CN114641502B - New anti-TIGIT antibody - Google Patents
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- CN114641502B CN114641502B CN202080075905.9A CN202080075905A CN114641502B CN 114641502 B CN114641502 B CN 114641502B CN 202080075905 A CN202080075905 A CN 202080075905A CN 114641502 B CN114641502 B CN 114641502B
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
- A61K2039/507—Comprising a combination of two or more separate antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
Abstract
The present invention provides TIGIT monoclonal antibodies, in particular humanized monoclonal antibodies that specifically bind to TIGIT with high affinity. The invention also provides functional monoclonal antibodies that are cross-reactive to TIGIT in humans, cynomolgus monkeys and mice. The invention also provides amino acid sequences, cloning or expression vectors, host cells, and methods for expressing or isolating the antibodies of the invention. The epitope of the antibody is identified. Therapeutic compositions comprising the antibodies of the invention are also provided. The invention also provides methods of treating cancer and other diseases using anti-TIGIT antibodies.
Description
Technical Field
The present invention relates generally to anti-TIGIT antibodies and compositions thereof, and therapies for treating tumors or inflammatory disorders using anti-TIGIT antibodies.
Background
TIGIT (T Cell immune receptor with Ig and ITIM domains), also known as Vstm3, WUCAM, is an inhibitory receptor expressed on NK and cd8+ T cells and on the cd4+ T Cell subclass 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, imcity, no. 40, no.4, pp.569-581,2014. It has been identified as a member of the CD28 family based on the genetic 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, resulting in immunosuppression (Stanietsky, n. Et al, proc.Natl. Acad. Sci.U.S. A.106,17858-17863,2019.; stenel, 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 bind simultaneously to the co-stimulatory 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, allowing equilibrium to favor CD 226-mediated activation signal migration, thus 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, wellstock J, filer w.mamm genome.29 (11-12): 694-702, 2018.). TIGIT is upregulated in Cancer and inflammatory disease and identified as a failure marker (Chauvin JM, pagliano, fourcade J, sun Z, wang H, sander C et al, J Clin Invest2015; 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, humim, yang Y et al, cancer Cell 4; 26:923-37.) identical to other failure markers such as PD-1, lang 3 and TIM3 (flury M, belkinna AC, proctor EA et al, arthris rheomol 2018R; 70 (4): 566-577; omentin R, baken W, lawang et al, lang et al, cancer Cell 2016, 5; 14, 7). 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): 355-362;Niclas C.Blessin,Ronald Simon et AL, dis markers.2019;2019: 5160565), and that blocking both PD-1 and TIGIT can reverse immunosuppression in tumor tissue (hunt AL, maxwell R, therodos D, belcaid Z et AL, oncoimmunology.2018may 24;7 (8)). Overall TIGIT can be used as a single agent or in combination with other immunomodulators, a promising therapeutic target for tumor immunotherapy.
Disclosure of Invention
The present invention provides isolated antibodies, particularly monoclonal antibodies against TIGIT or fully human antagonist antibodies.
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 a sequence identical to SEQ ID NO:7, an amino acid sequence that is at least 70%, 80%, 90%, 95% or 99% homologous to the sequence of seq id no; and
b) A light chain variable region having a sequence identical to SEQ ID NO:8, an amino acid sequence which is at least 70%, 80%, 90%, 95% or 99% homologous to the sequence of seq id no,
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 the amino acid sequence of SEQ ID NO: 7; and
b) A light chain variable region having the amino acid sequence of SEQ ID NO:8, the amino acid sequence of the gene,
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 CDR1, CDR2 and CDR3 sequences,
Wherein the heavy chain variable region CDR3 sequence comprises SEQ ID NO:5 and conservative modification 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) Comprising SEQ ID NO:1, a heavy chain variable region CDR1;
b) Comprising SEQ ID NO:3, a heavy chain variable region CDR2;
c) Comprising SEQ ID NO:5, a heavy chain variable region CDR3;
d) Comprising SEQ ID NO:2, a light chain variable region CDR1;
e) Comprising SEQ ID NO:4, a light chain variable region CDR2;
f) Comprising SEQ ID NO:6, a light chain variable region CDR3;
Wherein the antibody or antigen binding fragment specifically binds to TIGIT.
The antibodies of the invention may be chimeric antibodies.
The antibodies of the invention may be humanized antibodies.
The antibodies of the invention may be fully human antibodies.
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 present invention provides a cloning or expression vector comprising a nucleic acid molecule encoding said 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 invention provides a method comprising culturing the host cell described above and isolating the antibody;
wherein the antibody is prepared by immunization of a rat with human TIGIT protein.
In another aspect, the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment of said antibody described in the invention and one or more pharmaceutically acceptable excipients, diluents or carriers.
The invention also provides a method of preparing 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 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 SEQ ID NO:6, CDR3 sequences; and
(b) Expressing the altered antibody sequence as a protein.
The present invention also provides a method of 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 of the invention as described above.
The invention also provides a combination method for preventing or treating a tumor or inflammatory disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen binding fragment of the invention described above, 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 manufacture of a medicament for the prevention or treatment of a tumour 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, malignant melanoma of skin or eyes, 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 broadly relates 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 fields of antibody therapeutics and diagnostics, and can be used in conjunction with antibodies that react with a variety of different targets.
The present 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 the group consisting of:
(i) And SEQ ID NO:1, the HCDR1 set forth in (c) has at least 90% sequence identity to the HCDR1;
(ii) And SEQ ID NO:3, HCDR2 having at least 90% sequence identity; and
(iii) And SEQ ID NO:5, HCDR3 having at least 90% sequence identity to the HCDR3 set forth in 5;
b) One or more light chain CDRs (LCDR) selected from the group consisting of:
(i) And SEQ ID NO:2, the LCDR1 set forth in (c) has at least 90% sequence identity;
(ii) And SEQ ID NO:4, LCDR2 having at least 90% sequence identity; and
(iii) And SEQ ID NO:6, LCDR3 having at least 90% sequence identity;
or (b)
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) Comprising SEQ ID NO:1 or an amino acid addition, deletion or substitution of an amino acid that differs by no more than 2 amino acids from the amino acid sequence derived from said HCDR1;
(ii) Comprising SEQ ID NO:3 or an amino acid addition, deletion or substitution of an HCDR2 that differs from the amino acid sequence derived from said HCDR2 by no more than 2 amino acids; and
(iii) Comprising SEQ ID NO:5 or an amino acid addition, deletion or substitution of HCDR3 that differs from the amino acid sequence derived from said HCDR3 by no more than 2 amino acids;
b) One or more light chain CDRs (LCDR) selected from the group consisting of:
(i) Comprising SEQ ID NO:2 or an LCDR1 that differs from the amino acid sequence from said LCDR1 by no more than 2 amino acids;
(ii) Comprising SEQ ID NO:4 or an amino acid addition, deletion or substitution of an LCDR2 that differs from the amino acid sequence from said LCDR2 by no more than 2 amino acids; and
(iii) Comprising SEQ ID NO:6 or an amino acid addition, deletion or substitution of LCDR3 that differs from the amino acid sequence from said LCDR3 by no more than 2 amino acids; or (b)
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) Comprising SEQ ID NO: HCDR1 of 1;
(ii) Comprising SEQ ID NO: HCDR2 of 3; and
(iii) Comprising SEQ ID NO: HCDR3 of 5; and/or
(B) The VL comprises:
(i) Comprising SEQ ID NO: LCDR1 of 2;
(ii) Comprising SEQ ID NO: LCDR2 of 4; and
(iii) Comprising 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 has an amino acid sequence of at least 85%, 90% or 95% identity; or (b)
(iii) And SEQ ID NO:7 an amino acid sequence having one or more amino acid additions, deletions and/or substitutions; and/or
(B) A light chain variable region comprising:
(i) SEQ ID NO:8 'amino acid sequence'
(ii) And SEQ ID NO:8 has an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical; or (b)
(iii) And SEQ ID NO:8 with 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 the heavy chain variable region set forth in SEQ ID NO:8, and a light chain variable region as set forth in seq id no.
In certain embodiments, the isolated antibody or antigen binding fragment thereof disclosed herein is a chimeric, humanized or fully human or rat antibody. Preferably, the antibody is a fully human monoclonal antibody.
In certain embodiments, the isolated antibodies or antigen binding fragments thereof disclosed herein comprise 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, an amino acid sequence of seq id no;
(ii) And SEQ ID NO:11 has an amino acid sequence that is at least 85%, 90% or 95% identical; or (b)
(iii) And SEQ ID NO:11 to an amino acid sequence having one or more amino acid additions, deletions and/or substitutions; and/or
(B) A light chain comprising:
(i) SEQ ID NO:12, an amino acid sequence of seq id no;
(ii) And SEQ ID NO:12 has an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical; or (b)
(iii) And SEQ ID NO:12 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 polypeptide comprising SEQ ID NO:11 and a heavy chain comprising SEQ ID NO: 12.
In certain aspects, the 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 aspects, the 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 as disclosed herein.
In certain aspects, the 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 aspects, the 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 aspects, the disclosure relates to a method of modulating an immune response in a subject, the method comprising administering an antibody or antigen binding fragment thereof disclosed herein to the subject, so as to modulate an immune response in the subject, optionally the immune response is TIGIT-related.
In certain aspects, the 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 disclosed herein or a pharmaceutical composition.
In certain instances, the present 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 manufacture of a medicament for the treatment or prevention of 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 present 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, which are useful for treating diseases including cancer.
The foregoing is a summary and thus contains, by necessity, simplifications, generalizations, and omissions of detail; accordingly, 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.
FIG. 2a shows ELISA results for binding to human TIGIT; 2b shows ELISA results binding to human CD 28; 2c shows ELISA results binding to human PD-1; 2d shows ELISA results binding to human ICOS; 2e shows ELISA results binding to human CTLA 4; 2f shows ELISA results binding to human PVRIG.
Figure 3 shows cynomolgus 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 of Jurkat cells expressing human TIGIT.
FIG. 8 shows an activation assay for 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 curve of W3645-2.131.4-hIgG1L 3.
FIG. 12 shows serum stability analysis of W3645-2.131.4-hIgG1L 3.
FIG. 13 shows the results of CT26 isogenic model.
FIG. 14 shows the results of the C57 (hTIGIT)/MC 38 model.
Fig. 15 shows the proportion of cd4+ and cd8+ T cells in the spleen.
Figure 16 shows the ratio of cd4+ and cd8+ T cells in TIL.
Figure 17 shows Treg ratios in spleen and TIL.
Fig. 18 shows the proportion of M1 population in TIL.
Figure 19 shows intracellular IFNg after 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 terms "TIGIT", "T cell immune receptor with Ig and ITIM domains", also referred to herein as Vstm3 and WUCAM, are one inhibitory receptor expressed on NK and cd8+ T cells and on the cd4+ T cell subclass including immunosuppressive tregs.
The term "antibody" as referred to herein includes whole antibodies and any antigen-binding fragment (i.e., an "antigen-binding portion") or single chain thereof. "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 CH3. 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 one domain CL. The VH and VL regions may be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), 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 carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. CDR boundaries of antibodies are defined or identified by the Kabat numbering system.
The term "antibody" as used in this disclosure refers to an immunoglobulin or fragment or derivative thereof, and encompasses any polypeptide comprising an antigen binding site, whether it is produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, non-specific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutant, 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 amino acids responsible for specific binding between the antibody and antigen. In the case of large antigens, the antigen binding fragment may bind to only a portion of the antigen. The portion of the antigen molecule responsible for 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 only of VH domains, but still retain some of the antigen-binding function of the intact antibody.
Consistent with the foregoing, the term "epitope" defines an antigenic determinant that is specifically bound/recognized by a binding fragment as defined above. The binding fragments may specifically bind to/interact with conformational or sequential epitopes that are 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 which come together on the molecular surface as 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 is folded into a three-dimensional structure, these residues come together on the surface of the molecule to form an epitope. Instead, a continuous or linear epitope is made up of two or more discrete amino acid residues that are present in a single linear segment of a polypeptide chain.
The term "cross-reactive" refers to the binding of an antigenic fragment described herein to the same target molecule in humans, monkeys and/or rats (mice or rats). Thus, "cross-reactivity" should be understood as being an intergeneric reactivity with the same molecule X expressed in a different species, but not with molecules other than X. The 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, e.g., 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 "treatment method" refer to both therapeutic treatment and prophylactic/preventative measures. A person in need of treatment may include an individual already having a particular medical condition and an individual who may ultimately have the condition.
The term "conservative modification" 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 amino acid sequence. Such conservative sequence modifications include nucleotide and amino acid substitutions, additions, and deletions. Modifications may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions 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.
Examples
Example 1: material and antibody production
1. Universal material
Antigens were purchased from vendors or prepared by themselves (see table 1 for details). W364-hPro1.ECD. His is the extracellular domain of human TIGIT with a C-terminal polyhistidine tag (NP-776160.2); 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 with C-terminal polyhistidine tag (NP-001139797.1); W364-mPro1.ECD. HFc is the extracellular domain of mouse TIGIT (NP-001139797.1) carrying 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 antigen and ligand
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 (WBP 364-BMK1; tiragolumab, U.S. patent No. 20170088613 A1/4.1D3), anti-TIGIT antibodies (WBP 364-BMK4; U.S. patent nos. 20160176963A1, 22G 2) and anti-TIGIT antibodies (WBP 364-BMK6; WO2017059095A1, MAB 10) were first codon optimized for mammalian expression and then synthesized by GENEWIZ (su, china). The DNA segment is then subcloned into a pcDNA expression vector with the constant region of human IgG1 or IgG 4.
TABLE 3 information on reference antibodies
3. Hybridoma sequencing and construction of self-made discovered antibodies
The anti-TIGIT antibodies were found by the present institution after immunization with human TIGIT (Omni rats). First, total RNA of hybridoma cell samples was extracted according to the instructions of the TaKaRa MiniBEST general RNA extraction kit. The RNA was then converted to cDNA using a SMART RACE cDNA amplification kit from Clonetech. Heavy and light chain DNA sequences were amplified from 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 confirm the monoclonality of the hybridoma cell samples, the amino acid sequences of VH and VL domains were codon optimized for mammalian expression and then synthesized by GENEWIZ (su, china). The DNA segment is then subcloned into a pcDNA expression vector with the constant region of human IgG1 or IgG 4.
4. Expression and purification of antibodies
Plasmids containing VH and VL genes were co-transfected into Expi293 cells (thermosusher, a 14635). The cells were cultured for 5 days according to the manufacturer's recommended protocol. After the supernatant was verified by SDS-PAGE, the antibody was purified using a protein A column (GE Healthcare, cat.175438). The concentration of purified Fc-tagged protein was determined by absorbance at 280 nm. 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 1260 affinity HPLC. mu.L of antibody solution was loaded onto a TSKgel SuperSW3000 column using 50mM sodium phosphate, 0.15M NaCl,pH 7.0 buffer. The run time was 20 minutes. Peak retention time on the column was monitored at 280 nm. The data were analyzed using ChemStation software (V2.99.2.0). All antibodies were required to be >90% pure.
5. Cell pool/line preparation
5.1 cell pool/line for binding to assayed expression targets
Cell lines expressing mouse TIGIT (W364-flpincho. Mpro1. Fl) and cynomolgus monkey TIGIT (W364-flpincho. 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. 48-72 hours after transfection, the transfected cells were cultured in hygromycin-containing medium for selection and assayed for TIGIT expression. Cell pools expressing mouse and cynomolgus TIGIT were then obtained.
Cell lines expressing human TIGIT (W364-chok 1.Hpro1. Fl.2a11) and cell pools (W364-293 f. Hpro1. Fl) were generated. Briefly, CHO-K1 or 293F cells were transfected with pcDNA3.3 expression vectors containing full-length human TIGIT (NP-776160.2), respectively, using Lipofectamine 2000 transfection kit according to the manufacturer's protocol. 48-72 hours after transfection, the transfected cells were cultured in medium containing blasticidin for selection and assayed 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 with NFAT-luciferase reporter (W364-Jurkat. HPro1.NFAT. 2D11) were generated by transfecting Jurkat cells overexpressing NFAT-luciferase reporter 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 selection and 4. Mu.g/mL blasticidin.
HT1080.2A11 (also known as HT1080.OKT3.scFv.2A11) is produced by the present institution by transfecting HT1080 tumor cells with a pSB plasmid encoding single chain Fv of OKT3 (anti-CD 3 antibody).
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 accession number 60548.
6. Hybridoma antibody production
6.1 immunization
4 OMT rats of 6-8 weeks of age were vaccinated 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 once every two weeks via the footpad, subcutaneous, and intraperitoneal routes. Serum titers were measured by ELISA. When the serum titer was high enough (. Gtoreq.1:24, 300), the animals with the highest titers were last boosted with protein in sterile PBS without adjuvant. After 2-4 days (48-96 hours), animals were euthanized and lymph nodes or spleens were used for cell fusion.
6.2 serum titre detection
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 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 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 (Bethy, A110-236P) secondary antibody for 1 hour. After washing TMB substrate was added, the interaction was terminated by 2M HCl. 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 titres (ELISA binding to human/mouse TIGIT)
6.3 hybridoma production
Lymph nodes and spleens from immunized animals 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 with pronase solution, 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 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 96-well plates. The fused cells were subjected to 5% CO at 37 DEG C 2 The incubator is maintained for 10-14 days.
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 exposed to 5% CO at 37℃ 2 The incubator is maintained for 7-8 days. Each single colony visible was picked into a 96-well plate containing DMEM medium supplemented with 10% fbs. After 2-3 days, the 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 confirmation of the monoclonal nature of the positive clone, the amino acid sequences of VH and VL domains without PTM sites were codon optimized for mammalian expression and then synthesized by GENEWIZ (su, china). The DNA segment is then subcloned into a pcDNA expression vector with the constant region of human IgG1 or human IgG 4. Plasmids containing VH and VL genes were co-transfected into Expi293 cells (Thermo Fisher, a 14635). And 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 purified Fc-tagged protein was determined by absorbance at 280 nm. 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 1260 affinity HPLC. mu.L of antibody solution was loaded onto a TSKgel SuperSW3000 column using 50mM sodium phosphate, 0.15M NaCl,pH 7.0 buffer. The run time was 20 minutes. Peak retention time on the column was monitored at 280 nm. The data were analyzed using ChemStation software (V2.99.2.0). All antibodies were required to be >90% pure.
6.6. Antibody sequences
Through primary and secondary binding screens and TIGIT/CD155 blocking and reporter assays, 51 positive cell lines were selected for subcloning. After confirmation of purified subclone antibodies, 10 antibodies were selected for sequencing and subsequent human IgG conversion. The final lead antibodies were 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
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Example 2: in vitro characterization of lead antibodies
1. Human TIGIT binding (FACS)
293F cells expressing human TIGIT (W364-293F. HPro1.FL. Cells, 1X 10) 5 Individual cells/well) were incubated with serial dilutions of W3645-2.131.4-hIgG1L3 (5-fold, 8 concentrations starting from 100 nM) for 1 hour at 4 ℃. After washing with 1 XPBS/1% BSA, a 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 antibodies were used as negative controls. 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, cantoII) and analyzed by flowjo.7. Cell-bound EC were obtained using GraphPad prism.6 software using four-parameter nonlinear analysis 50 Values.
The binding of W3645-2.131.4-hIgG1L3 to WBP64-293F.hPro1.FL. Cell is shown in FIG. 1. The WBP3645 leader antibody can be used in an EC of 0.17nM 50 Dose-dependently binding to human TIGIT on cell surface, EC of reference antibodies WBP364-BMK1 (Genentech) and WBP364-BMK4 (BMS) 50 Is 0.35nM to 0.38nM. The data is shown in fig. 1.
2. Cross-family binding (ELISA)
Flat bottom 96-well plates treated with non-tissue cultures were pre-coated overnight at 4 ℃ with 1.0 μg/mL institutional human CD28 ECD, human CTLA4 (cytotoxic T-lymphocyte-associated protein 4) ECD, human ICOS (inducible T-cell co-stimulators) ECD, human PD-1 (programmed death protein 1) protein ECD, human PVRIG (CD 112 receptor) ECD and human TIGIT ECD. 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. Color development was performed by dispensing 100. Mu.L of TMB substrate, and then stopped by 100. Mu.L of 2N HCl. Absorbance was read at 450nm using a microplate spectrophotometer (MD, M5 e) and the data was analyzed by Prism 6.
TIGIT has been identified as a member of the CD28 family based on the genetic structure. ELISA binding results of WBP3645 leader antibody (W3645-2.131.4-hIgG 1L 3) with 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 is shown in fig. 2-f. From the results of the ELISA binding assay, W3645-2.131.4-hIgG1L3 specifically bound to TIGIT and did not cross-react with human CD28, PD-1, ICOS, CTLA4 and PVRIG.
3. Cross species binding (FACS)
3.1 cynomolgus monkey TIGIT binding
FLPin CHO cells expressing cynomolgus TIGIT (W364-FLPin CHO. Cynopro1.FL. Cells, 1X 10) 5 Individual cells/well) were incubated with serial dilutions of W3645-2.131.4-hIgG1L3 (5-fold, 8 spots starting from 100 nM) for 1 hour at 4 ℃. After washing with 1 XPBS/1% BSA, a 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 antibodies were used as negative controls. The cells were then washed and resuspended in 1 XPBS/1% BSA. The MFI of the cells was measured by flow cytometry (BD, cantoII) and analyzed by FlowJo.7. Cell-bound EC were obtained using GraphPad prism.6 software using four-parameter nonlinear analysis 50 Values.
W3645-2.131.4-hIgG1L3 with EC of 0.05nM 50 Cross-binding to FLPin-CHO cells expressing cynomolgus TIGIT. The data is shown in fig. 3.
3.2 mouse TIGIT binding
Flp-in CHO cells expressing mouse TIGIT (W364-FLPin CHO. Mousepro1.FL. Cells (1X 10) 5 Individual cells/well) were incubated with serial dilutions of W3645-2.131.4-hIgG1L3 (5-fold, 8 spots starting from 100 nM) for 1 hour at 4 ℃. After washing with 1 XPBS/1% BSA, a 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. An anti-human TIGIT antibody W364-BMK6 was used as a positive control. Human IgG1 isotype antibodies were used as negative controls. 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 EC were obtained using GraphPad prism.6 software using four-parameter nonlinear analysis 50 Values.
W3645-2.131.4-hIgG1L3 with EC of 0.14nM 50 Cross-binding to FLP-in-CHO cells expressing mouse TIGIT. BMK6 is a reference antibody (Astella) that cross-binds mouse TIGIT. The data is shown in fig. 4.
4. Affinity with human TIGIT (SPR)
The binding affinity of TIGIT antibodies to antigens was detected by SPR (surface plasmon resonance) assay using Biacore 8K. Antibodies were captured on a CM5 sensor chip (GE) with anti-human IgG Fc antibodies immobilized. Different concentrations of human TIGIT extracellular domain were injected onto the sensor chip at a flow rate of 30 μl/min for a binding period of 180s, followed by 3600s dissociation. After each binding cycle, the chip was regenerated with 10mM glycine (pH 1.5). The sensorgram for the blank surface and buffer channel was 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.
W3645-2.131.4-hIgG1L3 had an affinity KD of 7.50E-10M for the soluble antigen (see data in Table 9).
TABLE 9 affinity based on protein by SPR
5. Affinity with cell surface human TIGIT (FACS)
The binding affinity of the antibodies to cell surface TIGIT was measured by FACS analysis. CHOK1 cells overexpressing human TIGIT (W364-CHOK 1. HPro1.2A11) were treated at 5X10 4 The density of individual cells/ml was transferred to a 96-well U-shaped bottom plate. 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 in the dark at 4 ℃ for 0.5 hours. Cells were then washed once and resuspended in 1 XPBS/1% BSA and analyzed by flow cytometry. The fluorescence intensity was converted into bound molecules/cells on the basis of quantitative beads (QuantumTM MESF kit, bangs Laboratories, inc.).
The affinity of the antibody to cell surface human TIGIT, as measured by FACS, was KD of 2.70E-11M. The data are shown in table 10.
TABLE 10 affinity based on cells by FACS
6. Ligand competition assay
The binding of CD155 (PVR, poliovirus receptor) to TIGIT expressing cells was blocked by antibodies, as determined by flow cytometry. Briefly, serial dilutions of 1E5 293F (TIGIT+) cells with TIGIT or hIgG1 isotype control antibodies and 2. Mu.g/mL carrymFc-tagged cd155.ecd was incubated at 4 ℃ for 60 min. After washing twice with cold PBS (wash buffer) supplemented with 1% bsa, cell surface bound ligands were detected by incubating the cells with PE-conjugated anti-mFc antibodies for 30 minutes at 4 ℃. Cells were washed twice in the same buffer and MFI (mean fluorescence intensity) of stained cells was measured using FACS Canto II cytometry (BD Biosciences). Background fluorescence was established using wells containing no antibody or only secondary antibody, cell-bound ICs were obtained using GraphPad Prism software, using four-parameter nonlinear regression analysis 50 Values.
The blocking of binding of human CD155 (PVR) to cells expressing human TIGIT by antibodies was determined by flow cytometry. The lead Ab blocks binding of human TIGIT to ligand CD155, IC 50 0.14nM. The data is shown in fig. 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 (BPS, cat No. 60548) overexpressing CD155 and TCR activators were used as target cells. When these two cells are co-cultured, NFAT luciferase reporter signal in effector cells is inhibited with binding of TIGIT on Jurkat cells and ligand CD155 on CHO cells. By blocking the binding of TIGIT to CD155 with an anti-TIGIT antibody, luciferase signal is increased. Briefly, cells that overexpress CD155 and TCR activator were seeded on white plates at a density of 2E 4/well overnight. Serial dilutions of antibodies and TIGIT-overexpressing Jurkat/NFAT reporter cells (2E 4/well) were then incubated with target cells at 37℃and 5% CO 2 The following co-culture was performed for 5 to 6 hours. After incubation, ONE-Glo luciferase reagent (Promega, catalog number E6130) was added to the cells and NFAT activity was measured by Envision (PE) using luminescence. Curve fitting and EC acquisition using GraphPad prism.6 software using four parameter nonlinear analysis 50 Values.
In the Jurkat NFAT-luciferase reporter cell line over-expressing hTIGIT, the WBP3645 leader antibody may stimulate the NFAT pathway, EC 50 0.92nM. The data are shown in the figure6.
8. Activation assay of Jurkat cells expressing human TIGIT (IL-2 ELISA)
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 cell. HT1080 is a tumor cell line that endogenously expresses human CD 155. After 2 days of incubation of W364-Jurkat. HPro1.NFAT.2D11 (1E 4/well) with HT1080.OKT3scFV.A10 (1E 4/well), IL2 secretion in the medium was determined by ELISA (capture antibody: purified mouse anti-hIL 2 monoclonal IgG2A clone 5355, R&D, MAB602; detection of antibodies: biotinylated anti-hIL 2 antibody, R&D, BAF202; recombinant human IL2 standard: r is R&D, 202-IL-050). Curve fitting and EC acquisition using GraphPad prism.6 software using four parameter nonlinear analysis 50 Values.
The WBP3645 guide antibody can enhance IL2 release of Jurkat cells over expressing hTIGIT, EC 50 0.29nM. The data is shown in fig. 7.
Activation assay for CD8+ T cells
Co-culture assays of CD8+ T with CD155 expressing tumor cells were performed to assess whether the TIGIT antibodies were able to enhance CD8+ T activity. Briefly, cd8+ T cells (1×10 5 Individual cells/well) with 20nM concentration of antibody and HT1080 cells transfected with OKT3ScFv (1X 10) 4 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-hIFNγ antibody, thermo, M701B; recombinant IFNg standard: peproTech, 300-02-250).
The WBP3645 lead antibody W3645-2.131.4-hIgG1L3 can enhance IFNg release in a CD8+ T activation assay. The data is shown in fig. 8.
NK killing assay
The effect of anti-human TIGIT antibodies on NK cell-mediated lysis of cells expressing CD155 was evaluated in vitro. Briefly, CD155 (PVR) expressing P815 cells (mouse mast cell tumor cell line) as target cells were preloaded with bat da (PE, AD 0116) and exposed to human NK cells in the presence of various concentrations (10 nM, 2nM, 0.4nM, 0.08nM, 0.016 nM) of anti TIGIT antibodies. W364-BMK1 was used as positive control and human IgG1 isotype control was used as negative control. After 2-4 hours of incubation, the supernatant was collected and mixed with europium buffer. Cytotoxicity was detected by Envision (PE, ex337/Em 615) using TRF (time resolved fluorescence) and the data presented with Prism 6.
The WBP3645 lead antibody can enhance the killing activity of NK on cells over expressing PVR in a dose-dependent manner, and has better efficacy than BMK. The data is shown in fig. 9.
ADCC (antibody-dependent cell-mediated cytotoxicity) assay
To test for ADCC effect, engineered human TIGIT expressing cells (W364-chok 1.Hpro 1.2a11) were pre-incubated with TIGIT antibodies at various concentrations for 30 min in 96-well plates, then at 20:1 effector cell/target cell ratio freshly isolated PBMCs (peripheral blood mononuclear cells) were added. The plates were incubated at 37℃with 5% CO 2 Incubate for 4 hours. Target cell lysis was determined by LDH-based cytotoxicity detection kit. Absorbance was read at 492nm using a microplate spectrophotometer (MD, spectromax, M5 e). Curve fitting was performed using GraphPad prism.6 software using four parameter non-linear analysis and EC50 values were obtained.
W3645-2.131.4-hIgG1L3 lyses cells that overexpress hTIGIT by antibody-dependent cell-mediated cytotoxicity with an EC50 of 0.0019nM. The data is shown in fig. 10.
12. Evaluation of thermal stability by DSF
Tm of WBP3645 pilot antibodies was studied using the quantsudio 7Flex real-time PCR system (Applied Biosystems). mu.L of antibody solution was mixed with 1. Mu.L of 62.5 XSYPRO 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 derivative of the fluorescence change at different temperatures is calculated and the maximum is defined as the melting temperature Tm. If the protein has multiple unfolding transitions, the first two Tms, designated Tm1 and Tm2, respectively, are reported. Data collection and Tm calculation were performed automatically by the operating software (quantskio real-time PCR software v 1.3).
Using Quantum studio TM 7 Flex real-time PCR System (Applied Biosystems) study of the T of the WBP3645 Pilot antibody m . Good thermal stability was observed by DSF testing. The data of the software calculations are shown in table 11 and fig. 11.
TABLE 11 thermal stability of W3645-2.131.4-hIgG1L3
13. Serum stability assessment
To assess the serum stability of antibodies, serum was freshly prepared from the blood of healthy human donors. The antibodies were gently mixed with serum, aliquoted into 5 tubes and incubated at 37 ℃. Aliquots were then withdrawn at indicated time points: day 0, day 1, day 4, day 7 and day 14, and the aliquots were flash frozen in liquid nitrogen and stored at-80 ℃ until use. The binding capacity detected by ELISA was used to assess the serum stability of the antibodies. Absorbance was read at 450nm using a microplate spectrophotometer (MD, spectromax, M5 e). Curve fitting was performed using GraphPad prism.6 software using four parameter non-linear analysis and EC50 values were obtained.
In the serum stability test, W3645-2.131.4-hIgG1L3 was stable in serum at 37℃for at least 2 weeks. The data is shown in fig. 12.
Example 3: in vivo characterization
1. Rodent efficacy studies
1.1 CT26 isogenic model
In BALB/c mice, a WBP3645 pilot antibody efficacy study was tested in CT-26 model. Female BALB/c mice (Beijing Vital 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 the presence of 10% fetal bovine bloodIn RPMI 1640 medium containing 100U/mL penicillin and 100 μg/mL streptomycin, the culture was incubated at 37℃and 5% CO 2 Is in the cell incubator of the air.
For the treatment model, each mouse was treated with CT-26 tumor cells (3.0X10 at the armpit of the right forearm 5 ) Subcutaneous inoculation. When the average tumor volume reached approximately 55mm 3 At this time, animals were randomly divided into different groups: control-PBS; PD-1Ab 3mg/kg; WBP364-BMK6.UIgG4.SPK 10mg/kg; W3645-2.131.4-hIgG1L3 10mg/kg; WBP364-BMK6.UIgG4.SPK+PD-1Ab 10+3mg/kg; w3645-2.131.4. HIgG1L3+PD-1Ab10+3mg/kg and received a first antibody injection 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 performed following guidelines approved by the institutional review board (Institutional Animal Care and Use Committee) (IACUC) for animal care and use of the Shanghai biological model (Shanghai Bio-model), following guidelines of the laboratory animal care assessment Association (Association for Assessment and Accreditation of Laboratory Animal Care) (AAALAC).
Tumor volume was calculated 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 two-way ANOVA Bonferroni post hoc test of 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 is less than 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 fig. 13.
1.2 C57 (hTIGIT)/MC 38 model
In vivo efficacy studies of W3645-2.131.4.HIgG1L3 were tested in MC38 model using h-TIGIT mice. Female hTIGIT mice (Nanjing GemPharmatech co., LTD) 6-8 weeks old were used in the study.
In this in vivo efficacy model, each mouse was treated with MC38 tumor cells (5.0x10 at the right forearm axilla 5 ) Subcutaneous inoculation. When the average tumor volume reached approximately 65mm 3 At this time, animals were randomly divided into different groups: medium-PBS; PD-1Ab 0.3mg/kg; WBP364-BMK410mg/kg; W3645-2.131.4-hIgG1L3 10mg/kg; WBP 364-BMK4+PD-1Ab10+0.3 mg/kg; w3645-2.131.4. UIgG1L3+PD-1Ab10+0.3 mg/kg, then received a first antibody injection and was treated intraperitoneally with PBS or antibody twice weekly 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 performed following guidelines approved by the institutional review board (Institutional Animal Care and Use Committee) (IACUC) for animal care and use of the Shanghai biological model (Shanghai Bio-model), following guidelines of the laboratory animal care assessment Association (Association for Assessment and Accreditation of Laboratory Animal Care) (AAALAC).
Tumor volume was calculated 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 two-way ANOVA Bonferroni post hoc test of Prism software, p<0.05 was considered statistically significant.
In vivo efficacy studies of W3645-2.131.4.HIgG1L3 were tested in MC38 model using h-TIGIT mice. WBP364-BMK4 was used as a reference control. In this model, WBP3645 lead antibody as a single agent was unable to inhibit tumor growth in the MC38 model. The WBP3645 lead antibody combined with the PD-1 antibody showed a synergistic effect in the C57 (hTIGIT)/MC 38 efficacy model. The data is shown in fig. 14. 1.3 Ex vivo study of MC38 model
In an in vivo efficacy study of the C57 (hTIGIT)/MC 38 model (see 3.7.1.2), spleen and tumor tissue were isolated from 4 groups of mice (n=4 from a total of 8) using different treatments for cell population profiling. The 4 groups are: control-PBS; PD-1 antibody 0.3mg/kg; W3645-2.131.4-hIgG1L3 10mg/kg; a combination of 0.3mg/kg of anti-PD-1 antibody and 10mg/kg of W3645-2.131.4-hIgG1L 3. In this study, populations of cd4+ T cells, cd8+ T cells, treg, SPL (splenic lymphocytes) M1 and TIL (tumor infiltrating lymphocytes) were analyzed, as well as T cell function.
First, spleen lymphocytes from each mouse were isolated by gentle milling, filtration with a 70 μm filter and washing twice with 15mL PBS. Tumor-infiltrating lymphocytes were isolated from tumor tissue following the procedure of the mouse tumor dissociation kit (Miltenyi Biotec Cat:130-096-730). After the cells are isolated, some of them are fixed for staining. Intracellular markers were stained after fixation and permeation with buffer (BD, 51-2090 KZ). Information for 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, 5533035, apc coupled); mouse F4/80 antibody (eBioscience, 11-4801-82, FITC-conjugated); mouse MHC-II (eBioscience, 47-5321-82, APC coupled). Stained cells were detected by FACS (BD, cantoII) and data were analyzed using flowjo.7 and Prism 6.
For the evaluation of T cell function in TIL, intracellular IFNg was detected after stimulation. Briefly, 1E6 cells isolated from tumor tissue were treated with 0.5x cell stimulation mix (Invitrogen, 4333766) and 1x Golgi Stop (BD 51-2092K 2) 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, 5533035, apc coupled); mouse IFNg antibody (BD, 561479, apc-cy7 conjugated). Stained cells were detected by FACS (BD, cantoII) and data were analyzed using flowjo.7 and Prism 6.
In the study of 4.3.1.2, tissues of 4 groups of animals were obtained for ex vivo analysis. They are the following group: PBS; PD-1 antibodies; W3645-2.131.4-hIgG1L3; and a combination of PD-1 antibody with W3645-2.131.4-hIgG1L 3. The population of cd4+ T cells and 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 fig. 15.
The population of cd4+ T cells in cd4+ TIL was significantly increased in the anti-PD-1 antibodies and in the combination compared to the control-PBS group, and the data is shown in figure 16.
The population of Treg cells in splenic cd4+ T cells was significantly reduced (P < 0.05) in anti-PD-1 antibodies and in the combination compared to the control-PBS group. No significant differences were observed between the different groups for cd25+/foxp3+ Treg in cd4+ TIL. The data is shown in fig. 17.
By staining with F4/80 and MHCII, the number of M1 populations in TIL was significantly increased in the combined group compared to the PBS group. The data is shown in fig. 18.
A higher intracellular IFNg was observed in the combined group compared to the other groups after the mixture stimulation, but there was no significant difference for cd4+ cells and cd8+ cells in TIL. The data is shown in fig. 19.
2. Pharmacokinetic and acute toxicity assessment on cynomolgus monkeys
Pharmacokinetic characterization on cynomolgus monkeys
This study was performed to determine the pharmacokinetics of W3645 following a single intravenous bolus dose of 30mg/kg in naive male cynomolgus monkeys. Two animals were dosed once with W3645-2.131.4-hIgG1L3 at a dose of 30mg/kg, respectively, by intravenous bolus administration. The formulation was formulated in PBS. PK blood samples were collected before 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 before dosing, 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 before dosing, 24h, D3, D7, D14, D21, and D28.
This study was performed to determine the pharmacokinetics of WBP3645 final lead antibody following a single intravenous bolus dose of 30mg/kg in naive male cynomolgus monkeys. Two animals were dosed once with W3645-2.131.4-hIgG1L3 at a dose of 30mg/kg, respectively, by intravenous bolus administration. The formulation was formulated in PBS. PK blood samples were collected before 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 the serum sample was determined by ELISA.
Data were analyzed using Phoenix WinNonlin software (version 8.1, pharsight, mountain View, CA). The linear/logarithmic trapezoidal rule is applied in obtaining PK parameters.
A summary of PK parameters is listed in table 12 below.
TABLE 12 PK data for W3645-2.131.4-hIgG1L3 at a dose of 30mg/Kg
Note that: due to ADA effects, the pharmacokinetic parameters for G1-1 and G1-2 were calculated without time points on day 12, day 14, day 21 and day 28, data not shown.
Sequence listing
<110> Shanghai pharmaceutical 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
<210> 1
<211> 12
<212> PRT
<213> artificial sequence
<220>
<223> HCDR1
<400> 1
Gly Gly Ser Ile Thr Ser Ser Ser Tyr Tyr Trp Gly
1 5 10
<210> 2
<211> 14
<212> PRT
<213> artificial sequence
<220>
<223> LCDR1
<400> 2
Thr Gly Thr Ser Asn Asp Val Gly Gly Tyr Asn Tyr Val Ser
1 5 10
<210> 3
<211> 16
<212> PRT
<213> artificial sequence
<220>
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<400> 3
Ser Ile Phe Phe Ser Gly Ile Thr Tyr Tyr Asn Pro Ser Leu Lys Ser
1 5 10 15
<210> 4
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> LCDR2
<400> 4
Asp Val Ser Asn Arg Arg Thr
1 5
<210> 5
<211> 14
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<213> artificial sequence
<220>
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Arg Gly Leu Gly Gly Ile Leu Ala Ser Gly Tyr Phe Asp Tyr
1 5 10
<210> 6
<211> 11
<212> PRT
<213> artificial sequence
<220>
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<400> 6
Ser Ser Tyr Thr Ser Ile Ser Thr Leu Val Val
1 5 10
<210> 7
<211> 124
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<220>
<223> VH
<400> 7
Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Gly Ser Ile Thr Ser Ser
20 25 30
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
115 120
<210> 8
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<212> PRT
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<400> 8
Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
1 5 10 15
Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Asn Asp Val Gly Gly Tyr
20 25 30
Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Arg Leu
35 40 45
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
100 105 110
<210> 9
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<212> DNA
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<220>
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caactccagc tgcaagagtc gggcccagga ctggtaaagc cttcggagac cctgtccctc 60
acctgcgctg tctctggtgg ctccatcacc agtagtagtt actactgggg ctggatccgc 120
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 ca 372
<210> 10
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<212> DNA
<213> artificial sequence
<220>
<223> VL
<400> 10
cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccata 60
tcctgcactg gaaccagcaa tgacgttggt ggttacaact atgtctcctg gtaccaacaa 120
catccaggca aagccccccg actcttgatt tttgatgtca gtaatcgacg cacagggatt 180
tccaatcgct tctctggctc caagtctggc atcacggcct ccctgaccat ctctgggctc 240
caggctgagg acgaggctga ctattactgc agttcatata caagcatcag cactctcgtg 300
gtattcggcg gagggaccaa gctgaccgtc cta 333
<210> 11
<211> 450
<212> PRT
<213> artificial sequence
<220>
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<400> 11
Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Gly Ser Ile Thr Ser Ser
20 25 30
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
115 120 125
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
130 135 140
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
165 170 175
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
<211> 217
<212> PRT
<213> artificial sequence
<220>
<223> light chain
<400> 12
Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
1 5 10 15
Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Asn Asp Val Gly Gly Tyr
20 25 30
Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Arg Leu
35 40 45
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
acctgcgctg tctctggtgg ctccatcacc agtagtagtt actactgggg ctggatccgc 120
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
tggcaggagg ggaatgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1320
acacagaaga gcctctccct gtctctgggt 1350
<210> 14
<211> 651
<212> DNA
<213> artificial sequence
<220>
<223> light chain
<400> 14
cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccata 60
tcctgcactg gaaccagcaa tgacgttggt ggttacaact atgtctcctg gtaccaacaa 120
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 (15)
1. 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 CDR1 sequence is SEQ ID NO:1, wherein the amino acid sequence is shown in the specification,
Wherein the heavy chain variable region CDR2 sequence is SEQ ID NO:3, the amino acid sequence is shown in the specification,
wherein the heavy chain variable region CDR3 sequence is SEQ ID NO:5, the amino acid sequence shown in the specification,
wherein the light chain variable region CDR1 sequence is SEQ ID NO:2, and a polypeptide sequence represented by the following formula (2);
wherein the light chain variable region CDR2 sequence is SEQ ID NO:4, the amino acid sequence shown in the specification,
wherein the light chain variable region CDR3 sequence is SEQ ID NO:6, the amino acid sequence is shown in the specification,
wherein the antibody or antigen binding fragment specifically binds to TIGIT.
2. An antibody or antigen-binding fragment thereof, comprising:
a) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 7; and
b) A light chain variable region having the amino acid sequence of SEQ ID NO:8, and a sequence of amino acids.
3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment specifically binds to human, cynomolgus monkey and mouse TIGIT.
4. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody is a chimeric, humanized, fully human or rat antibody.
5. The antibody or antigen binding fragment thereof of claim 4, wherein the antibody comprises a human IgG constant domain.
6. The antibody or antigen binding fragment thereof of claim 5, wherein the human IgG constant domain is a human IgG1 or IgG4 constant domain.
7. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of claims 1-6.
8. A cloning or expression vector comprising the nucleic acid molecule of claim 7.
9. A host cell comprising one or more cloning or expression vectors according to claim 8.
10. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1 to 6, the method comprising culturing the host cell of claim 9 and isolating the antibody or antigen-binding fragment thereof.
11. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 6 and one or more pharmaceutically acceptable excipients, diluents and carriers.
12. 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 SEQ ID NO:5, a CDR3 sequence; and
(ii) A light chain variable region antibody sequence comprising SEQ ID NO:2, the CDR1 sequence of SEQ ID NO:4 and SEQ ID NO:6, CDR3 sequences; and
(b) The antibody sequences are expressed as proteins.
13. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 in the manufacture of a medicament for the prevention or treatment of a tumor or inflammatory disease.
14. The use of claim 13, 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, uterine cancer, ovarian cancer, liver cancer, gastrointestinal cancer, glioblastoma, cervical cancer, bladder cancer, and rectal cancer.
15. The use of claim 14, wherein the melanoma is selected from cutaneous or intraocular malignant melanoma.
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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
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Patent 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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