CN114641502A - Novel anti-TIGIT antibody - Google Patents
Novel anti-TIGIT antibody Download PDFInfo
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- CN114641502A CN114641502A CN202080075905.9A CN202080075905A CN114641502A CN 114641502 A CN114641502 A CN 114641502A CN 202080075905 A CN202080075905 A CN 202080075905A CN 114641502 A CN114641502 A CN 114641502A
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
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Abstract
The present invention provides TIGIT monoclonal antibodies, particularly humanized monoclonal antibodies that specifically bind to TIGIT with high affinity. The invention also provides functional monoclonal antibodies having cross-reactivity to TIGIT in humans, cynomolgus monkeys and mice. The invention also provides amino acid sequences of the antibodies of the invention, cloning or expression vectors, host cells and methods for expressing or isolating the antibodies. Epitopes of the antibodies were identified. Therapeutic compositions comprising the antibodies of the invention are also provided. The invention also provides methods of using anti-TIGIT antibodies to treat cancer and other diseases.
Description
Technical Field
The present invention relates generally to anti-TIGIT antibodies and compositions thereof, and therapies for treating tumors or inflammatory diseases using anti-TIGIT antibodies.
Background
TIGIT (T Cell immunoreceptor with Ig and ITIM domains), also known as Vstm3, WUCAM, is an inhibitory receptor expressed on NK and CD8+ T cells and the subset of 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 Nature of Sciences of science of State, J.10, J.J.armouron et al, J.10, J.P.10, J.10, J.P.P.10, J.10, J.P.10, J.P.P.P.10, J.P.10, J.P.P.P.P.P.P.P.10, J.P.P.P.P.P.10, J.P.10, J.10, J.P.10, J.10, J.P.P.P.P.P.10, J.10, J.P.P.P.10, J.10, J.P.P.10, J.P.P.P.P.P.P.P.P.P.P.10, J.10, J.P.P.P.P.P.P.P.P.P.P.10, J.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.. It has been identified as a member of the 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 tumor and antigen presenting cell expressed, leading to immunosuppression (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 simultaneously bind to the costimulatory molecule CD226, leading to NK and T cell activation. Blocking antibodies to TIGIT disrupt TIGIT's binding to its ligand and block its inhibitory signal, shifting the equilibrium towards activation signals favorable to CD226 mediation, thereby inducing 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, Wellock J, Fieder W.Mamm genome.29(11-12):694 702, 2018.). TIGIT is upregulated in Cancer and inflammatory diseases and is identified as a marker for failure (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; 26: 923-37.; Johnston J., Tsu H.J., Cang Y et al; 26: 923-37.; identical to other markers for failure such as PD-1, LAG3 and 3 (Flury M, Betina AC, Prolker EA et al, Arthritis Rheumatour April; 70 MB 574; Bantlman W57566; Paul 12, W577, W7, W12, W7, W12.; M). Recently, more and more studies have demonstrated that TIGIT is expressed in parallel with PD-1 (Josefsson SE, Beiske K, Blaker YN et AL, Cancer Immunol Res.2019Mar; 7(3): 355-; Niclauss C.Blessin, Ronald Simon et AL, Dis markers.2019; 2019:5160565), and that blockade of both PD-1 and TIGIT in tumor tissues can reverse immunosuppression (Hung AL, Maxwell R, Theodros D, Belchad Z et AL, oncoimmunology.2018May 24; 7 (8)). Generally, TIGIT can be used as a single agent or in combination with other immunomodulators, and is a promising therapeutic target for tumor immunotherapy.
Disclosure of Invention
The invention provides isolated antibodies, particularly monoclonal antibodies or fully human antagonist antibodies against TIGIT.
In one aspect, the invention provides an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment binds to human, cynomolgus monkey, and mouse TIGIT.
The present invention provides an antibody or antigen-binding fragment thereof comprising:
a) a heavy chain variable region having an amino acid sequence substantially identical to SEQ ID NO: 7, at least 70%, 80%, 90%, 95%, or 99% homologous; and
b) a light chain variable region having an amino acid sequence substantially identical to SEQ ID NO: 8, which is at least 70%, 80%, 90%, 95% or 99% homologous,
wherein the antibody or antigen binding fragment specifically binds to TIGIT.
In various embodiments, the antibody or antigen-binding fragment thereof comprises:
a) a heavy chain variable region having SEQ ID NO: 7; and
b) a light chain variable region having SEQ ID NO: 8 in a sequence selected from the group consisting of SEQ ID NO,
wherein the antibody or antigen binding fragment specifically binds to TIGIT.
In another aspect, the invention provides an antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences; and a light chain variable region comprising the CDR1, CDR2, and CDR3 sequences,
wherein the heavy chain variable region CDR3 sequence comprises SEQ ID NO: 5 and conservative modifications thereof,
wherein the antibody or antigen binding fragment specifically binds to TIGIT.
Preferably, wherein the light chain variable region CDR3 sequence comprises SEQ ID NO: 6 and conservative modifications thereof.
Preferably, wherein the heavy chain variable region CDR2 sequence comprises SEQ ID NO: 3 and conservative modifications thereof.
Preferably, wherein the light chain variable region CDR2 sequence comprises SEQ ID NO: 4 and conservative modifications thereof.
Preferably, wherein the heavy chain variable region CDR1 sequence comprises SEQ ID NO: 1 and conservative modifications thereof.
Preferably, wherein the light chain variable region CDR1 sequence comprises SEQ ID NO: 2 and conservative modifications thereof.
A preferred antibody or antigen-binding fragment thereof, comprising:
a) comprises the amino acid sequence of SEQ ID NO: 1 heavy chain variable region CDR 1;
b) comprises SEQ ID NO: 3, CDR 2;
c) comprises SEQ ID NO: 5, CDR 3;
d) comprises SEQ ID NO: 2, CDR 1;
e) comprises the amino acid sequence of SEQ ID NO: 4 CDR 2;
f) comprises the amino acid sequence of SEQ ID NO: 6 light chain variable region CDR 3;
wherein the antibody or antigen binding fragment specifically binds to TIGIT.
The antibody of the invention may be a chimeric antibody.
The antibody of the invention may be a humanized antibody.
The antibody of the invention may be a fully human antibody.
The antibody of the invention may be a rat antibody.
In another 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 a 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 another aspect, the present invention provides a method comprising culturing the above-described host cell and isolating the antibody;
wherein the antibody is prepared by immunization in rats with human TIGIT protein.
In another aspect, the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment of said antibody as described herein and one or more pharmaceutically acceptable excipients, diluents or carriers.
The present invention also provides a method of making an anti-TIGIT antibody or antigen-binding fragment thereof, the method comprising:
(a) providing:
(i) a heavy chain variable region antibody sequence comprising SEQ ID NO: 1, the CDR1 sequence of SEQ ID NO: 3 and the CDR2 sequence of SEQ ID NO: 5, a CDR3 sequence; and/or
(ii) A light chain variable region antibody sequence comprising SEQ ID NO: 2, the CDR1 sequence of SEQ ID NO: 4 and the CDR2 sequence of SEQ ID NO: 6, a CDR3 sequence; and
(b) expressing the altered antibody sequence as a protein.
The present invention also provides a method for preventing or treating a disease associated with bone loss in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment as described above in the present invention.
The invention also provides a combination method of preventing or treating a tumour or an inflammatory disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment as described above in the invention, and administering to the subject a therapeutically effective amount of an immune checkpoint antibody.
The invention also provides the use of the antibody or antigen-binding fragment thereof in the preparation of a medicament for the prevention or treatment of a tumor or inflammatory disease.
The cancer is selected from melanoma, renal cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, liver cancer, gastrointestinal cancer, glioblastoma, cervical cancer, bladder cancer and rectal cancer.
These and other objects are provided by the present disclosure, which is broadly directed to compounds, methods, compositions, and articles of manufacture that provide antibodies with improved potency. The benefits provided by the present disclosure are broadly applicable to the field of antibody therapeutics and diagnostics, and can be used in conjunction with antibodies that react with a variety of different targets.
The invention provides isolated antibodies, particularly monoclonal antibodies or fully human antagonist antibodies, against TIGIT.
In one aspect, the invention provides an isolated antibody or antigen binding fragment thereof, wherein the antibody or antigen binding fragment binds to human, cynomolgus monkey and/or mouse TIGIT with high affinity.
In certain embodiments, an isolated antibody or antigen-binding fragment thereof described herein comprises:
A) one or more heavy chain cdrs (hcdr) selected from:
(i) and SEQ ID NO: 1 HCDR1 having at least 90% sequence identity HCDR 1;
(ii) and SEQ ID NO: 3 HCDR2 having at least 90% sequence identity to the HCDR2 set forth in fig. 3; and
(iii) and SEQ ID NO: 5 HCDR3 having at least 90% sequence identity HCDR 3;
B) one or more light chain cdrs (lcdr) selected from:
(i) and SEQ ID NO: 2 LCDR1 having at least 90% sequence identity LCDR 1;
(ii) and SEQ ID NO: 4, LCDR2 having at least 90% sequence identity to LCDR2 set forth in fig. 4; and
(iii) and SEQ ID NO: LCDR3 set forth in 6 having an LCDR3 of at least 90% sequence identity;
or
C) One or more HCDRs of a) and one or more LCDRs of B).
In certain embodiments, an isolated antibody or antigen-binding fragment thereof described herein comprises:
A) one or more heavy chain cdrs (hcdr) selected from the group consisting of:
(i) comprises the amino acid sequence of SEQ ID NO: 1 or HCDR1 which differs from the amino acid sequence from said HCDR1 by no more than 2 amino acids by an amino acid addition, deletion or substitution;
(ii) comprises the amino acid sequence of SEQ ID NO: 3 or HCDR2 which differs from the amino acid sequence from said HCDR2 by no more than 2 amino acids addition, deletion or substitution; and
(iii) comprises the amino acid sequence of SEQ ID NO: 5 or HCDR3 which differs from the amino acid sequence from said HCDR3 by no more than 2 amino acids addition, deletion or substitution;
B) one or more light chain cdrs (lcdr) selected from:
(i) comprises the amino acid sequence of SEQ ID NO: 2 or LCDR1 which differs from the amino acid sequence from said LCDR1 by no more than 2 amino acids of an amino acid addition, deletion or substitution;
(ii) comprises the amino acid sequence of SEQ ID NO: 4 or LCDR2 which differs from the amino acid sequence from said LCDR2 by no more than 2 amino acids of addition, deletion or substitution of an amino acid; and
(iii) comprises SEQ ID NO: 6 or LCDR3 which differs from the amino acid sequence from said LCDR3 by no more than 2 amino acids of addition, deletion or substitution of an amino acid; or
C) One or more HCDRs of a) and one or more LCDRs of B).
In certain embodiments, an isolated antibody or antigen-binding fragment thereof described herein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein
(A) The VH comprises:
(i) comprises the amino acid sequence of SEQ ID NO: HCDR1 for 1;
(ii) comprises the amino acid sequence of SEQ ID NO: HCDR2 of 3; and
(iii) comprises the amino acid sequence of SEQ ID NO: HCDR3 of 5; and/or
(B) The VL comprises:
(i) comprises the amino acid sequence of SEQ ID NO: LCDR1 of 2;
(ii) comprises SEQ ID NO: LCDR2 of 4; and
(iii) comprises SEQ ID NO: LCDR3 of 6.
In certain embodiments, an isolated antibody or antigen-binding fragment thereof described herein comprises:
(A) a heavy chain variable region comprising:
(i) SEQ ID NO: 7;
(ii) and SEQ ID NO: 7 an amino acid sequence having at least 85%, 90% or 95% identity; or
(iii) And SEQ ID NO: 7 has one or more amino acid additions, deletions and/or substitutions compared to the amino acid sequence; and/or
(B) A light chain variable region comprising:
(i) SEQ ID NO: 8 'amino acid sequence'
(ii) And SEQ ID NO: 8, an amino acid sequence having at least 85%, at least 90%, or at least 95% identity; or
(iii) And SEQ ID NO: 8 compared to an amino acid sequence having one or more amino acid additions, deletions and/or substitutions.
In certain embodiments, an isolated antibody or antigen-binding fragment thereof described herein comprises SEQ ID NO: 7 and SEQ ID NO: 8, or a light chain variable region as set forth in fig. 8.
In certain embodiments, an isolated antibody or antigen-binding fragment thereof disclosed herein is a chimeric, humanized, or fully human antibody or a rat antibody. Preferably, the antibody is a fully human monoclonal antibody.
In certain embodiments, the isolated antibody or antigen-binding fragment thereof 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.
In certain embodiments, an isolated antibody or antigen-binding fragment thereof described herein comprises:
(A) a heavy chain comprising:
(i) SEQ ID NO: 11;
(ii) and SEQ ID NO: 11, an amino acid sequence having at least 85%, 90%, or 95% identity; or
(iii) And SEQ ID NO: 11 has one or more amino acid additions, deletions and/or substitutions; and/or
(B) A light chain comprising:
(i) SEQ ID NO: 12;
(ii) and SEQ ID NO: 12 an amino acid sequence having at least 85%, at least 90%, or at least 95% identity; or
(iii) And SEQ ID NO: 12 compared to an amino acid sequence having one or more amino acid additions, deletions and/or substitutions.
In certain embodiments, an isolated antibody or antigen-binding fragment thereof described herein comprises a heavy chain variable region comprising SEQ ID NO: 11 and a light chain comprising SEQ ID NO: 12, light chain.
In certain instances, the present disclosure relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a heavy chain variable region and/or a light chain variable region of an isolated antibody disclosed herein.
In certain instances, the present disclosure relates to a vector comprising a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof disclosed herein.
In certain instances, the present disclosure relates to a host cell comprising an expression vector disclosed herein.
In certain instances, the present disclosure relates to a pharmaceutical composition comprising at least one antibody or antigen-binding fragment thereof disclosed herein and a pharmaceutically acceptable carrier.
In certain instances, the present disclosure relates to a method of making an anti-TIGIT antibody or antigen-binding fragment thereof, the method comprising expressing the antibody or antigen-binding fragment thereof in a host cell disclosed herein, and isolating the antibody or antigen-binding fragment thereof from the host cell.
In certain instances, the present disclosure relates to a method of modulating an immune response in a subject, the method comprising administering to the subject an antibody or antigen-binding fragment thereof disclosed herein, such that an immune response is modulated in the subject, optionally the immune response is TIGIT-associated.
In certain instances, the present disclosure relates to a method of inhibiting tumor cell growth in a subject, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof or pharmaceutical composition disclosed herein.
In certain instances, the disclosure relates to a method of treating or preventing a disease including a proliferative disorder (e.g., cancer) in a subject, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof or pharmaceutical composition disclosed herein.
In certain instances, the disclosure relates to the use of an antibody or antigen-binding fragment thereof disclosed herein for the preparation of a medicament for treating or preventing a disease, including a proliferative disorder (e.g., cancer).
In certain embodiments, the cancer is selected from melanoma, renal cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, liver cancer, gastrointestinal cancer, glioblastoma, cervical cancer, bladder cancer, and rectal cancer.
In certain instances, the disclosure relates to kits or devices and related methods utilizing the antibodies or antigen-binding portions thereof disclosed herein and the pharmaceutical compositions disclosed herein that are useful for treating diseases, including cancer.
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 described herein and/or other subject matter will become apparent in the teachings set forth herein. This 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.
Drawings
Figure 1 shows a human TIGIT binding assay by FACS.
Figure 2a shows ELISA results binding to human TIGIT; 2b shows the results of ELISA binding to human CD 28; 2c shows the results of ELISA binding to human PD-1; 2d shows ELISA results binding to human ICOS; 2e shows ELISA results binding to human CTLA 4; 2f shows the results of ELISA binding to human PVRIG.
Figure 3 shows a cynomolgus monkey TIGIT binding assay by FACS.
Figure 4 shows a mouse TIGIT binding assay.
Figure 5 shows a human TIGIT ligand competition assay by FACS.
FIG. 6 shows the reporter gene assay for W3645-2.131.4-hIgG1L 3.
Fig. 7 shows an activation assay for Jurkat cells expressing human TIGIT.
Figure 8 shows the activation assay of CD8+ T cells.
Figure 9 shows NK-killing assay.
FIG. 10 shows the ADCC effect of W3645-2.131.4-hIgG1L 3.
FIG. 11 shows the melting curves of W3645-2.131.4-hIgG1L 3.
FIG. 12 shows the serum stability analysis of W3645-2.131.4-hIgG1L 3.
Fig. 13 shows the results of the CT26 isogenic model.
FIG. 14 shows the results of the C57(hTIGIT)/MC38 model.
Figure 15 shows the proportion of CD4+ and CD8+ T cells in the spleen.
FIG. 16 shows the proportion of CD4+ and CD8+ T cells in TIL.
Figure 17 shows Treg ratios in spleen and TIL.
FIG. 18 shows the proportion of M1 populations in the TIL.
FIG. 19 shows intracellular IFNg following stimulation in TIL.
Detailed Description
In order that the 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 immunoreceptor with Ig and ITIM domains", also known as Vstm3 and WUCAM, referred to herein is an inhibitory receptor expressed on NK and CD8+ T cells and on a subset of 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 chain thereof. An "antibody" refers to a protein or antigen-binding portion thereof comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains CH1, CH2, and CH 3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises a domain CL. The VH and VL regions may be further subdivided into hypervariable regions, termed Complementarity Determining Regions (CDRs), interspersed with more conserved regions, termed Framework Regions (FRs). 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, FR 4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The CDR boundaries of an antibody are defined or identified by the Kabat numbering system.
The term "antibody" as used in this disclosure refers to an immunoglobulin or a fragment or derivative thereof, and encompasses any polypeptide comprising an antigen binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, 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, these fragments will comprise antigen binding fragments.
The terms "antigen-binding fragment," "antigen-binding domain," and "binding fragment" refer to the portion of an antibody molecule that comprises the amino acids responsible for specific binding between an antibody and an antigen. In the case of large antigens, the antigen-binding fragment may bind only a portion of the antigen. The portion of the antigenic molecule responsible for the specific interaction with the antigen-binding fragment is referred to as an "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, so-called Fd antibody fragments consist of only VH domains, but still retain some of the antigen-binding function of the intact antibody.
In line with the above, the term "epitope" defines an antigenic determinant, which is specifically bound/recognized by a binding fragment as defined above. The binding fragment can specifically bind to/interact with a conformation or contiguous epitope that is unique to the target structure. For polypeptide antigens, conformational or discontinuous epitopes are characterized by the presence of two or more discrete amino acid residues that 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 segments of one or more polypeptide chains. When the polypeptide chain folds into a three-dimensional structure, these residues come together on the surface of the molecule to form an epitope. In contrast, a continuous or linear epitope is composed of two or more discrete amino acid residues that are present in a single linear segment of a polypeptide chain.
The term "cross-reactivity" refers to the binding of an antigenic fragment described herein to the same target molecule in humans, monkeys, and/or rodents (mice or rats). Thus, "cross-reactivity" should be understood as inter-species reactivity with the same molecule X expressed in different species, but not with molecules other than X. Cross-species specificity of monoclonal antibodies that recognize monkey and/or murine (mouse or rat) TIGIT can be determined, for example, by FACS analysis.
As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, and the like. The terms "patient" or "subject" are used interchangeably unless otherwise indicated.
The terms "treatment" and "method of treatment" refer to both therapeutic treatment and prophylactic/preventative measures. The person in need of treatment may include individuals already having a particular medical condition as well as individuals who may ultimately have the condition.
The term "conservative modifications" refers to nucleotide and amino acid sequence modifications that do not significantly affect or alter the binding characteristics of an antibody encoded by or containing the nucleotide sequence. Such conservative sequence modifications include nucleotide and amino acid substitutions, additions and deletions. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with 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).
Unless otherwise specified, the experimental methods in the following examples are conventional methods.
Examples
Example 1: materials and antibody production
1. Universal material
The antigen was purchased from a vendor or prepared by itself (see details in table 1). W364-hpro1.ecd.his is the extracellular domain of human TIGIT (NP _776160.2) with a C-terminal poly-histidine tag; W364-hPro1.ECD. hFc is the extracellular domain of human TIGIT (NP-776160.2) carrying the Fc region of human IgG1 at the C-terminus; w364-mro 1.ecd.his is the extracellular domain of mouse TIGIT (NP _001139797.1) with a C-terminal poly-histidine tag; W364-mPro1.ECD. hFc is the extracellular domain of mouse TIGIT (NP-001139797.1) with an Fc region of human IgG1 at the C-terminus; w364-hpro1l1.ecd. hfc is the extracellular domain of human CD155(NP _006496.3) bearing the Fc region of human IgG1 at the C-terminus; W364-hPro1L1.ECD. mFc is the extracellular domain of human CD155 (NP-006496.3) bearing the Fc region of mouse IgG1 at the C-terminus.
TABLE 1 antigens and ligands
The general study materials and their sources are listed in table 2 below.
TABLE 2 general cell lines and reagents
Construction of expression vector for BMK antibody
The amino acid sequences encoding the variable domains of anti-TIGIT antibodies (WBP364-BMK 1; Tiragolumab, U.S. Pat. No. 5, 20170088613A1/4.1D3), anti-TIGIT antibodies (WBP364-BMK 4; U.S. Pat. No. 20160176963A1, 22G2) and anti-TIGIT antibodies (WBP364-BMK 6; WO2017059095A1, MAB10) were first codon optimized for mammalian expression and then synthesized by GENEWIZ (Suzhou, China). The DNA segment was then subcloned into pcDNA expression vectors carrying human IgG1 or IgG4 constant regions.
TABLE 3 information on reference antibody
3. Sequencing of hybridomas and construction of self-made antibodies
The anti-TIGIT antibody was discovered by the mechanism after immunization with human TIGIT (Omni rats). Total RNA was first extracted from hybridoma cell samples following the instructions of the TaKaRa MiniBEST universal RNA extraction kit. The RNA was then converted to cDNA using the SMART RACE cDNA amplification kit from Clonetech. The heavy and light chain DNA sequences were amplified from the cDNA using 30 cycles of PCR, each cycle using 94 ℃ denaturation for 30 seconds, 60 ℃ annealing for 30 seconds, and then 72 ℃ extension for 30 seconds. The PCR product was then subcloned into a TA-cloning vector and then sent to GENEWIZ for sequencing. After sequencing data confirmed the monoclonality of the hybridoma cell samples, the amino acid sequences of the VH and VL domains were codon optimized for mammalian expression and then synthesized by GENEWIZ (suzhou, china). The DNA segment was then subcloned into pcDNA expression vectors carrying human IgG1 or IgG4 constant regions.
4. Expression and purification of antibodies
Plasmids containing the VH and VL genes were co-transfected into Expi293 cells (Thermofisiher, A14635). The cells were cultured for 5 days according to the manufacturer's recommended protocol. After validation of the supernatant by SDS-PAGE, the antibody was purified using a protein a column (GE Healthcare, cat.175438). The concentration of the purified Fc-tagged protein was determined by absorbance at 280 nm. The size and purity were confirmed by SDS-PAGE and SEC-HPLC, respectively; then stored at-80 ℃. The purity of the antibodies was tested by SEC-HPLC using Agilent 1260Infinity HPLC. 50 μ L of the antibody solution was injected onto a TSKgel SuperSW3000 column using 50mM sodium phosphate, 0.15M NaCl, pH 7.0 buffer. The run time was 20 minutes. The peak retention time on the column was monitored at 280 nm. Data were analyzed using ChemStation software (V2.99.2.0). All antibodies are required to be > 90% pure.
5. Preparation of cell pools/lines
5.1 cell pools/lines expressing targets for binding assays
Cell lines expressing mouse TIGIT (W364-flinchho. mpro1.fl) and cell lines expressing cynomolgus monkey TIGIT (W364-flinchho. cynopro1.fl) were generated. Briefly, Flp-In-CHO cells were transfected with pcDNA5 expression vectors encoding full-length mouse TIGIT (NP-001139797.1) and cynomolgus monkey TIGIT (XP-015300911.1) using the Lipofectamine 2000 transfection kit according to the manufacturer's protocol. At 48-72 hours post-transfection, the transfected cells were cultured in hygromycin-containing medium for selection and tested for TIGIT expression. A pool of cells expressing mouse and cynomolgus TIGIT was then obtained.
Cell lines (W364-CHOK1.hPro1.FL.2A11) and cell pools (W364-293F. hpro1.FL) expressing human TIGIT were generated. Briefly, CHO-K1 or 293F cells were transfected with pcDNA3.3 expression vector containing full-length human TIGIT (NP-776160.2), respectively, using the Lipofectamine 2000 transfection kit, following the manufacturer's protocol. At 48-72 hours post-transfection, the transfected cells were cultured in a medium containing blasticidin for selection and tested for TIGIT expression. Monoclonal cell lines expressing human TIGIT were obtained by limiting dilution.
5.2 cells expressing target/ligand for functional assays
Jurkat cells overexpressing human TIGIT (W364-Jurkat. hPro1.NFAT.2D11) having NFAT-luciferase reporter gene were generated by transfecting Jurkat cells overexpressing the NFAT-luciferase reporter gene with pSBbi-RB-W364-hPro1.FL (pSBbi-RB plasmid encoding human TIGIT). The cells were cultured in complete RPMI1640 medium containing 10% FBS, 0.5mg/mL hygromycin B for screening, and 4. mu.g/mL blasticidin.
HT1080.2A11 (also known as HT1080.OKT3.scFv.2A11) was produced by the mechanism by transfecting HT1080 tumor cells with pSB plasmid encoding OKT3 (anti-CD 3 antibody) single chain Fv.
P815-PVR was generated by transfecting P815 cells with pCDNA3.1 encoding human CD155(PVR, NP-006496.3).
CD155/TCR activator-CHO recombinant cell line was purchased from BPS Bioscience under catalog number 60548.
6. Production of hybridoma antibodies
6.1 immunization
4 OMT rats 6-8 weeks old were immunized with 40. mu.g protein (W364-hPro1.ECD. his)/animal. The adjuvant mixture comprises Adju-Phos, CpG-ODN or Titer-Max. The animals were injected biweekly by footpad, subcutaneous, intraperitoneal route. Serum titers were measured by ELISA. When serum titers were high enough (. gtoreq.1: 24, 300), animals with the highest titers were finally boosted with protein in sterile PBS without adjuvant. After 2-4 days (48-96 hours), animals were euthanized and lymph nodes or spleen were used for cell fusion.
6.2 serum Titer assay
Serum antibody titers against the provided antigens were measured using ELISA assays. Plates (Nunc) were coated with 100. mu.L of antigen (W364-hPro1.ECD. hFc) at a concentration of 1. mu.g/mL overnight at 4 ℃ and then blocked with blocking buffer (1 XPBS/2% BSA) for 1 hour at room temperature. Rat serum was diluted 1:3 in blocking buffer starting from a 1:100 dilution and incubated for 1 hour at room temperature. The plates were then washed and subsequently incubated with goat anti-rat IgG-Fc-HRP (Bethyyl, A110-236P) secondary antibody for 1 hour. After washing, TMB substrate was added and the interaction was stopped by 2M HCl. The absorbance at 450nm was read using a microplate reader (Molecular Device). Serum titers were determined using more than three times the negative value as a standard.
After immunization, 2 OMT rats were selected for fusion, and the serum titers of these two OMT rats before fusion are shown in table 4.
TABLE 4 serum titers (ELISA binding to human/mouse TIGIT)
6.3 hybridoma Generation
Lymph nodes and spleen from immunized animals were homogenized and filtered to remove blood clots and cellular debris. Sp2/0 myeloma cells in logarithmic growth were collected and centrifuged. B cells and Sp2/0 myeloma cells were treated with pronase solutions, respectively, and the reaction was terminated by FBS. Cells were washed and counted. B cells were mixed with Sp2/0 myeloma cells in a 1:1 ratio in the electrofusion solution. The electrofusion is performed according to an electrofusion procedure. The fused cells were resuspended in DMEM medium (hypoxanthine-aminopterin-thymidine medium) supplemented with 20% FBS and 1 xhat, and then transferred to a 96-well plate. (ii) fusing the cells at 37 ℃ with 5% CO2Holding 10-14 in incubatorAnd (5) day.
6.4 antibody screening and subcloning
Positive clones were screened by cell-based binding and ligand competition assays. Positive hybridoma cells in logarithmic growth were counted and 200-300 cells were added to 1.5mL of semi-solid HAT medium. The cells were gently mixed in a vortex shaker for 5-10 seconds and then seeded in 6-well plates. The plates were incubated at 37 ℃ 5% CO2The culture is maintained in the incubator for 7-8 days. Each visible single colony was picked into a 96-well plate containing DMEM medium supplemented with 10% FBS. After 2-3 days, cell supernatants were collected and screened again to obtain positive hybridoma clones.
6.5 hybridoma sequencing and antibody Generation
Positive clones were sent to GENEWIZ for sequencing. After confirming the monoclonality of the positive clones, the amino acid sequences of the VH and VL domains without the PTM site were codon optimized for mammalian expression and then synthesized by GENEWIZ (suzhou, china). The DNA segment was then subcloned into a pcDNA expression vector with constant regions of human IgG1 or human IgG4. Plasmids containing the VH and VL genes were co-transfected into Expi293 cells (Thermo Fisher, A14635). Then cultured for 5 days according to the manufacturer's recommended protocol. After confirmation of the supernatant by SDS-PAGE, the antibody was purified using a protein a column (GE Healthcare, cat.175438). The concentration of the purified Fc-tagged protein was determined by absorbance at 280 nm. The size and purity were confirmed by SDS-PAGE and SEC-HPLC, respectively; then stored at-80 ℃. The purity of the antibodies was tested by SEC-HPLC using Agilent 1260Infinity HPLC. 50 μ L of the antibody solution was injected onto a TSKgel SuperSW3000 column using 50mM sodium phosphate, 0.15M NaCl, pH 7.0 buffer. The run time was 20 minutes. The peak retention time on the column was monitored at 280 nm. Data were analyzed using ChemStation software (V2.99.2.0). All antibodies are required to be > 90% pure.
6.6. Antibody sequences
51 positive cell lines were selected for subcloning by primary and secondary binding screens and TIGIT/CD155 blocking and reporter assays. After confirmation of the purified subcloned antibodies, 10 antibodies were selected for sequencing and subsequent human IgG transformation. The final lead antibody was identified and the sequences are shown in tables 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 and light chains
Example 2: in vitro characterization of lead antibodies
1. Human TIGIT combination (FACS)
293F cells (W364-293F. hPro1.FL. cells, 1X 10) expressing human TIGIT5Individual cells/well) with W3645-2.131.4-hIgG1L3 in serial dilutions (from100nM start dilution, 5 fold, 8 concentrations) was incubated at 4 ℃ for 1 hour. After washing with 1 XPBS/1% BSA, the secondary antibody R-PE-labeled goat anti-human IgG antibody (1:150) was added and incubated with the cells in the dark at 4 ℃ 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 XPBS/1% BSA. The MFI (median fluorescence intensity) of the cells was measured by flow cytometry (BD, cantonii) and analyzed by flowjo.7. Cell-bound ECs were obtained using four-parameter nonlinear analysis using GraphPad prism.6 software50The value is obtained.
The binding results of W3645-2.131.4-hIgG1L3 on WBP64-293F.hPro1.FL. pool are shown in FIG. 1. The WBP3645 leader antibody can have an EC of 0.17nM50Human TIGIT, reference antibodies WBP364-BMK1(Genentech) and WBP364-BMK4(BMS) with dose-dependent binding to cell surface EC50Is 0.35nM to 0.38 nM. The data are shown in figure 1.
2. Cross-family binding (ELISA)
Non-tissue culture treated flat bottom 96 well plates were pre-coated overnight at 4 ℃ with human CD28 ECD, human CTLA4 (cytotoxic T-lymphocyte-associated protein 4) ECD, human ICOS (inducible T-cell co-stimulator) ECD, human PD-1 (programmed death protein 1) protein ECD, human PVRIG (CD112 receptor) ECD, and human TIGIT ECD, homemade by the 1.0 μ g/mL institution. After blocking with 2% BSA, 100. mu.L of 10-fold titrated antibody from 1nM to 0.01nM was added to each well and incubated for 1 hour at ambient temperature (22 ℃). After removal of unbound antibody, HRP-labeled goat anti-human IgG antibody was added to the wells and incubated for 1 hour. The development was performed by dispensing 100. mu.L of TMB substrate and then stopped by 100. mu.L of 2N HCl. The absorbance was read at 450nm using a microplate spectrophotometer (MD, M5e) and the data was analyzed by Prism 6.
TIGIT has been identified as a member of the CD28 family based on genetic structure. The ELISA binding results of the WBP3645 leader antibody (W3645-2.131.4-hIgG1L3) to the TIGIT paralogs CD28, PD-1, ICOS, CTLA4 are shown in FIGS. 2-a to 2-e. Binding to PVRIG, which shares a ligand for CD112 with TIGIT, was also tested by ELISA. The data are shown in fig. 2-f. From the results of the ELISA binding assay, W3645-2.131.4-hIgG1L3 binds specifically to TIGIT with no cross-reactivity with human CD28, PD-1, ICOS, CTLA4 and PVRIG.
3. Cross species binding (FACS)
3.1 Macaca fascicularis TIGIT binding
FLPin CHO cells (W364-FLPin CHO. cynoPro1.FL. cells, 1X 10. cells) expressing cynomolgus monkey TIGIT5Individual cells/well) were incubated with serial dilutions of W3645-2.131.4-hIgG1L3 (5-fold dilution from 100nM, 8 points) at 4 ℃ for 1 hour. After washing with 1 XPBS/1% BSA, the secondary antibody R-PE-labeled goat anti-human IgG antibody (1:150) was added and incubated with the cells in the dark at 4 ℃ 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 XPBS/1% BSA. The MFI of the cells was measured by flow cytometry (BD, cantonii) and analyzed by flowjo.7. Cell-bound ECs were obtained using four-parameter nonlinear analysis using GraphPad prism.6 software50The value is obtained.
W3645-2.131.4-hIgG1L3 with an EC of 0.05nM50Cross-binding to FLPin-CHO cells expressing Macaca fascicularis TIGIT. The data are shown in figure 3.
3.2 mouse TIGIT binding
Flp-in CHO cells (W364-FLPin CHO. mousePro1.FL. cells) (1X 10) expressing mouse TIGIT5Individual cells/well) were incubated with serial dilutions of W3645-2.131.4-hIgG1L3 (5-fold dilution from 100nM, 8 points) at 4 ℃ for 1 hour. After washing with 1 XPBS/1% BSA, the secondary antibody R-PE-labeled goat anti-human IgG antibody (1:150) was added and incubated with the cells in the dark at 4 ℃ for 1 hour. Anti-human TIGIT antibody 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 XPBS/1% BSA. The MFI of the cells was measured by flow cytometry and analyzed by flowjo.7. Cell-bound ECs were obtained using four-parameter nonlinear analysis using GraphPad prism.6 software50The value is obtained.
W3645-2.131.4-hIgG1L3 with an EC of 0.14nM50Cross-binding to FLP-in-CHO cells expressing mouse TIGIT. BMK6 is a reference antibody (Astellas) that cross-binds mouse TIGIT. The data are shown in figure 4.
4. Affinity to human TIGIT (SPR)
Binding affinity of TIGIT antibody to antigen was detected by SPR (surface plasmon resonance) assay using Biacore 8K. Antibodies were captured on CM5 sensor chip (GE) against an anti-human IgG Fc antibody immobilized. Different concentrations of human TIGIT extracellular domain were injected onto the sensor chip at a flow rate of 30 μ L/min for a binding period of 180s, followed by dissociation for 3600 s. After each binding cycle, the chip was regenerated with 10mM glycine (pH 1.5). The sensorgrams for the blank surface and buffer channel were subtracted from the test sensorgram. Experimental data were fitted with a 1:1 model using Langmiur analysis. The molar concentration of W364-hPro1.ECD. his (Sino) was calculated using a molecular weight of 20 kDa.
The affinity KD of W3645-2.131.4-hIgG1L3 for soluble antigen was 7.50E-10M (see data in Table 9).
TABLE 9 protein-based affinity by SPR
5. Affinity to cell surface human TIGIT (FACS)
Binding affinity of the antibody to TIGIT on the cell surface was measured by FACS analysis. CHOK1 cells (W364-CHOK1.hPro1.2A11) overexpressing human TIGIT were cultured at 5X104The density of individual cells/ml was transferred to a 96-well U-shaped bottom plate. The test antibodies were serially diluted in wash buffer (1 XPBS/1% BSA) and incubated with the cells for 1 hour at 4 ℃. The secondary antibody goat anti-human IgG Fc FITC (2.5 moles FITC per mole IgG) was added and incubated for 0.5 hours at 4 ℃ in the dark. Then will beCells were washed once and resuspended in 1 XPBS/1% BSA and analyzed by flow cytometry. The fluorescence intensity was converted to bound molecules/cells on the basis of quantitative beads (QuantumTM MESF kit, Bangs Laboratories, Inc.).
The affinity of the antibody to human TIGIT on the cell surface as measured by FACS was 2.70E-11M KD. The data are shown in table 10.
TABLE 10 cell-based affinity by FACS
6. Ligand competition assay
Binding of CD155(PVR, poliovirus receptor) to TIGIT expressing cells was blocked by the antibody, as determined by flow cytometry. Briefly, 1E 5293F (TIGIT +) cells were incubated with serial dilutions of TIGIT or hIgG1 isotype control antibody and 2 μ g/mL of CD155.ECD with mFc tag for 60 minutes at 4 ℃. After two washes 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 min at 4 ℃. Cells were washed twice in the same buffer and MFI (mean fluorescence intensity) of stained cells was measured using a FACS Canto II cytometer (BD Biosciences). Background fluorescence was established using wells containing no antibody or secondary antibody only, cell-bound IC was obtained using GraphPad Prism software using four-parameter non-linear regression analysis50The value is obtained.
Blocking of binding of human CD155(PVR) to cells expressing human TIGIT by antibodies was determined by flow cytometry. The leader Ab blocks the binding of human TIGIT and ligand CD155, IC50It was 0.14 nM. The data are shown in figure 5.
NFAT reporter assay
In this assay, W364-jurkat.hpro1.nfat.2d11 (Jurkat cells expressing human TIGIT/NFAT reporter) was used as effector cells; CHO cells overexpressing CD155 and TCR activator (BPS, Cat. No. 60548) were used as target cells. When these two cells were co-cultured, NFAT luciferase reporter signal was inhibited in effector cells with binding of TIGIT on Jurkat cells and ligand CD155 on CHO cells. Luciferase signal was increased by blocking the binding of the TIGIT and CD155 with anti-TIGIT antibody. Briefly, cells overexpressing CD155 and TCR activator were seeded overnight on white plates at a density of 2E 4/well. The serially diluted antibodies and TIGIT-overexpressing Jurkat/NFAT reporter cells (2E 4/well) were then combined with target cells at 37 ℃ and 5% CO2The following co-cultivation was carried out for 5 to 6 hours. After incubation, ONE-Glo luciferase reagent (Promega, cat No. E6130) was added to the cells, and NFAT activity was measured by envision (pe) using luminescence. Curve fitting and EC acquisition Using four parameter nonlinear analysis Using GraphPad prism.6 software50The value is obtained.
WBP3645 lead antibody can stimulate NFAT pathway, EC, in a Jurkat NFAT-luciferase reporter cell line overexpressing hTIGIT50It was 0.92 nM. The data is shown in figure 6.
8. Activation assay (IL-2ELISA) of Jurkat cells expressing human TIGIT
In this assay, W364-jurkat.hpro1.nfat.2d11 (Jurkat cells expressing human TIGIT/NFAT reporter gene) was used as effector cells; ht1080.okt3scfv.a10 was used as target cells. HT1080 is a tumor cell line endogenously expressing human CD155. After 2 days incubation of W364-Jurkat. hPro1.NFAT.2D11(1E 4/well) with HT1080.OKT3scFV.A10(1E 4/well), secretion of IL2 in the medium was determined by ELISA (capture antibody: purified mouse anti-hIL 2 monoclonal IgG2A clone 5355, R&D, MAB 602; detecting an antibody: biotinylated anti-hIL 2 antibody, R&D, BAF 202; recombinant human IL2 standard: r&D, 202-IL-050). Curve fitting and EC acquisition Using four parameter nonlinear analysis Using GraphPad prism.6 software50The value is obtained.
The WBP3645 leader antibody can enhance IL2 release, EC, from Jurkat cells overexpressing hTIGIT50It was 0.29 nM. The data are shown in figure 7.
Activation assay for CD8+ T cells
CD8+ T and CD155 expressing tumor cells were performedTo assess whether the TIGIT antibody is capable of enhancing CD8+ T activity. Briefly, CD8+ T cells (1X 10)5Individual cells/well) with antibody at a concentration of 20nM and HT1080 cells (1X 10) transfected with OKT3ScFv4/well) co-culture. After 5 days of incubation IFNg production in the medium was determined by ELISA (capture Ab: human IFNg Mab clone 2G1, Thermo, M700A; detection Ab: biotinylated anti-hIFN. gamma. antibody, Thermo, M701B; recombinant IFNg standard: PeproTech, 300-02-250).
The WBP3645 leader antibody W3645-2.131.4-hIgG1L3 can enhance IFNg release in a CD8+ T activation assay. The data is shown in figure 8.
NK killing assay
The effect of anti-human TIGIT antibodies on NK cell-mediated lysis of CD 155-expressing cells was evaluated in vitro. Briefly, CD155(PVR) -expressing P815 cells (mouse mast cell tumor cell line) as target cells were pre-loaded with BATDA (PE, AD0116) and exposed to human NK cells in the presence of anti-TIGIT antibodies at various concentrations (10nM, 2nM, 0.4nM, 0.08nM, 0.016 nM). W364-BMK1 was used as a positive control, and a human IgG1 isotype control was used as a negative control. After incubation for 2-4 hours, the supernatant was collected and mixed with europium buffer. Cytotoxicity was detected by Envision (PE, Ex337/Em615) using TRF (time resolved fluorescence) and data presented with Prism 6.
The WBP3645 lead antibody can dose-dependently enhance NK killing activity on PVR-overexpressing cells, with better potency than BMK. The data are shown in figure 9.
ADCC (antibody-dependent cell-mediated cytotoxicity) assay
To test ADCC effects, engineered TIGIT expressing human cells (W364-chok1.hpro1.2a11) were preincubated with various concentrations of TIGIT antibody in 96-well plates for 30 minutes, followed by 20: 1 ratio of effector cells/target cells freshly isolated PBMC (peripheral blood mononuclear cells) were added. The plates were incubated at 37 ℃ and 5% CO2The mixture was incubated for 4 hours. Target cell lysis was determined by LDH-based cytotoxicity detection kit. The blots were read at 492nm using a microplate spectrophotometer (MD, SpectraMax, M5e)And (4) luminosity. Curve fitting was performed using GraphPad prism.6 software using four parameter nonlinear analysis and EC50 values were obtained.
W3645-2.131.4-hIgG1L3 can lyse hTIGIT-overexpressing cells by antibody-dependent cell-mediated cytotoxicity, with 0.0019nM EC 50. The data is shown in figure 10.
12. Thermal stability evaluation by DSF
Tm of WBP3645 leader antibody was studied using QuantStudio 7Flex real-time PCR system (Applied Biosystems). mu.L of the antibody solution was mixed with 1. mu.L of 62.5X SYPRO Orange solution (Invitrogen) and transferred to 96-well plates (Biosystems). The plate was heated from 26 ℃ to 95 ℃ at a rate of 0.9 ℃/min and the resulting fluorescence data collected. The negative derivatives of the change in fluorescence at different temperatures were calculated and the maximum was defined as the melting temperature Tm. If the protein has multiple unfolding transitions, the first two Tm's are reported, designated Tm1 and Tm2, respectively. Data collection and Tm calculation were automated by operating software (Quantstudio real-time PCR software v 1.3).
Using QuantstrudioTM7Flex real-time PCR System (Applied Biosystems) to study T of WBP3645 leader antibodym. Good thermal stability was observed by DSF testing. The data calculated by the software are shown in table 11 and fig. 11.
TABLE 11 thermal stability of W3645-2.131.4-hIgG1L3
13. Serum stability assessment
To evaluate the serum stability of the antibodies, serum was freshly prepared from the blood of healthy human donors. Antibodies were gently mixed with serum, aliquoted into 5 tubes and incubated at 37 ℃. Aliquots were then drawn at the indicated time points: days 0, 1, 4, 7 and 14, and the aliquots were flash frozen in liquid nitrogen and stored at-80 ℃ until use. The serum stability of the antibodies was assessed using the binding capacity detected by ELISA. The absorbance was read at 450nm using a microplate spectrophotometer (MD, SpectraMax, M5 e). Curve fitting was performed using GraphPad prism.6 software using four parameter nonlinear analysis and EC50 values were obtained.
In the serum stability assay, W3645-2.131.4-hIgG1L3 was stable in serum at 37 ℃ for at least 2 weeks. The data is shown in figure 12.
Example 3: in vivo characterization
1. Study of rodent efficacy
1.1 CT26 isogenic model
The WBP3645 lead antibody efficacy study was tested in the CT-26 model in BALB/c mice. Female BALB/c mice (Beijing vitamin River Laboratory Animal Technology Co., LTD) 6-8 weeks old were used in the study. CT-26 cells were cultured in vitro as monolayer cultures in RPMI1640 medium containing 10% fetal bovine serum, 100U/mL penicillin and 100. mu.g/mL streptomycin at 37 ℃ and 5% CO2In a cell culture chamber.
For the treatment model, each mouse was treated with CT-26 tumor cells (3.0X 10) at the right forearm axilla5) Subcutaneous inoculation. When the average tumor volume reached approximately 55mm3At time, animals were randomized into different groups: control-PBS; PD-1Ab 3 mg/kg; WBP364-BMK6.uIgG4.SPK 10 mg/kg; W3645-2.131.4-hIgG1L 310 mg/kg; WBP364-BMK6.uIgG4.SPK + PD-1Ab 10+3 mg/kg; w3645-2.131.4.hIgG1L3+ PD-1Ab 10+3mg/kg and received a first injection of antibody followed by intraperitoneal treatment with PBS or antibody twice weekly for a total of 5 injections. The day of the first injection was taken as day 0. For all tumors studied, mice were weighed and tumor growth was measured twice weekly using calipers. All procedures related to Animal handling, Care and treatment in this study were conducted in compliance with the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of the Shanghai Biomodel (Shanghai Bio-model) under the approval of the laboratory Animal Care and Use CommitteeAssociation for Association and Acceptance of Laboratory Animal Care (AAALAC).
Tumor volume using the formula (1/2 (length x width)2) To calculate. Results are expressed as mean and standard error (mean ± SEM). Data were analyzed using a two-way ANOVA Bonferroni post hoc test with Prism software, p<0.05 was considered statistically significant.
CT26 is a model of PD-1 resistance in which the highest TGI of PD-1 antibodies was below 50% (data not shown). As a single agent, the WBP3645 lead antibody showed better efficacy in this model than the W364-BMK6 and PD-1 antibodies. When combined with the-PD-1 antibody, the WBP3645 lead antibody showed a better synergistic effect than W364-BMK 6. The data is shown in figure 13.
1.2C 57(hTIGIT)/MC38 model
In vivo efficacy studies of W3645-2.131.4.hIgG1L3 were tested in the MC38 model using h-TIGIT mice. Female hTIGIT mice (Nanjing GemPharmatech Co., LTD) of 6-8 weeks of age were used in the study.
In this in vivo model of function, each mouse was treated with MC38 tumor cells (5.0X 10) at the right forearm axilla5) Subcutaneous inoculation. When the average tumor volume reached approximately 65mm3At the time, animals were randomized into different groups: medium-PBS; PD-1Ab 0.3 mg/kg; WBP364-BMK410 mg/kg; W3645-2.131.4-hIgG1L 310 mg/kg; WBP364-BMK4+ PD-1Ab 10+0.3 mg/kg; w3645-2.131.4. uggg 1L3+ PD-1Ab 10+0.3mg/kg, then received a first injection of antibody and was treated intraperitoneally twice weekly with PBS or antibody for a total of 6 injections. The day of the first injection was taken as day 0. For all tumors studied, mice were weighed and tumor growth was measured twice weekly using calipers. All procedures related to Animal handling, Care and treatment in this study were conducted in compliance with the Association for the Assessment and certification of Laboratory Animal Care (Association for Association and acceptance of Laboratory Animal Care) in accordance with the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of the Shanghai Biomodel (Shanghai Bio-model)(AAALAC).
Tumor volume using the formula (1/2 (length x width)2) To calculate. Results are expressed as mean and standard error (mean ± SEM). Data were analyzed using a two-way ANOVA Bonferroni post hoc test with Prism software, p<0.05 was considered statistically significant.
In vivo efficacy studies of W3645-2.131.4.hIgG1L3 were tested in the MC38 model using h-TIGIT mice. WBP364-BMK4 was used as a reference control. In this model, WBP3645 lead antibody as a single agent failed to inhibit tumor growth in the MC38 model. The WBP3645 lead antibody in combination with the PD-1 antibody showed a synergistic effect in the C57(hTIGIT)/MC38 efficacy model. The data are shown in figure 14. 1.3 Ex vivo study of MC38 model
In an in vivo efficacy study of the C57(hTIGIT)/MC38 model (see 3.7.1.2), spleen and tumor tissues were isolated from 4 groups of mice (n-4, from a total of 8) with different treatments for cell population profiling. These 4 groups were: control-PBS; PD-1 antibody 0.3 mg/kg; W3645-2.131.4-hIgG1L 310 mg/kg; and a combination of 0.3mg/kg of anti-PD-1 antibody and W3645-2.131.4-hIgG1L 310 mg/kg. In this study, populations of M1 and TIL (tumor infiltrating lymphocytes) in CD4+ T cells, CD8+ T cells, tregs, SPL (splenic lymphocytes) were analyzed, as well as T cell function.
Splenic lymphocytes from each mouse were first isolated by gentle milling, filtration through a 70 μm filter and washed twice with 15mL PBS. Tumor infiltrating lymphocytes were isolated from tumor tissue following the protocol of the mouse tumor dissociation kit (Miltenyi Biotec Cat: 130-. After the cells are isolated, some of them are fixed for staining. After fixation and permeation with buffer (BD,51-2090KZ), intracellular markers were stained. Information for the antibodies used for cell population profiling is shown below. 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, cantonii) and data were analyzed using flowjo.7 and Prism 6.
For the assessment of T cell function in TIL, intracellular IFNg was detected after stimulation. Briefly, 1E6 cells isolated from tumor tissue were treated with a 0.5X cell stimulation mixture (Invitrogen,4333766) and 1X Golgi Stop (BD 51-2092K2) for 9 hours. Subsequently, the cells were fixed and permeabilized, and then stained with the following antibodies. 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, cantonii) and data were analyzed using flowjo.7 and Prism 6.
In the 4.3.1.2 study, tissues from 4 groups of animals were obtained for ex vivo analysis. They are of the following group: PBS; a PD-1 antibody; W3645-2.131.4-hIgG1L 3; and the combination of the PD-1 antibody with W3645-2.131.4-hIgG1L 3. The population of CD4+ T cells as well as CD8+ T cells in the spleen was significantly increased in the combination group compared to the control-PBS group (p < 0.05). The data is shown in figure 15.
Population numbers of CD4+ T cells in CD45+ TIL were significantly increased in the anti-PD-1 antibody and combination group compared to the control-PBS group, and the data are shown in figure 16.
The population of Treg cells among the CD4+ T cells of the spleen was significantly reduced in the anti-PD-1 antibody and combination group compared to the control-PBS group (P < 0.05). No significant differences were observed between the different groups for CD25+/FoxP3+ tregs in CD4+ TIL. The data are shown in figure 17.
By staining with F4/80 and MHCII, the number of M1 populations in TILs of the combination group was significantly increased compared to the PBS group. The data is shown in fig. 18.
Higher intracellular IFNg was observed in the combination group after mixture stimulation compared to the other groups, but there was no significant difference for CD4+ cells and CD8+ cells in TIL. The data is shown in figure 19.
2. Pharmacokinetic and acute toxicity assessment on cynomolgus monkeys
Pharmacokinetic characterization on cynomolgus monkeys
This study was conducted to determine the pharmacokinetics of W3645 in naive male cynomolgus monkeys after a single intravenous bolus administration of 30 mg/kg. Two animals were dosed once with W3645-2.131.4-hIgG1L3 at a dose of 30mg/kg, by intravenous bolus administration. The formulations were formulated in PBS. PK blood samples were collected prior to dosing, 0.25h, 0.5h, 1h, 4h, 8h, 24h, D3, D5, D7, D10, D12, D14, D21 and D28. Anti-drug antibody (ADA) samples were collected prior to dosing, D14 and D28. Serum concentrations of W3645 and ADA in serum samples were determined by ELISA. Samples for hematological and clinical chemistry testing were collected prior to dosing, 24h, D3, D7, D14, D21 and D28.
This study was conducted to determine the pharmacokinetics of WBP3645 final lead antibody in naive male cynomolgus monkeys after a single intravenous bolus dose of 30 mg/kg. Two animals were dosed once with W3645-2.131.4-hIgG1L3 at 30mg/kg by intravenous bolus injection. The formulations were formulated in PBS. PK blood samples were collected prior to dosing, 0.25h, 0.5h, 1h, 4h, 8h, 24h, D3, D5, D7, D10, D12, D14, D21 and D28. The concentration of W3645-2.131.4-hIgG1L3 in serum samples was determined by ELISA.
Data were analyzed using Phoenix WinNonlin software (version 8.1, Pharsight, Mountain View, CA). Linear/logarithmic trapezoidal rule was applied in obtaining PK parameters.
A summary of PK parameters is listed in table 12 below.
TABLE 12.30 mg/Kg dose PK data for W3645-2.131.4-hIgG1L3
Note: pharmacokinetic parameters for G1-1 and G1-2 were calculated without time points at day 12, 14, 21 and 28 due to ADA effects, data not shown.
Sequence listing
<110> Shanghai Yaoming Biotechnology Co., Ltd
<120> a novel anti-TIGIT antibody
<130> AJ3297PCT2010CN
<150> PCT/CN2019/118522
<151> 2019-11-14
<160> 14
<170> PatentIn version 3.3
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Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
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Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
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Ser Gly Ser Lys Ser Gly Ile Thr Ala Ser Leu Thr Ile Ser Gly Leu
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Ser Thr Leu Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
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catccaggca aagccccccg actcttgatt tttgatgtca gtaatcgacg cacagggatt 180
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Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
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Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
35 40 45
Trp Ile Gly Ser Ile Phe Phe Ser Gly Ile Thr Tyr Tyr Asn Pro Ser
50 55 60
Leu Lys Ser Arg Val Thr Val Ser Val Asp Thr Ser Lys Asn Gln Phe
65 70 75 80
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr
85 90 95
Cys Ala Arg Arg Gly Leu Gly Gly Ile Leu Ala Ser Gly Tyr Phe Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
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Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
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Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
145 150 155 160
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
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Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
180 185 190
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn
195 200 205
Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser
210 215 220
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
260 265 270
Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr
290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser
355 360 365
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Leu Gly
450
<210> 12
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<213> Artificial sequence
<220>
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Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
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20 25 30
Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Arg Leu
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Leu Ile Phe Asp Val Ser Asn Arg Arg Thr Gly Ile Ser Asn Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Ile Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ile
85 90 95
Ser Thr Leu Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 110
Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu
115 120 125
Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe
130 135 140
Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val
145 150 155 160
Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys
165 170 175
Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser
180 185 190
His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
195 200 205
Lys Thr Val Ala Pro Thr Glu Cys Ser
210 215
<210> 13
<211> 1350
<212> DNA
<213> Artificial sequence
<220>
<223> heavy chain
<400> 13
caactccagc tgcaagagtc gggcccagga ctggtaaagc cttcggagac cctgtccctc 60
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cagcccccag ggaaggggct ggagtggatt gggagtatct tttttagtgg gatcacctac 180
tacaacccgt ccctcaagag tcgagtcacc gtatccgtag acacgtccaa gaaccagttc 240
tccctgaaac tgagctctgt gaccgccgca gacacggcta tttattactg tgcgagacgg 300
ggacttgggg gaattctcgc ctccggctac tttgactact ggggccaggg aaccctggtc 360
accgtctcct cagcgtcgac caagggccca tccgtcttcc ccctggcgcc ctgctccagg 420
agcacctccg agagcacagc cgccctgggc tgcctggtca aggactactt ccccgaaccg 480
gtgacggtgt cgtggaactc aggcgccctg accagcggcg tgcacacctt cccggctgtc 540
ctacagtcct caggactcta ctccctcagc agcgtggtga ccgtgccctc cagcagcttg 600
ggcacgaaga cctacacctg caacgtagat cacaagccca gcaacaccaa ggtggacaag 660
agagttgagt ccaaatatgg tcccccatgc ccaccatgcc cagcacctga gttcctgggg 720
ggaccatcag tcttcctgtt ccccccaaaa cccaaggaca ctctcatgat ctcccggacc 780
cctgaggtca cgtgcgtggt ggtggacgtg agccaggaag accccgaggt ccagttcaac 840
tggtacgtgg atggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagttc 900
aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaacggc 960
aaggagtaca agtgcaaggt ctccaacaaa ggcctcccgt cctccatcga gaaaaccatc 1020
tccaaagcca aagggcagcc ccgagagcca caggtgtaca ccctgccccc atcccaggag 1080
gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta ccccagcgac 1140
atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1200
gtgctggact ccgacggctc cttcttcctc tacagcaggc taaccgtgga caagagcagg 1260
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<212> DNA
<213> Artificial sequence
<220>
<223> light chain
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catccaggca aagccccccg actcttgatt tttgatgtca gtaatcgacg cacagggatt 180
tccaatcgct tctctggctc caagtctggc atcacggcct ccctgaccat ctctgggctc 240
caggctgagg acgaggctga ctattactgc agttcatata caagcatcag cactctcgtg 300
gtattcggcg gagggaccaa gctgaccgtc ctaggtcagc ccaaggctgc cccctcggtc 360
actctgttcc cgccctcctc tgaggagctt caagccaaca aggccacact ggtgtgtctc 420
ataagtgact tctacccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 480
aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 540
agctacctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag ctgccaggtc 600
acgcatgaag ggagcaccgt ggagaagaca gtggccccta cagaatgttc a 651
Claims (20)
1. An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment binds to human, cynomolgus monkey and mouse TIGIT.
2. An antibody or antigen-binding fragment thereof, comprising:
a) a heavy chain variable region having an amino acid sequence substantially identical to SEQ ID NO: 7, at least 70%, 80%, 90%, 95%, or 99% homologous; and
b) a light chain variable region having an amino acid sequence substantially identical to SEQ ID NO: 8, which is at least 70%, 80%, 90%, 95% or 99% homologous,
wherein the antibody or antigen binding fragment specifically binds to TIGIT.
3. An antibody or antigen-binding fragment thereof, comprising:
a) a heavy chain variable region having SEQ ID NO: 7; and
b) a light chain variable region having SEQ ID NO: 8 in a sequence selected from the group consisting of SEQ ID NO,
wherein the antibody or antigen binding fragment specifically binds to TIGIT.
4. An antibody or 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 SEQ ID NO: 5 and conservative modifications thereof,
wherein the antibody or antigen binding fragment specifically binds to TIGIT.
5. The antibody or antigen-binding fragment thereof of claim 4, wherein the light chain variable region CDR3 sequence comprises the amino acid sequence of SEQ ID NO: 6 and conservative modifications thereof.
6. The antibody or antigen-binding fragment thereof of claim 4 or 5, wherein the heavy chain variable region CDR2 sequence comprises the amino acid sequence of SEQ ID NO: 3 and conservative modifications thereof.
7. The antibody or antigen-binding fragment thereof of any one of claims 4 to 6, wherein the light chain variable region CDR2 sequence comprises the amino acid sequence of SEQ ID NO: 4 and conservative modifications thereof.
8. The antibody or antigen-binding fragment thereof of any one of claims 4 to 7, wherein the heavy chain variable region CDR1 sequence comprises the amino acid sequence of SEQ ID NO: 1 and conservative modifications thereof.
9. The antibody or antigen binding fragment thereof of any one of claims 4 to 8, wherein the light chain variable region CDR1 sequence comprises the amino acid sequence of SEQ ID NO: 2 and conservative modifications thereof.
10. The antibody or antigen binding fragment thereof of any one of claims 1 to 9, wherein the antibody is a chimeric, humanized, fully human or rat antibody.
11.A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.
12. A cloning or expression vector comprising the nucleic acid molecule of claim 11.
13. A host cell comprising one or more cloning or expression vectors of claim 12.
14. A method of making the antibody of any one of claims 1 to 10, comprising culturing the host cell of claim 13 and isolating the antibody.
15. The method of claim 14, wherein the antibody is prepared by immunization in rats with human TIGIT protein.
16. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 and one or more pharmaceutically acceptable excipients, diluents and carriers.
17. A method of making an anti-TIGIT antibody or antigen-binding fragment thereof, the method comprising:
(a) providing:
(i) a heavy chain variable region antibody sequence comprising SEQ ID NO: 1, the CDR1 sequence of SEQ ID NO: 3 and the CDR2 sequence of SEQ ID NO: 5, a CDR3 sequence; and/or
(ii) A light chain variable region antibody sequence comprising SEQ ID NO: 2, the CDR1 sequence of SEQ ID NO: 4 and the CDR2 sequence of SEQ ID NO: 6, a CDR3 sequence; and
(b) expressing the altered antibody sequence as a protein.
18. A method of preventing or treating a tumor or an inflammatory disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.
19. Use of an 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 a tumor or inflammatory disease.
20. The method of claim 18, the use of claim 19, wherein the tumor is selected from the group consisting of melanoma, renal cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, liver cancer, gastrointestinal cancer, glioblastoma, cervical cancer, bladder cancer, and rectal cancer.
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Citations (4)
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CN103073644A (en) * | 2012-12-31 | 2013-05-01 | 中国科学技术大学 | Specific anti-mouse TIGIT monoclonal antibody and preparation method, identification and application thereof |
CN107207594A (en) * | 2014-12-23 | 2017-09-26 | 百时美施贵宝公司 | For TIGIT antibody |
CN108368176A (en) * | 2015-10-01 | 2018-08-03 | 波滕扎治疗公司 | Anti- TIGIT antigen-binding proteins and its application method |
WO2019154415A1 (en) * | 2018-02-06 | 2019-08-15 | I-Mab | Antibodies to t cell immunoreceptor with ig and itim domains (tigit) and uses thereof |
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CN107148430B (en) * | 2014-08-19 | 2021-08-27 | 默沙东公司 | anti-TIGIT antibody |
JP6861418B2 (en) * | 2015-09-02 | 2021-04-28 | イッサム リサーチ デベロップメント カンパニー オブ ザ ヘブリュー ユニバーシティー オブ エルサレム リミテッド | Antibodies specific for human T cell immunoglobulin and ITIM domain (TIGIT) |
JP7109382B2 (en) * | 2016-03-04 | 2022-07-29 | ジェイエヌ バイオサイエンシーズ エルエルシー | Antibody to TIGIT |
US10759855B2 (en) * | 2016-12-02 | 2020-09-01 | Rigel Pharmaceuticals, Inc. | Antigen binding molecules to TIGIT |
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2020
- 2020-11-13 WO PCT/CN2020/128659 patent/WO2021093849A1/en active Application Filing
- 2020-11-13 CN CN202080075905.9A patent/CN114641502B/en active Active
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CN103073644A (en) * | 2012-12-31 | 2013-05-01 | 中国科学技术大学 | Specific anti-mouse TIGIT monoclonal antibody and preparation method, identification and application thereof |
CN107207594A (en) * | 2014-12-23 | 2017-09-26 | 百时美施贵宝公司 | For TIGIT antibody |
CN108368176A (en) * | 2015-10-01 | 2018-08-03 | 波滕扎治疗公司 | Anti- TIGIT antigen-binding proteins and its application method |
WO2019154415A1 (en) * | 2018-02-06 | 2019-08-15 | I-Mab | Antibodies to t cell immunoreceptor with ig and itim domains (tigit) and uses thereof |
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