >90%.

5. Cell Pool/Line Preparation

5.1 Target-expressing cell pool/line for binding assays

Mouse TIGIT-expressing cell line (W364-FlpinCHO. mpro1. FL) and cyno TIGIT-expressing cell line (W364-FlpinCHO. cynopro1. FL) were generated. Briefly, Flp-In-CHO cells were transfected with pcDNA5 expression vector encoding a full-length of mouse TIGIT (NP_001139797.1) and cynomolgus TIGIT (XP_015300911.1) using Lipofectamine 2000 transfection kit according to manufacturer’s protocol. At 48-72 hours post transfection, the transfected cells were cultured in medium containing hygromycin for selection and tested for TIGIT expression. Then mouse and cyno TIGIT-expressing cell pools were obtained.

Human TIGIT-expressing cell line (W364-CHOK1. hPro1. FL. 2A11) and cell pool (W364-293F. hpro1. FL) were generated. Briefly, CHO-K1 or 293F cells were transfected with pcDNA3.3 expression vector containing full length of human TIGIT (NP_776160.2) using Lipofectamine 2000 transfection kit according to manufacturer’s protocol, respectively. At 48-72 hours post transfection, the transfected cells were cultured in medium containing blasticidin for selection and tested for TIGIT expression. Human TIGIT-expressing cell line were obtained by limiting dilution.

5.2 Target/ligand expressing cells for functional assays

Human TIGIT over expressing Jurkat cells with NFAT-luciferase reporter gene (W364-Jurkat. hPro1. NFAT. 2D11) was generated by transfecting pSBbi-RB-W364-hPro1. FL (human TIGIT encoded pSBbi-RB plasmid) on NFAT-luciferase reporter gene over expressing Jurkat cells. The cells were cultured in complete RPMI1640 medium containing 10%FBS, and 0.5 mg/mL of Hygromycin B and 4 μg/ml of blasticidine as selection.

HT1080.2A11 (also called as HT1080. OKT3. scFv. 2A11) was in-house generated by transfecting OKT3 (anti-CD3 antibody) single chain Fv ecoded pSB plasmid on HT1080 tumor cells.

P815-PVR was generated by transfecting human CD155 (PVR, NP_006496.3) encoded pCDNA3.1 on P815 cells.

CD155 /TCR Activator -CHO Recombinant Cell line was purchased from BPS Bioscience Cat. #: 60548.

6. Generation of Hybridoma Antibody

6.1 Immunization

4 OMT rats at age of 6-8 weeks were immunized with 40μg protein (W364-hPro1. ECD. his) /animal. The adjuvant mixture includes Adju-Phos, CpG-ODN or Titer-Max. The animals were injected once every other week via footpad, subcutaneous, intra-peritoneal routes. The serum titer was measured by ELISA. When the serum titer was sufficiently high (≥1: 24, 300) , the animal with the highest titer were given a final boost with protein in sterilized PBS without adjuvant. After 2-4 days (48-96 hours) , the animals were euthanized and lymph nodes or spleen were used for cell fusion.

6.2 Serum titer detection

ELISA assay was used to measure serum antibody titers against antigen given. Plates (Nunc) were coated with 100 μL of antigen (W364-hPro1. ECD. hFc) at 1 μg/mL at 4 ℃ overnight, and then blocked with blocking buffer (1×PBS/2%BSA) for 1 h at room temperature. Rat serum was 1: 3 diluted starting at 1: 100 dilutions in blocking buffer and incubated for 1 h at room temperature. The plates were then washed and subsequently incubated with secondary antibody goat anti-rat IgG-Fc-HRP (Bethyl, A110-236P) for 1 h. After washing, TMB substrate was added and the interaction was stopped by 2M HCl. The absorbance at 450 nm was read using a microplate reader (Molecular Device) . Serum titer was determined at 3-folds background.

After immunization, 2 OMT rats were selected for fusion, the serum titer of these 2 OMT rats before fusion is shown in Table 4.

Table 4. Serum titer (ELISA binding to human/mouse TIGIT)

6.3 Hybridoma generation

Lymph nodes and spleen from immunized animal were homogenized and filtered to remove blood clots and cell debris. Sp2/0 myeloma cells in logarithmic growth were collected and centrifuged. B cells and Sp2/0 myeloma cells were treated separately with pronase solution and the reaction was stopped by FBS. The cells were washed, and counted. B cells were mixed with Sp2/0 myeloma cells at 1: 1 ratio in electric fusion solution. The electro-fusion was performed according to Electro-fusion procedures. The fused cells were resuspended in DMEM medium supplemented with 20%FBS and 1× HAT (hypoxanthine-aminopterin-thymidine medium) , and then transferred into 96-well plates. The fused cells were kept in a 37℃ 5%CO ₂ incubator for 10-14 days.

6.4 Antibody screening and subcloning

The positive clones were screened by cell based binding and ligand competiotion assays. The positive hybridoma cells in logarithmic growth were counted and 200-300 cells were added to 1.5 ml semi-solid-HAT media. The cells were mixed gently in vortex oscillators for 5-10 seconds and then seeded in 6-well plates. The plates were kept in a 37℃ 5%CO ₂ incubator for 7-8 days. Each visible single colony was picked into 96-well plates with DMEM medium supplemented with 10%FBS. After 2～3 days, the cell supernatant were collected and screened again for obtaining positive hybridoma clones.

6.5 Hybridoma sequencing and antibody generation

The positive clones were sent for GENEWIZ for sequencing. After the positive clones were confirmed monoclonality. The amino acid sequences of the VH and VL domains without PTM site were codon optimized for mammalian expression then synthesized by GENEWIZ (SuZhou, CHINA) . The DNA segments were then subcloned into pcDNA expression vectors with constant region of human IgG1 or human IgG4. The plasmids containing VH and VL gene were co-transfected into Expi293 cells (Thermo Fisher, A14635) . Then cultured for 5 days following the manufacturer suggested protocol. After the supernatants were confirmed by SDS-PAGE, the antibodies were purified with Protein a column (GE Healthcare, Cat. 175438) . The concentration of purified Fc-tagged proteins was determined by absorbance at 280 nm. The size and purity were confirmed by SDS-PAGE and SEC-HPLC, respectively; and then stored at -80℃. The purity of antibodies was tested by SEC-HPLC using Agilent 1260 Infinity HPLC. 50 μL of antibody solution was injected on a TSKgel SuperSW3000 column using 50 mM sodium phosphate, 0.15 M NaCl, pH 7.0 buffer. The running time was 20 min. Peak retention times on the column were monitored at 280 nm.Data was analyzed using ChemStation software (V2.99.2.0) . The purity of all antibodies is required >90%.

6.6. Antibody sequence

Through primary and secondary binding screening, as well as TIGIT/CD155 blocking and report gene assay, 51 positive cell lines were selected for subcloning. After confirmation of purified subcloning antibodies, 10 hits were selected for sequencing and followed with human IgG conversion. Final lead was identified and the sequence is shown in Table 5-8.

Table 5. CDR amino acid sequences

Table 6. Variable region amino acid sequences

Table 7. Variable region nucleotide sequences

Table 8. Full length sequences of heavy chain and light chain

Example 2: 3.6 In vitro Characterization of lead antibody

1. Human TIGIT binding (FACS)

Human TIGIT expressing 293F cells (W364-293F. hPro1. FL. cells, 1×10 ⁵ cells/well) were incubated with serially dilution of W3645-2.131.4-hIgG1L3 (diluted from 100 nM, 5 folds, 8 points) at 4℃ for 1 hour. After washing with 1×PBS/1%BSA, a secondary antibody, R-PE-labeled goat anti-human IgG (1: 150) , was added and incubated with cells at 4 ℃ in dark for 1 hour. Anti-human TIGIT antibodies W364-BMK1 and W364-BMK4 were used as positive controls. Human IgG1 isotype antibody was used as a negative control. The cells were then washed and resuspended in 1×PBS/1%BSA. MFI (Median Fluorescence Intensity) of the cells was measured by a flow cytometer (BD, CantoII) and analyzed by FlowJo. 7. Four-parameter non-linear analysis was used to obtain EC ₅₀ values for cell binding using GraphPad Prism. 6. software.

The binding result of W3645-2.131.4-hIgG1L3 on WBP64-293F. hPro1. FL. pool is shown in Figure 1. WBP3645 lead can dose dependently bind to cell surface human TIGIT with EC ₅₀ of 0.17 nM, EC ₅₀ of reference antibodies WBP364-BMK1 (Genentech) and WBP364-BMK4 (BMS) is 0.35nM to 0.38nM. Data is shown in Figure 1.

2. Cross-family binding (ELISA)

Non-tissue culture treated flat-bottom 96-well plates were pre-coated with 1.0 μg/mL in house made human CD28 ECD, human CTLA4 (Cytotoxic T-Lymphocyte-Associated protein 4) ECD, human ICOS (Inducible T-cell co-stimulator) ECD, human PD-1 (Programmed cell Death protein 1) protein ECD, human PVRIG (CD112 receptor) ECD and human TIGIT ECD overnight at 4℃. After 2%BSA blocking, 100 μL 10-fold titrated antibodies from 1 nM to 0.01 nM were added into each well and incubated for 1 hour at ambient temperature (22℃) . Following removal of the unbound antibodies, HRP-labeled goat anti-human IgG was added to the wells and incubated for 1 hour. The color was developed by dispensing 100 μL TMB substrate, and then stopped by 100 μL 2N HCl. The absorbance was read at 450 nm using a Microplate Spectrophotometer (MD, M5e) , data was analyzed by Prism6.

TIGIT has been identified as a member of CD28 family based on gene structure. ELISA binding results of WBP3645 lead (W3645-2.131.4-hIgG1L3) to TIGIT paralogs of CD28, PD-1, ICOS, CTLA4 is shown in Figure 2-a to 2-e. The binding to PVRIG is also tested by ELISA which share the ligand of CD112 with TIGIT. Data is shown in Figure 2-f. From the results of ELISA binding assay, W3645-2.131.4-hIgG1L3 is specifically binding to TIGIT without cross reactivity to human CD28, PD-1, ICOS, CTLA4 and PVRIG.

3. Cross-species binding (FACS)

3.1 Cynomolgus TIGIT binding

Cynomolgus TIGIT expressing FLP in CHO cells (W364-FLPin CHO. cynoPro1. FL. cells, 1×10 ⁵ cells/well) were incubated with serially dilution of W3645-2.131.4-hIgG1L3 (diluted from 100 nM, 5 folds, 8points) at 4℃ for 1 hour. After washing with 1×PBS/1%BSA, a secondary antibody, R-PE-labeled goat anti-human IgG (1: 150) , was added and incubated with cells at 4 ℃ in dark for 1 hour. Anti-human TIGIT antibodies W364-BMK1 and W364-BMK4 were used as positive controls. Human IgG1 isotype antibody was used as a negative control. The cells were then washed and resuspended in 1×PBS/1%BSA. MFI of the cells was measured by a flow cytometer (BD, CantoII) and analyzed by FlowJo. 7. Four-parameter non-linear analysis was used to obtain EC50 values for cell binding using GraphPad Prism. 6. software.

W3645-2.131.4-hIgG1L3 is cross bind to cynomolgus TIGIT expressing FLP in-CHO cells with EC ₅₀ of 0.05nM. Data is shown in Figure 3.

3.2 Mouse TIGIT binding

Mouse TIGIT expressing Flp-in CHO cells (W364-FLPin CHO. mousePro1. FL. cells (1×10 ⁵ cells/well) were incubated with serially dilution of W3645-2.131.4-hIgG1L3 (diluted from 100 nM, 5 folds, and 8 points) at 4℃ for 1 hour. After washing with 1×PBS/1%BSA, a secondary antibody, R-PE-labeled goat anti-human IgG (1: 150) , was added and incubated with cells at 4 ℃ for 1 hour. Anti-human TIGIT antibodies W364-BMK6 was used as a positive control. Human IgG1 isotype antibody was used as a negative control. The cells were then washed and resuspended in 1×PBS/1%BSA. MFI of the cells was measured by a flow cytometer and analyzed by FlowJo. 7. Four-parameter non-linear analysis was used to obtain EC ₅₀ values for cell binding using GraphPad Prism. 6. software.

W3645-2.131.4-hIgG1L3 is cross bind to mouse TIGIT expressing FLP-in-CHO cells with EC ₅₀ of 0.14 nM. BMK6 is a cross mouse TIGIT binding reference antibody (Astellas) . Data is shown in Figure 4.

4. Affinity to human TIGIT (SPR)

The binding affinity of TIGIT antibodies to the antigen was detected by SPR (Surface Plasmon Resonance) assay using Biacore 8K. Antibodies were captured on an anti-human IgG Fc antibody immobilized CM5 sensor chip (GE) . Extracellular domain of human TIGIT at different concentrations were injected over the sensor chip at a flow rate of 30 μL/min for an association phase of 180 s, followed by 3600 s dissociation. The chip was regenerated by 10 mM glycine (pH 1.5) after each binding cycle. The sensorgrams of blank surface and buffer channel were subtracted from the test sensorgrams. The experimental data was fitted by 1: 1 model using Langmiur analysis. Molecular weight of 20 kDa was used to calculate the molar concentration of W364-hPro1. ECD. his (Sino) .

The affinity KD (M) of W3645-2.131.4-hIgG1L3 to the soluble antigen is 7.50E-10 (see the data in Table 9) .

Table 9. Protein based affinity by SPR

5. Affinity to cell surface human TIGIT (FACS)

The binding affinity of antibodies to cell surface TIGIT was measured by FACS analysis. Human TIGIT over-expressing CHOK1 cells (W364-CHOK1. hPro1.2A11) were transferred in to 96-well U-bottom plates at a density of 5x104cells/ml. Tested antibodies were serially diluted in wash buffer (1×PBS/1%BSA) and incubated with cells at 4 ℃ for 1 h. The secondary antibody goat anti-human IgG Fc FITC (2.5 moles FITC per mole IgG) was added and incubated at 4 ℃ in the dark for 0.5 h. The cells were then washed once and re-suspended in 1×PBS/1%BSA, and analyzed by flow cytometry. Fluorescence intensity will be converted to bound molecules/cell based on the quantitative beads (QuantumTM MESF Kits, Bangs Laboratories, Inc. ) .

The affinity of antibodies to cell surface human TIGIT was measured by FACS with KD (M) of 2.70E-11. Data is shown in Table 10.

Table 10. Cell based affinity by FACS

Antibodies	Bmax (M)	KD (M)	r2
WBP364-BMK1. uIgG1K	3.90E-11	2.60E-10	1
WBP364-BMK4. uIgG1K	3.10E-11	8.40E-11	0.95
W3645-2.131.4-hIgG1L3	2.20E-11	2.70E-11	1

6 Ligands competition assay

The blockade of CD155 (PVR, Poliovirus Receptor) binding to TIGIT expressing cells by antibodies was determined by flow cytometry. Briefly, 1E5 293F (TIGIT+) cells were incubated for 60 minutes at 4℃ with serial dilutions of TIGIT or hIgG1 isotype control antibodies and 2 μg/mL mFc tagged CD155. ECD. After washing twice with cold PBS supplemented with 1%BSA (wash buffer) , cell surface bound ligands were detected by incubating the cells with PE conjugated anti-mFc antibody for 30 minutes at 4℃. Cells were washed twice in the same buffer and the MFI (mean fluorescence intensity) of stained cells was measured using a FACS Canto II cytometer (BD Biosciences) . Wells containing no antibody or secondary antibody only were used to establish background fluorescence. Four-parameter non-linear regression analysis was used to obtain IC ₅₀ values for cell binding using GraphPad Prism software.

The blockade of human CD155 (PVR) binding to human TIGIT expressing cells by antibodies was determined by flow cytometry. The lead Ab blocks the binding of human TIGIT to ligand CD155 with IC ₅₀ of 0.14 nM. Data is shown in Figure 5.

7 NFAT reporter gene assay

In this assay, W364-Jurkat. hPro1. NFAT. 2D11 (human TIGIT/NFAT report gene expressing Jurkat cells) were used as effector cells; CD155 and TCR activator over-expressing CHO cells (BPS, Cat#. 60548) were used as target cells. When these two cells are co-cultivated, NFAT luciferase reporter signal in effector cells was inhibited with the binding of TIGIT on Jurkat cells and the ligand CD155 on CHO cells. The luciferase signal was increase the by blocking the binding of TIGIT and CD155 with the anti-TIGIT antibodies. Briefly, CD155 and TCR activator overexpressing cells were seeded at the density of 2E4/well on the white plate overnight. Then serially diluted antibodies and TIGIT over expressing Jurkat/NFAT reporter cells (2E4/well) were co-cultured with target cells for 5 to 6 hours under 37℃ with 5%CO ₂. After incubation, ONE-Glo Luciferase reagent (Promega. Cat: #E6130) was added to the cells to measure NFAT activity with luminescence by Envision (PE) . Four-parameter non-linear analysis was used for curve fit and obtain EC ₅₀ values using GraphPad Prism. 6. software.

WBP3645 lead Ab can stimulate NFAT pathway in an hTIGIT-overexpressing Jurkat NFAT-luciferase reporter cell line with EC ₅₀ of 0.92 nM. Data is shown in Figure 6.

8. Human TIGIT-expressing Jurkat cell activation assay (IL-2 ELISA)

In this assay W364-Jurkat. hPro1. NFAT. 2D11 (human TIGIT/NFAT report gene expressing Jurkat cells) were used as effector cells; HT1080. OKT3scFv. A10 were used as target cells. HT1080 is a human CD155 endogenous expressing tumor cell line. After incubation W364-Jurkat. hPro1. NFAT. 2D11 (1E4/well) and HT1080. OKT3scFV. A10 (1E4/well) for 2 days, IL2 production in the culture medium was determined by ELISA (Capture Ab: Anti-hIL2 purified mouse monoclonal IgG2A clone 5355, R&D, MAB602; Detection Ab: Biotinylated anti-hIL2 antibody, R&D, BAF202; Standard recombinant human IL2: R&D, 202-IL-050) . Four-parameter non-linear analysis was used for curve fit and obtain EC ₅₀ values using GraphPad Prism. 6. software.

WBP3645 lead antibody can enhance IL2 release of hTIGIT overexpressing Jurkat cells with EC ₅₀ of 0.29nM. Data is shown in Figure 7.

9. CD8+T cell activation assay

The co-culture assay of CD8+T with CD155 expressing humor cells was performed to evaluate if the TIGIT antibodies could enhance CD8+T activity. Briefly, CD8+T cells (1×10 ⁵ cells/well) were co-cultured with antibodies at the concentration of 20 nM and HT1080 cells (1×10 ⁴/well) which were transfected with OKT3ScFv. After incubation for 5 days, IFNg production in the culture medium was determined by ELISA (Capture Ab: Human IFNg Mab clone 2G1, Thermo, M700A; Detection Ab: Biotinylated anti-hIFNγ antibody, Thermo, M701B; Standard recombinant IFNg: PeproTech, 300-02-250) .

WBP3645 lead antibody W3645-2.131.4-hIgG1L3 can enhance IFNg release in CD8+T activation assay. The data is shown in Figure 8.

10. NK killing assay

The effect of anti-human TIGIT antibody on NK-cell mediated lysis of CD155 expressing cells in vitro was assessed. Briefly, CD155 (PVR) expressing P815 cells (mouse mastocytoma cell line) as target cells were pre-loaded with BATDA (PE, AD0116) and the exposed to human NK cells in the presence anti-TIGIT antibodies under multiple concentrations (10nM, 2nM, 0.4nM, 0.08nM, 0.016nM) . W364-BMK1 was used as positive control and human IgG1 isotype control was used as negative control. After incubation for 2-4 hours, supernatant was collected and mixed with Europium buffer. Cytotoxicity was detected with TRF (time-resolved fluorescence) by Envision (PE, Ex337/Em615) , data was presented with Prism 6.

WBP3645 lead antibody can dose dependently enhance NK killing activity towards PVR overexpressing cells with better potency than BMKs. The data is shown in Figure 9.

11. ADCC (Antibody-Dependent Cell-mediated Cytotoxicity) assay

In order to test ADCC effect, engineered human TIGIT-expressing cells (W364-CHOK1. hPro1.2A11) and various concentrations of TIGIT antibodies were pre-incubated in 96-well plate for 30 minutes, and then fresh isolated PBMCs (Peripheral Blood Mononuclear Cells) were added at the effector/target ratio of 20: 1. The plate was incubated under 37℃ with 5%CO2 for 4 hours. Target cell lysis was determined by LDH-based cytotoxicity detection kit. The absorbance was read at 492 nM using a Microplate Spectrophotometer (MD, SpectraMax, M5e) . Four-parameter non-linear analysis was used for curve fit and obtain EC50 values using GraphPad Prism. 6. software.

W3645-2.131.4-hIgG1L3 can lyse hTIGIT overexpressing cells by antibody-dependent cell-mediated cytotoxicity with EC50 of 0.0019nM. Data is shown in Figure 10.

12. Thermal stability evaluation by DSF

Tm of WBP3645 lead antibody was investigated using QuantStudio 7 Flex Real-Time PCR system (Applied Biosystems) . 19 μL of antibody solution was mixed with 1 μL of 62.5 X SYPRO Orange solution (Invitrogen) and transferred to a 96 well plate (Biosystems) . The plate was heated from 26 ℃ to 95 ℃ at a rate of 0.9 ℃/min, and the resulting fluorescence data was collected. The negative derivatives of the fluorescence changes with respect to different temperatures were calculated, and the maximal value was defined as melting temperature Tm. If a protein has multiple unfolding transitions, the first two Tm were reported, named as Tm1 and Tm2. Data collection and Tm calculation were conducted automatically by the operation software (QuantStudio Real Time PCR software v1.3) .

T _m of WBP3645 lead antibody was investigated using QuantStudio ^TM 7 Flex Real-Time PCR system (Applied Biosystems) . Good thermal stability was observed by DSF test. Software calculated data is shown in Table 11 and Figure 11.

Table 11. Thermal stability of W3645-2.131.4-hIgG1L3

13. Serum stability evaluation

In order to evaluate the serum stability of the antibody, serum was freshly prepared from human blood of healthy donor. The antibody was gently mixed with serum and aliquot to 5 tubes and incubated under 37 ℃. Then aliquots were drawn at the indicated time points: 0 day, 1 day, 4 days, 7 days and 14 days, and the aliquots were quickly frozen into liquid nitrogen and store them at -80℃ until use. The binding ability by ELISA was used to evaluate the serum stability of the antibody. The absorbance was read at 450 nM using a Microplate Spectrophotometer (MD, SpectraMax, M5e) ; Four-parameter non-linear analysis was used for curve fit and obtain EC50 values using GraphPad Prism. 6. software.

In the serum stability test, W3645-2.131.4-hIgG1L3 is stable in serum for at least 2 weeks under 37 ℃. Data is shown in Figure 12.

Example 3: In vivo Characterization

1. Rodent efficacy study

1.1 CT26 syngeneic model

WBP3645 lead antibody efficacy study was tested in CT-26 model in BALB/c mice. Female BALB/c mice (Beijing Vital River Laboratory Animal Technology Co., LTD) of 6-8 week-old were used in the study. CT-26 cells were maintained in vitro as a monolayer culture in RPMI 1640 medium supplemented with 10%fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin at 37℃ in an atmosphere of 5%CO ₂ in air.

For the therapeutic model, each mouse was inoculated subcutaneously at the right for earm armpit with CT-26 tumor cells (3.0×10 ⁵) . When the average tumor volume reached approximately to 55 mm ³, animals were randomly grouped into different groups, Vehicle-PBS; PD-1 Ab 3 mg/kg; WBP364-BMK6. uIgG4. SPK 10 mg/kg; W3645-2.131.4-hIgG1L3 10 mg/kg; WBP364-BMK6. uIgG4. SPK+PD-1 Ab 10+3 mg/kg; W3645-2.131.4. hIgG1L3+PD-1 Ab 10+3 mg/kg, and received the first antibody injection, then treated with PBS or antibodies intraperitoneally twice a week for total 5 injections. The day of the first injection was considered as day 0. For all tumor studies, mice were weighed and tumor growth was measured twice a week using calipers. All the procedures related to animal handling, care and the treatment in the study were performed according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Bio-model following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) .

Tumor volume was calculated with the formula (1/2 (length × width ²) . The results were represented by mean and the standard error (Mean ± SEM) . Data were analyzed using Two way ANOVA Bonferroni posttests with Prism and p<0.05 was considered to be statistically significant.

CT26 is a PD-1 resistant model, the highest TGI of αPD-1 in this model is below 50% (data not shown) . As single agent, WBP3645 lead antibody showed better efficacy than W364-BMK6 and αPD-1 in this model. When in combination with αPD-1, WBP3645 lead antibody showed better synergistic effect than W364-BMK6. Data is shown in Figure 13.

1.2 C57 (hTIGIT) /MC38 model

In vivo efficacy study of W3645-2.131.4. hIgG1L3 was tested in MC38 model with h-TIGIT mice. Female hTIGIT mice (Nanjing GemPharmatech Co., LTD) of 6-8 week-old were used in the study.

In this in vivo efficacy model, each mouse was inoculated subcutaneously at the right forearm armpit with MC38 tumor cells (5.0×10 ⁵) . When the average tumor volume reached approximately to 65 mm ³, animals were randomly grouped into different groups, Vehicle-PBS; PD-1 Ab 0.3 mg/kg; WBP364-BMK4 10 mg/kg; W3645-2.131.4-hIgG1L3 10 mg/kg; WBP364-BMK4 + PD-1 Ab 10+0.3 mg/kg; W3645-2.131.4. uIgG1L3+PD-1 Ab 10+0.3 mg/kg and then received the first antibody injection with PBS or antibodies intraperitoneally twice a week for total 6 injections. The day of the first injection was considered as day 0. For all tumor studies, mice were weighed and tumor growth was measured twice a week using calipers. All the procedures related to animal handling, care and the treatment in the study were performed according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Bio-model following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) .

In vivo efficacy study of W3645-2.131.4. hIgG1L3 was tested in MC38 model with h-TIGIT mice. WBP364-BMK4 was used as reference control. In this model, WBP3645 lead antibody cannot inhibit tumor growth in MC38 model as single agent. WBP3645 lead antibody in combination with PD-1 antibody showed synergistic effect in C57 (hTIGIT) /MC38 efficacy model. Data is shown in Figure 14.

1.3 Ex vivo study of MC38 model

In the in vivo efficacy study of C57 (hTIGIT) /MC38 model (see 3.7.1.2) , spleen and tumor tissues were isolated from 4 groups of mice with different treatments (n=4 from total 8) for cell population profiling. These 4 groups are vehicle-PBS; PD-1 Ab 0.3 mg/kg; W3645-2.131.4-hIgG1L3 10 mg/kg and the combination of anti-PD-1 0.3mg/kg and W3645-2.131.4-hIgG1L3 10 mg/kg. In this study, the populations of CD4+T cells, CD8+ T cells, Tregs, M1 in SPL (Spleen lymphocytes) and TIL (Tumor Infiltrate Lymphocytes) were analyzed as well as the T cell function.

First of all, spleen lymphocytes from each mouse were isolated by gently grinding and filtered with 70uM filter, and washed with 15ml PBS twice. Tumor infiltrate lymphocytes were isolated from tumor tissue according the protocol of mouse tumor dissociation kit (Miltenyi Biotec Cat: 130-096-730) . After the cells were isolated, some of them were fixed for staining. The intracellular maker were stained after fixation and permeabilization with buffer (BD, 51-2090KZ) . The information of the antibodies used for cell population profiling is shown as follows. Mouse CD4 antibody (BD, 553729, FITC conjugated) ; Mouse CD25 antibody (BD, 558642, PE conjugated) ; mouse FoxP3 antibody (eBioscience, 17-5773-80, APC conjugated) ; mouse CD45 antibody (BD, 553079, FITC conjugated) ; mouse CD4 antibody (eBioscience, 12-0041-81, PE conjugated) ; mouse CD8 antibody (BD, 553035, APC conjugated) ; mouse F4/80 antibody (eBioscience, 11-4801-82, FITC conjugated) ; mouse MHC-II (ebioscience, 47-5321-82, APC conjugated) . Stained cells were detected by FACS (BD, CantoII) and the data was analyzed with FlowJo. 7. and Prism 6.

For T cell function evaluation in the TIL, intracellular IFNg was detected after stimulation. Briefly, 1E6 cells isolated from tumor tissue were treated with 0.5 x cell stimulation cocktail (Invitrogen, 4333766) and 1x Golgi Stop (BD 51-2092K2) for 9 hours. After that, the cells were the fixed and permeabilized then stained with the antibodies as follows. Mouse CD4 antibody (BD, 553729, FITC conjugated) ; Mouse CD8 antibody (BD, 553035, APC conjugated ) ; mouse IFNg antibody (BD, 561479, APC-cy7 conjugated) . Stained cells were detected by FACS (BD, CantoII) and the data was analyzed with FlowJo. 7. and Prism 6.

In the study of 4.3.1.2, 4 groups of animals were tissued for ex vivo analysis. They are group PBS; PD-1 antibody; W3645-2.131.4-hIgG1L3 and the combination of PD-1 antibody and W3645-2.131.4-hIgG1L3. The population of CD4+T cells as well as CD8+T cells were significantly increased (p<0.05) in the combo group comparing with vehicle-PBS group in spleen. Data is shown in Figure 15.

The population of CD4+ T cells was significantly increased in the anti-PD-1 and combo groups comparing with vehicle-PBS group in CD45+ TILs, data is shown in Figure 16.

The population of Treg cells was significantly decreased (P<0.05) in the anti-PD-1 and combo groups comparing with vehicle-PBS group in CD4+ T cells in spleen. No significant difference was observed between different groups for CD25+/FoxP3 +Tregs in CD4+ TILs. Data is shown in Figure 17.

The M1 population of combo group was significantly increased in TIL comparing with PBS group by staining with F4/80 and MHCII. Data is shown in Figure 18.

The intracellular IFNg of combo group was observed higher upon cocktail stimulation but no significant difference comparing with other groups in CD4+ cells and CD8+ cells in TILs. Data is shown in Figure 19.

2. Pharmacokinetics and acute toxicity assessment on cynomolgus monkey Pharmacokinetic characterization on cynomolgus monkey

This study was to determine the pharmacokinetics of W3645 in

male cynomolgus monkeys following a single intravenous bolus administration at 30 mg/kg. Two animals were administered with W3645-2.131.4-hIgG1L3 at 30mg/kg once by intravenous bolus administration, respectively. The formulations were formulated in PBS. PK blood samples were collected at pre-dose, 0.25h, 0.5h, 1h, 4h, 8h, 24h, D3, D5, D7, D10, D12, D14, D21, and D28. Antidrug antibody (ADA) samples were collected at pre-dose, D14 and D28. Serum concentrations of W3645 and ADA in serum samples were determined by ELISA. Samples for hematology and clinical chemistry tests were collected at pre-dose, 24h, D3, D7, D14, D21, and D28.

This study was to determine the pharmacokinetics of WBP3645 final lead in

male cynomolgus monkeys following a single intravenous bolus administration at 30 mg/kg. Two animals were administered with W3645-2.131.4-hIgG1L3 at 30mg/kg once by intravenous bolus administration, respectively. The formulations were formulated in PBS. PK blood samples were collected at pre-dose, 0.25h, 0.5h, 1h, 4h, 8h, 24h, D3, D5, D7, D10, D12, D14, D21, and D28. The concentrations of W3645-2.131.4-hIgG1L3 in serum samples were determined by ELISA.

Data is analyzed by using the Phoenix WinNonlin software (version 8.1, Pharsight, Mountain View, CA) . The linear/log trapezoidal rule was applied in obtaining the PK parameters.

The summary for PK parameters was listed in the Table 12 below.

Table 12. PK data of W3645-2.131.4-hIgG1L3 at dose of 30mg/Kg

Note: The pharmacokinetic parameters of G1-1 and G1-2 were calculated without the time points of day12, day14, day21 and day28 due to ADA effect, data not shown.

Claims

An antibody or an antigen binding fragment thereof, wherein the antibody or the antigen binding fragment binds to human, Cynomolgus and mouse TIGIT.
An antibody, or an antigen-binding fragment thereof, comprising:

a) a variable region of a heavy chain having an amino acid sequence that is at least 70%, 80%, 90%, 95%or 99%homologous to a sequence of SEQ ID NO: 7; and

b) a variable region of a light chain having an amino acid sequence that is at least 70%, 80%, 90%, 95%or 99%homologous to a sequence SEQ ID NO: 8,

wherein the antibody or the antigen binding fragment specifically binds to TIGIT.
An antibody or an antigen binding fragment thereof, comprising:

a) a variable region of a heavy chain having an amino acid sequence of SEQ ID NO: 7; and

b) a variable region of a light chain having an amino acid sequence of SEQ ID NO: 8 ,

wherein the antibody or the antigen binding fragment specifically binds to TIGIT.
An antibody, or an antigen binding fragment thereof, comprising:

a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences; and

a light chain variable region comprising CDR1, CDR2, and CDR3 sequences,

wherein the heavy chain variable region CDR3 sequence comprises an amino acid sequence of SEQ ID NO: 5, and conservative modifications thereof,

wherein the antibody or the antigen binding fragment specifically binds to TIGIT.
The antibody or the antigen binding fragment thereof according to claim 4, wherein the light chain variable region CDR3 sequence comprises an amino acid sequence of SEQ ID NO: 6, and conservative modifications thereof.
The antibody or the antigen binding fragment thereof according to claim 4 or 5, wherein the heavy chain variable region CDR2 sequence comprises an amino acid sequence of SEQ ID NO: 3, and conservative modifications thereof.
The antibody or the antigen binding fragment thereof according to any of claims 4 to 6, wherein the light chain variable region CDR2 sequence comprises an amino acid sequence of SEQ ID NO: 4, and conservative modifications thereof.
The antibody or the antigen binding fragment thereof according to any of claims 4 to 7, wherein the heavy chain variable region CDR1 sequence comprises an amino acid sequence of SEQ ID NO: 1, and conservative modifications thereof.
The antibody or the antigen binding fragment thereof according to any of claims 4 to 8, wherein the light chain variable region CDR1 sequence comprises an amino acid sequence of SEQ ID NO: 2, and conservative modifications thereof.
The antibody or the antigen binding fragment thereof according to any one of claims 1 to 9, wherein the antibody is chimeric, humanized, fully human, or rat antibody.
A nucleic acid molecule encoding the antibody or the antigen binding fragment thereof according to any one of claims 1 to 10.
A cloning or expression vector comprising the nucleic acid molecule of claim 11.
A host cell comprising one or more cloning or expression vectors of claim 12.
A process for the production of the antibody of any one of claims 1 to 10, comprising culturing the host cell of claim 13 and isolating the antibody.
The process of claims 14, wherein the antibody is prepared through immunization in a rat with human TIGIT protein.
A pharmaceutical composition comprising the antibody or the antigen binding fragment thereof according to any one of claims 1 to 10, and one or more of a pharmaceutically acceptable excipient, a diluent and a carrier.
A method for preparing an anti-TIGIT antibody or an antigen-binding fragment thereof comprising:

(a) providing:

(i) a heavy chain variable region antibody sequence comprising a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO: 3; and a CDR3 sequence of SEQ ID NO: 5; and/or

(ii) a light chain variable region antibody sequence comprising a CDR1 sequence of SEQ ID NO: 2, a CDR2 sequence of SEQ ID NO: 4, and a CDR3 of SEQ ID NO: 6; and

(b) expressing the altered antibody sequence as a protein.
A method of preventing or treating tumor or inflammatory disease comprising administering to a subject in need a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.
Use of the antibody or antigen binding fragment thereof according to any one of claims 1 to 10 in the manufacture of a medicament for the prevention or treatment of tumor or inflammatory disease.
The method of any one of claims 18, the use of claim 19, wherein the cancer is selected from a group consisting of melanoma, renal cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, liver cancer, gastrointestinal cancer, glioblastoma, cervical cancer, bladder cancer, and rectal cancer.