CN114989300A - Application of anti-TIGIT antibody in treatment of tumor or cancer - Google Patents

Abstract

The invention discloses the use of an anti-TIGIT antibody or antigen-binding fragment in the treatment of a tumor or cancer, the method of treatment comprising administering to a patient in need thereof an effective amount of an anti-TIGIT antibody or antigen-binding fragment.

Description

Application of anti-TIGIT antibody in treatment of tumor or cancer

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to an application of an anti-TIGIT antibody or an antigen binding fragment in treating tumors or cancers.

Background

TIGIT (T cell immunoreceptors with Ig and ITIM domains) is a T cell immunoreceptor with immunoglobulin (Ig) and tyrosine inhibitor motif (ITIM) domains, which is expressed predominantly on activated T cells and NK cells (Yu, X., et al (2009) 'The surface protein TIGIT substitutions T cell activation by The generating of a physical immunological signaling reliability cells.' Nature immunology 10(1): 48-57.). TIGIT structures are shown to comprise an extracellular immunoglobulin domain, a type I transmembrane region and two ITIM motifs. TIGIT is part of a co-stimulatory network consisting essentially of the activating receptor CD226 and the inhibitory receptor TIGIT on T cells, and the ligands CD155 (also known as PVR, a poliovirus receptor protein encoded by the PVR gene in humans) and CD112 expressed on the surface of APCs, tumor cells, infected cells. Binding of TIGIT to PVR or CD112 causes TIGIT cytosolic Tyr225 to be phosphorylated, and binding of TIGIT to cell adaptive growth factor receptor binding protein 2(GRB 2). GRB2 may recruit SHIP1 to inhibit phosphatidylinositol trikinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling. In addition, phosphorylated TIGIT recruits SHIP1 via Beta arrestin 2(β -arrestin2) and disrupts nuclear factor KB (NF-KB) activation by blocking self-ubiquitination of TNF receptor-related factor 6(TRAF6), a series of signaling ultimately leads to suppression of T cell or NK cell function and inhibition of cytokine production. PVR is a ligand for both TIGIT and CD226 molecules. Upon binding to CD112 or CD155, Ser329 and Tyr322 of the intracellular domain of CD226 are phosphorylated; ser329 phosphorylation promotes activation of Protein Kinase (PKC) and the binding of CD226 to lymphocyte-associated antigen 1(LFA 1). LFA1 was then used for TYN-mediated Tyr322 phosphorylation and CD 226-mediated downstream signaling. A series of signal transduction ultimately leads to activation of T cell or NK cell functions, promoting cytokine production. The interaction between TIGIT molecules present on the surface of T cells or NK cells and CD226 molecules also occurs in that TIGIT molecules can directly perturb CD226 to form normal dimers, thereby disrupting the normal physiological function of CD 226. TIGIT and CD226 act as balance ends, and through PVR, the body's immunity is skillfully regulated through the co-stimulation and co-inhibition signal transmission.

TIGIT is a very potential inhibitory target in tumor immunotherapy and can provide a new means for tumor immunotherapy.

Disclosure of Invention

The invention discloses a method or use of an anti-TIGIT antibody or antigen-binding fragment for treating a tumor or cancer. In some embodiments, an anti-TIGIT antibody or antigen-binding fragment agent is used to treat a tumor or cancer.

In some embodiments, the anti-TIGIT antibody or antigen-binding fragment comprises at least one or more of HCDR1 shown in SEQ ID No. 1, HCDR2 shown in SEQ ID No. 2, HCDR3 shown in SEQ ID No. 3, LCDR1 shown in SEQ ID No. 4, LCDR2 shown in SEQ ID No. 5, LCDR3 shown in SEQ ID No. 6.

In some embodiments, the anti-TIGIT antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID No. 1, HCDR2 shown in SEQ ID No. 2, HCDR3 shown in SEQ ID No. 3, LCDR1 shown in SEQ ID No. 4, LCDR2 shown in SEQ ID No. 5, and LCDR3 shown in SEQ ID No. 6.

In some embodiments, the heavy chain variable region of the anti-TIGIT antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID No. 7, or an amino acid sequence having at least 80% identity compared to the sequence set forth in SEQ ID No. 7, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence set forth in SEQ ID No. 7.

In some embodiments, the light chain variable region of the anti-TIGIT antibody or antigen binding fragment comprises the amino acid sequence set forth in SEQ ID No. 8, or an amino acid sequence having at least 80% identity compared to the sequence set forth in SEQ ID No. 8, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence set forth in SEQ ID No. 8.

In some embodiments, the heavy chain variable region of the anti-TIGIT antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID No. 7, or an amino acid sequence having at least 80% identity compared to the sequence set forth in SEQ ID No. 7, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence set forth in SEQ ID No. 7; the light chain variable region of the anti-TIGIT antibody or antigen binding fragment comprises the amino acid sequence set forth in SEQ ID No. 8, or an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID No. 8, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID No. 8.

In some embodiments, the heavy chain variable region of the anti-TIGIT antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID No. 7 and the light chain variable region of the anti-TIGIT antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID No. 8.

In some embodiments, the heavy chain of the anti-TIGIT antibody comprises the amino acid sequence set forth in SEQ ID No. 9, or an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID No. 9, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID No. 9.

In some embodiments, the light chain of the anti-TIGIT antibody comprises the amino acid sequence set forth in SEQ ID No. 10, or an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID No. 10, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID No. 10.

In some embodiments, the heavy chain of the anti-TIGIT antibody comprises the amino acid sequence set forth in SEQ ID No. 9, or an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID No. 9, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID No. 9; the light chain of the anti-TIGIT antibody comprises the amino acid sequence shown in SEQ ID NO. 10, or an amino acid sequence with at least 80% of identity compared with the sequence shown in SEQ ID NO. 10, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 10.

In some embodiments, the anti-TIGIT antibody is antibody h10D8OF or h10D8OFKF, the heavy chains of antibodies h10D8OF and h10D8OFKF comprise the amino acid sequence shown as SEQ ID No. 9, and the light chains of antibodies h10D8OF and h10D8OFKF comprise the amino acid sequence shown as SEQ ID No. 10; the antibodies h10D8OF and h10D8OFKF contain two heavy chains of identical sequence and two light chains of identical sequence, respectively.

In some embodiments, the anti-TIGIT antibody (e.g., antibody h10D8OFKF) or antigen binding fragment has a fucosylation level of 0-10%. In some embodiments, the anti-TIGIT antibody (e.g., antibody h10D8OFKF) or antigen binding fragment has a fucosylation level of 0-5%. In some embodiments, the anti-TIGIT antibody (e.g., antibody h10D8OFKF) or antigen binding fragment has a level of fucosylation of about 0, about 0.1%, about 0.5%, about 0.8%, about 1%, about 1.3%, about 1.6%, about 2.1%, 2.9%, about 3%, about 3.3%, 3.8%, about 4%, about 4.2%, 4.3%, about 4.6%, about 5%, or a range between any two of these values (including the endpoints) or any value therein. In some embodiments, the anti-TIGIT antibody (e.g., antibody h10D8OFKF) or antigen binding fragment does not bind fucose. In some embodiments, the anti-TIGIT antibody (e.g., antibody h10D8OFKF) or antigen-binding fragment has an enhanced ADCC effect (antibody-dependent cell-mediated cytotoxicity).

The anti-TIGIT antibody or the antigen binding fragment can be expressed in CHO cells or HEK293 cells by genetic engineering and obtained by purification; purification can be carried out by conventional methods, for example, by first centrifuging the cell suspension and collecting the supernatant, and then centrifuging again to further remove impurities. Methods such as ProteinA affinity columns and ion exchange columns can be used to purify antibody proteins.

In some embodiments, the low fucosylated or afucosylated anti-TIGIT antibody or antigen binding fragment is expressed by an alpha- (1,6) -fucosyltransferase gene knock-out cell line. In some embodiments, the antibody h10D8OFKF is expressed by an α - (1,6) -fucosyltransferase gene knock-out cell line, e.g., an α - (1,6) -fucosyltransferase gene knock-out CHO cell.

In some embodiments, the method or use comprises: administering to a patient in need thereof an effective amount of an anti-TIGIT antibody or antigen-binding fragment. In some embodiments, the anti-TIGIT antibody is antibody h10D8OF or h10D8 OFKF. In some embodiments, the effective dose of anti-TIGIT antibody or antigen-binding fragment administered is about 9mg to 1200mg per dose.

In some embodiments, the patient has a tumor or cancer. In some embodiments, tumors and cancers include, but are not limited to, hematologic cancers, solid tumors. In some embodiments, hematologic cancers include, but are not limited to, leukemia, lymphoma, and myeloma. In some embodiments, the leukemia includes Acute Lymphocytic Leukemia (ALL), Acute Myelogenous Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), and myeloproliferative disease/tumor (MPDS). In some embodiments, the lymphoma includes hodgkin's lymphoma, indolent and aggressive non-hodgkin's lymphoma, burkitt's lymphoma, and follicular lymphoma (both small and large). In some embodiments, the myeloma comprises Multiple Myeloma (MM), giant cell myeloma, heavy chain myeloma, and light chain or bense-jones myeloma. In some embodiments, the solid tumor comprises breast cancer, ovarian cancer, pancreatic cancer, prostate cancer, melanoma, colorectal cancer, colon cancer, lung cancer, head and neck cancer, bladder cancer, esophageal cancer, liver cancer, and kidney cancer. In some embodiments, the tumors and cancers are pathologically confirmed locally advanced or metastatic malignant solid tumors that have not been treated effectively.

In some embodiments, the invention discloses a method for treating a tumor or cancer in a patient in need thereof comprising administering an effective amount of an anti-TIGIT antibody or antigen-binding fragment, wherein the effective amount of the anti-TIGIT antibody or antigen-binding fragment administered is about 9mg to 1200mg per treatment cycle. In some embodiments, a treatment cycle is 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, or a range between any two of these values (including the endpoints) or any value therein. In some embodiments, the anti-TIGIT antibody is antibody h10D8OF or h10D8 OFKF.

In some embodiments, the anti-TIGIT antibody may be formulated as a pharmaceutical composition and administered to a patient in a variety of forms suitable for the chosen route of administration, e.g., parenteral, intravenous (iv), intramuscular, topical, or subcutaneous. In some embodiments, the anti-TIGIT antibody may be infused intravenously. The amount of anti-TIGIT antibody administered will depend on the nature of the drug, the extent to which the internalization, transport and release of the drug is triggered at the cell surface, as well as the disease being treated and the condition of the patient (e.g., age, sex, body weight, etc.).

In some embodiments, each administration of the anti-TIGIT antibody (e.g., antibody h10D8OF or h10D8OFKF) or antigen binding fragment is 0.01mg/kg to 26mg/kg or a formulation comprising such dose of the anti-TIGIT antibody or antigen binding fragment. In some embodiments, the anti-TIGIT antibody or antigen-binding fragment is about 0.01mg/kg, about 0.02mg/kg, about 0.03mg/kg, about 0.06mg/kg, about 0.08mg/kg, about 0.1mg/kg, about 0.2mg/kg, about 0.3mg/kg, about 0.5mg/kg, about 0.9mg/kg, about 1mg/kg, about 1.6mg/kg, about 2mg/kg, about 2.5mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg per administration, About 18mg/kg, about 19mg/kg, about 20mg/kg, about 21mg/kg, about 22mg/kg, about 23mg/kg, about 24mg/kg, about 25mg/kg, about 26mg/kg, or a range between any two of these values (including the endpoints) or any value therein, or a formulation containing this dose of the anti-TIGIT antibody or antigen binding fragment. In some embodiments, the administration is 1 time for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, or about 7 weeks.

In some embodiments, the present invention discloses a method for treating a tumor or cancer, the method comprising: about 0.01mg/kg, about 0.02mg/kg, about 0.03mg/kg, about 0.06mg/kg, about 0.08mg/kg, about 0.1mg/kg, about 0.2mg/kg, about 0.3mg/kg, about 0.5mg/kg, about 0.9mg/kg, about 1mg/kg, about 1.6mg/kg, about 2mg/kg, about 2.5mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, about 3mg/kg, about 6mg/kg, about, About 20mg/kg, about 21mg/kg, about 22mg/kg, about 23mg/kg, about 24mg/kg, about 25mg/kg, about 26mg/kg of the anti-TIGIT antibody or antigen-binding fragment, or a range between any two of these values (including the endpoints) or any value therein, or a formulation containing this dose of the anti-TIGIT antibody or antigen-binding fragment.

In some embodiments, the present invention discloses a method for treating a tumor or cancer, the method comprising: about 0.01mg/kg, about 0.02mg/kg, about 0.03mg/kg, about 0.06mg/kg, about 0.08mg/kg, about 0.1mg/kg, about 0.2mg/kg, about 0.3mg/kg, about 0.5mg/kg, about 0.9mg/kg, about 1mg/kg, about 1.6mg/kg, about 2mg/kg, about 2.5mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, about 3mg/kg, about 6mg/kg, about, About 20mg/kg, about 21mg/kg, about 22mg/kg, about 23mg/kg, about 24mg/kg, about 25mg/kg, about 26mg/kg of antibody h10D8OF, or a formulation containing this dose of antibody h10D8 OF.

In some embodiments, the present invention discloses a method for treating a tumor or cancer, the method comprising: about 0.01mg/kg, about 0.02mg/kg, about 0.03mg/kg, about 0.06mg/kg, about 0.08mg/kg, about 0.1mg/kg, about 0.2mg/kg, about 0.3mg/kg, about 0.5mg/kg, about 0.9mg/kg, about 1mg/kg, about 1.6mg/kg, about 2mg/kg, about 2.5mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, about 3mg/kg, about 6mg/kg, about, About 20mg/kg, about 21mg/kg, about 22mg/kg, about 23mg/kg, about 24mg/kg, about 25mg/kg, about 26mg/kg of antibody h10D8OFKF, or a formulation containing such a dose of antibody h10D8 OFKF.

In some embodiments, the invention discloses a method of treating a tumor or cancer comprising administering to a patient in need thereof an effective amount of an anti-TIGIT antibody or antigen-binding fragment (or formulation); wherein the effective amount of the anti-TIGIT antibody or antigen-binding fragment is 9mg to 1200mg (or a formulation comprising such a dose of the anti-TIGIT antibody or antigen-binding fragment) given in a single administration. The dosage schedule and mode of administration depends on the risk of benefit assessment and general clinical practice guidelines for the anti-TIGIT antibody or antigen-binding fragment (or formulation) in certain patient populations.

In some embodiments, the effective amount of the anti-TIGIT antibody or antigen-binding fragment administered per treatment cycle by the patient is 9mg to 1200mg (or a formulation containing such dose of the anti-TIGIT antibody or antigen-binding fragment).

In some embodiments, the effective amount of the anti-TIGIT antibody (e.g., antibody h10D8OF or h10D8OFKF) or antigen-binding fragment administered to the patient per treatment cycle is about 9mg, about 10mg, about 18mg, about 26mg, about 30mg, about 38mg, about 45mg, about 60mg, about 70mg, about 80mg, about 100mg, about 120mg, about 180mg, about 200mg, about 250mg, about 290mg, about 300mg, about 330mg, about 380mg, about 400mg, about 434mg, about 480mg, about 500mg, about 567mg, about 580mg, about 600mg, about 700mg, about 800mg, about 900mg, or a range between any two of these values (inclusive) or any value therein, or a formulation containing such dose of the anti-TIGIT antibody or antigen-binding fragment. In some embodiments, one treatment cycle is administered 1 time from 1 week to 7 weeks. In some embodiments, an effective amount of the anti-TIGIT antibody or antigen-binding fragment is about 10mg to 900mg, or a formulation comprising such a dose, per treatment cycle administered; wherein a treatment cycle is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, or a range between any two of these values (inclusive), or any value therein. In some embodiments, one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In some embodiments, the effective amount of the anti-TIGIT antibody or antigen-binding fragment administered to the patient per treatment cycle is from about 10mg to about 300mg, or a formulation containing such a dose of the anti-TIGIT antibody or antigen-binding fragment; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In some embodiments, the effective amount of the anti-TIGIT antibody or antigen-binding fragment administered to the patient per treatment cycle is about 10mg, about 30mg, about 40mg, about 50mg, about 78mg, about 82mg, about 96mg, about 100mg, about 124mg, about 143mg, about 157mg, about 180mg, about 196mg, about 200mg, about 226mg, about 240mg, about 260mg, about 286mg, about 300mg, or a range between any two of these values (including the endpoints) or any value therein, or a formulation comprising such a dose of the anti-TIGIT antibody or antigen-binding fragment; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In some embodiments, the effective amount of the anti-TIGIT antibody or antigen-binding fragment administered to the patient per treatment cycle is about 300mg to 600mg, or a formulation containing such a dose of the anti-TIGIT antibody or antigen-binding fragment; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In some embodiments, the effective amount of the anti-TIGIT antibody or antigen-binding fragment administered to the patient per treatment cycle is about 300mg, about 355mg, about 360mg, about 380mg, about 390mg, about 400mg, about 420mg, about 460mg, about 480mg, about 500mg, about 520mg, about 540mg, about 580mg, about 600mg, or a range between any two of these values (including the endpoints) or any value therein, or a formulation comprising such a dose of the anti-TIGIT antibody or antigen-binding fragment; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In some embodiments, the effective amount of the anti-TIGIT antibody or antigen-binding fragment administered to the patient per treatment cycle is about 600mg to 900mg, or a formulation comprising this dose of the anti-TIGIT antibody or antigen-binding fragment; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In some embodiments, the effective amount of the anti-TIGIT antibody or antigen-binding fragment administered to the patient per treatment cycle is about 600mg, about 630mg, about 642mg, about 660mg, about 680mg, about 700mg, about 734mg, about 750mg, about 780mg, about 790mg, about 800mg, about 820mg, about 844mg, about 878mg, about 900mg, or a range between any two of these values (including the endpoints) or any value therein, or a formulation containing such a dose of the anti-TIGIT antibody or antigen-binding fragment; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.

In some embodiments, the effective amount of anti-TIGIT antibody (e.g., antibody h10D8OF or h10D8OFKF) or antigen-binding fragment administered to the patient per treatment cycle is about 9mg to 13mg, or a formulation containing such a dose of anti-TIGIT antibody or antigen-binding fragment; such as about 10mg, for 1 dose. In some embodiments, the effective amount of the anti-TIGIT antibody or antigen-binding fragment administered to the patient per treatment cycle is about 10mg, or a formulation containing such a dose, of the anti-TIGIT antibody or antigen-binding fragment; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.

In some embodiments, the effective amount of anti-TIGIT antibody (e.g., antibody h10D8OF or h10D8OFKF) or antigen-binding fragment administered to the patient per treatment cycle is about 25mg to 33mg, or a formulation containing such a dose of anti-TIGIT antibody or antigen-binding fragment; e.g., about 30mg, for 1 dose. In some embodiments, the effective amount of the anti-TIGIT antibody or antigen-binding fragment administered to the patient per treatment cycle is about 30mg, or a formulation containing such a dose, of the anti-TIGIT antibody or antigen-binding fragment; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.

In some embodiments, the effective amount of anti-TIGIT antibody (e.g., antibody h10D8OF or h10D8OFKF) or antigen-binding fragment administered to the patient per treatment cycle is about 89mg to 120mg, or a formulation containing such a dose of anti-TIGIT antibody or antigen-binding fragment; such as about 100mg, for 1 dose. In some embodiments, the effective amount of anti-TIGIT antibody or antigen-binding fragment administered to the patient per treatment cycle is about 100mg, or a formulation containing such a dose of anti-TIGIT antibody or antigen-binding fragment; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.

In some embodiments, the effective amount of anti-TIGIT antibody (e.g., antibody h10D8OF or h10D8OFKF) or antigen-binding fragment administered to the patient per treatment cycle is about 289mg to 314mg, or a formulation containing such dose of anti-TIGIT antibody or antigen-binding fragment; such as about 300mg, for 1 dose. In some embodiments, the effective amount of anti-TIGIT antibody or antigen-binding fragment administered to the patient per treatment cycle is about 300mg, or a formulation containing such a dose of anti-TIGIT antibody or antigen-binding fragment; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.

In some embodiments, the effective amount of anti-TIGIT antibody (e.g., antibody h10D8OF or h10D8OFKF) or antigen-binding fragment administered to the patient per treatment cycle is about 390mg to 410mg, or a formulation containing such dose of anti-TIGIT antibody or antigen-binding fragment; such as about 400mg, for 1 dose. In some embodiments, the effective amount of anti-TIGIT antibody or antigen-binding fragment administered to the patient per treatment cycle is about 400mg, or a formulation containing such a dose of anti-TIGIT antibody or antigen-binding fragment; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.

In some embodiments, the effective amount of an anti-TIGIT antibody (e.g., antibody h10D8OF or h10D8OFKF) or antigen-binding fragment administered to the patient per treatment cycle is about 580mg to 740mg, or a formulation containing such a dose of anti-TIGIT antibody or antigen-binding fragment; such as about 600mg, for 1 dose. In some embodiments, the effective amount of anti-TIGIT antibody or antigen-binding fragment administered to the patient per treatment cycle is about 600mg, or a formulation containing such a dose of anti-TIGIT antibody or antigen-binding fragment; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.

In some embodiments, the patient is administered an effective amount of an anti-TIGIT antibody (e.g., antibody h10D8OF or h10D8OFKF) or antigen binding fragment of about 748mg to 833mg, or a formulation comprising this dose of anti-TIGIT antibody or antigen binding fragment, per treatment cycle; such as about 800mg, for 1 dose. In some embodiments, the effective amount of anti-TIGIT antibody or antigen-binding fragment administered to the patient per treatment cycle is about 800mg, or a formulation containing such a dose of anti-TIGIT antibody or antigen-binding fragment; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.

In some embodiments, the effective amount of anti-TIGIT antibody (e.g., antibody h10D8OF or h10D8OFKF) or antigen-binding fragment administered to the patient is about 872mg to 943mg per treatment cycle, or a formulation containing this dose of anti-TIGIT antibody or antigen-binding fragment; such as about 900mg, for 1 administration. In some embodiments, the effective amount of anti-TIGIT antibody or antigen-binding fragment administered to the patient per treatment cycle is about 900mg, or a formulation containing such a dose of anti-TIGIT antibody or antigen-binding fragment; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.

In some embodiments, the effective amount of an anti-TIGIT antibody (such as antibody h10D8OF or h10D8OFKF) or antigen binding fragment administered is 9mg to 600mg once every 3 weeks. In some embodiments, an effective amount of anti-TIGIT antibody is administered once every 3 weeks of about 9mg, about 10mg, about 16mg, about 18mg, about 30mg, about 60mg, about 100mg, about 180mg, about 200mg, about 300mg, about 400mg, about 500mg, or about 600 mg. In some embodiments, an effective amount of the anti-TIGIT antibody or antigen-binding fragment administered is about 10mg, about 30mg, about 100mg, about 300mg, or about 600mg once every 3 weeks. In some embodiments, an effective amount of the anti-TIGIT antibody or antigen-binding fragment administered is about 600mg to 1200mg once every 3 weeks. In some embodiments, the effective amount of the anti-TIGIT antibody or antigen-binding fragment administered is about 700mg, about 800mg, 900mg, or about 1200 once every 3 weeks.

In some embodiments, the patient is administered the anti-TIGIT antibody or antigen-binding fragment (or formulation) once per treatment cycle. In some embodiments, the anti-TIGIT antibody or antigen-binding fragment (or formulation) is administered multiple times, e.g., 2, 3, 4, or 5 times, per treatment cycle. In some embodiments, the patient is administered only 1 or 4 times per treatment cycle.

In some embodiments, the patient receives one treatment cycle of treatment. In some embodiments, the patient is treated with multiple (e.g., 2, 3, or 4) treatment cycles. In some embodiments, the patient receives treatment until the condition is alleviated and no treatment is required.

In some embodiments, the present invention discloses a method for treating a tumor or cancer, the method comprising: administering to a patient in need thereof about 9mg to 30mg, about 30mg to 100mg, about 100mg to 300mg, about 300mg to 600mg, about 600mg to 900mg, or about 900mg to 1200mg, such as about 9mg, about 10mg, about 18mg, about 20mg, about 30mg, about 60mg, 100mg, about 180mg, about 200mg, about 300mg, about 400mg, about 500mg, about 600mg, about 900mg, or about 1200mg (or a range between any two of these values (inclusive of the endpoints) or any value therein), or a formulation comprising the dose of the anti-TIGIT antibody or antigen binding fragment. In some embodiments, the anti-TIGIT antibody is antibody h10D8 OF. In some embodiments, the anti-TIGIT antibody is the antibody h10D8 OFKF.

In some embodiments, the anti-TIGIT antibody or antigen-binding fragment is administered about 9mg once every 3 weeks. In some embodiments, the anti-TIGIT antibody or antigen-binding fragment is administered at about 10mg once every 3 weeks. In some embodiments, the anti-TIGIT antibody or antigen-binding fragment is administered at about 18mg once every 3 weeks. In some embodiments, about 20mg of the anti-TIGIT antibody or antigen-binding fragment is administered once every 3 weeks. In some embodiments, the anti-TIGIT antibody or antigen-binding fragment is administered at about 30mg once every 3 weeks. In some embodiments, the anti-TIGIT antibody or antigen-binding fragment is administered about 60mg once every 3 weeks. In some embodiments, the anti-TIGIT antibody or antigen-binding fragment is administered at about 100mg once every 3 weeks. In some embodiments, about 300mg of the anti-TIGIT antibody or antigen-binding fragment is administered once every 3 weeks. In some embodiments, about 600mg of the anti-TIGIT antibody or antigen-binding fragment is administered once every 3 weeks. In some embodiments, the anti-TIGIT antibody or antigen-binding fragment is administered about 900mg once every 3 weeks. In some embodiments, the anti-TIGIT antibody or antigen-binding fragment is administered at about 1200mg once every 3 weeks.

In some embodiments, the patient is relieved of symptoms after a single dose administration. In some embodiments, after a single dose administration, the patient has not had the expected relief of symptoms, and about 9mg to 1200mg or about 10mg to 900mg of the anti-TIGIT antibody or antigen-binding fragment is administered to the patient until the patient's symptoms are relieved.

In some embodiments, the anti-TIGIT antibody or antigen-binding fragment (or formulation) is administered by subcutaneous (s.c.) injection, intraperitoneal (i.p.) injection, parenteral injection, intraarterial injection, or intravenous (i.v.) injection, among others. In some embodiments, the anti-TIGIT antibody or antigen-binding fragment (or formulation) is administered by infusion. In some embodiments, the anti-TIGIT antibody or antigen-binding fragment (or formulation) is administered as a bolus.

In some embodiments, the anti-TIGIT antibody or antigen-binding fragment (or formulation) is administered by intravenous (i.v.) infusion (i.e., intravenous infusion). In some embodiments, the intravenous infusion duration is about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 81 minutes, about 87 minutes, about 90 minutes, about 95 minutes, or a range between any two of these values (including the endpoints) or any value therein. In some embodiments, the duration of intravenous infusion is greater than or equal to 60 minutes.

In some embodiments, the anti-TIGIT antibody or antigen-binding fragment (or formulation) is used in combination with other therapeutic approaches to treat tumors or cancers, such as chemotherapy, radiation therapy, immunotherapy, hormonal therapy, targeted therapy, biological therapy, surgical therapy, and the like. In some embodiments, the anti-TIGIT antibody or antigen-binding fragment (or formulation) is used in combination with other tumor or cancer therapeutic agents to treat a tumor or cancer, such as hormones, antibodies to treat a tumor or cancer, and the like.

In another aspect, the invention discloses the use of an anti-TIGIT antibody or antigen-binding fragment for the manufacture of a medicament for the treatment of a tumor or cancer. In some embodiments, the agent for treating a tumor or cancer comprises an anti-TIGIT antibody or antigen-binding fragment. In some embodiments, the anti-TIGIT antibody is antibody h10D8 OF. In some embodiments, the anti-TIGIT antibody is the antibody h10D8 OFKF. In some embodiments, the antibody h10D8OFKF fucosylation level is 0-10%.

In another aspect, the invention also discloses a kit comprising an anti-TIGIT antibody or antigen-binding fragment (or formulation) and instructions for administering the anti-TIGIT antibody or antigen-binding fragment (or formulation) to a patient in need thereof. In some embodiments, the anti-TIGIT antibody is antibody h10D8 OF. In some embodiments, the anti-TIGIT antibody is the antibody h10D8 OFKF. In some embodiments, the antibody h10D8OFKF fucosylation level is 0-10%.

In another aspect, the invention also discloses a pharmaceutical composition suitable for injection, such as a bolus pharmaceutical composition or an infusion (instillation) pharmaceutical composition, comprising the anti-TIGIT antibody or antigen-binding fragment. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (herein water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic or Phosphate Buffered Saline (PBS), ethanol, solvents or dispersion media of polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), and suitable mixtures thereof. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutically acceptable carrier may comprise an antibacterial and/or antifungal agent, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some embodiments, the pharmaceutically acceptable carrier may comprise isotonic agents, such as sugars, polyalcohols (such as mannitol, sorbitol), sodium chloride. In some embodiments, the pharmaceutical composition comprises at least 0.1% of an anti-TIGIT antibody or antigen-binding fragment. The percentage of antibody can vary and is between about 2% to 90% of the weight of a given dosage form. The amount of anti-TIGIT antibody or antigen-binding fragment in such therapeutically useful pharmaceutical compositions can be an effective amount for administration. In some embodiments, the anti-TIGIT antibody is antibody h10D8 OF. In some embodiments, the anti-TIGIT antibody is the antibody h10D8 OFKF. In some embodiments, the antibody h10D8OFKF is expressed by an α - (1,6) -fucosyltransferase gene knock-out cell line. In some embodiments, the antibody h10D8OFKF is expressed by α - (1,6) -fucosyltransferase gene knockout CHO cells.

On the other hand, the invention also discloses a preparation method of the pharmaceutical composition, which comprises the following steps: the anti-TIGIT antibodies or antigen binding fragments described herein are each mixed with a pharmaceutically acceptable carrier suitable for injection (e.g., water for injection, physiological saline, etc.). Methods of mixing the above anti-TIGIT antibodies or antigen binding fragments with a pharmaceutically acceptable carrier are generally known in the art.

The anti-TIGIT antibodies or antigen binding fragments (or formulations) of the present invention can be used in the treatment of tumors or cancer.

Drawings

Figures 1A and 1B show that anti-TIGIT antibodies inhibit proliferation of tumor cells; wherein Isotype IgG 30mg/kg represents group G1, h10D8OF 30mg/kg represents group G2, h10D8OFKF 10mg/kg represents group G3, h10D8OFKF 30mg/kg represents group G4, and Tiragolumab 30mg/kg represents group G5.

Term(s) for

Unless otherwise specified, each of the following terms shall have the meaning set forth below.

Definition of

It should be noted that the term "an" entity refers to one or more of the entities, e.g., "an antibody" should be understood as one or more antibodies, and thus the terms "a" (or "an"), "one or more" and "at least one" may be used interchangeably herein.

The terms "comprising" or "including" as used herein mean that the compositions and methods and the like include the recited elements, such as components or steps, but not excluding others. By "consisting essentially of … …" is meant that the compositions and methods exclude other elements that have a fundamental effect on the combined characteristics, but do not exclude elements that do not materially affect the composition or method. "consisting of … …" means excluding elements not specifically recited.

The term "polypeptide" is intended to encompass both the singular "polypeptide" and the plural "polypeptide" and refers to a molecule composed of monomers of amino acids linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any single chain or multiple chains of two or more amino acids and does not refer to a particular length of the product. Thus, included within the definition of "polypeptide" are peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other term used to refer to two or more amino acid chains, and the term "polypeptide" may be used in place of, or in alternation with, any of the above terms. The term "polypeptide" is also intended to refer to the product of post-expression modification of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or non-naturally occurring amino acid modification. The polypeptide may be derived from a natural biological source or produced by recombinant techniques, but it need not be translated from a specified nucleic acid sequence, and it may be produced in any manner, including chemical synthesis.

"amino acid" refers to an organic compound containing both amino and carboxyl groups, such as an alpha-amino acid, which may be encoded by a nucleic acid, either directly or in the form of a precursor. A single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The same amino acid is encoded by different codons and is referred to as "degeneracy of the genetic code". Amino acids include natural amino acids and unnatural amino acids. Natural amino acids include alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y) and valine (val, V).

"conservative amino acid substitution" refers to the substitution of one amino acid residue with another amino acid residue having a side chain (R group) of similar chemical nature (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of the protein. Examples of classes of amino acids containing chemically similar side chains include: 1) aliphatic side chain: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chain: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chain: phenylalanine, tyrosine and tryptophan; 5) basic side chain: lysine, arginine and histidine; 6) acidic side chain: aspartic acid and glutamic acid.

The number of amino acids of a "conservative amino acid substitution of VL, VH" can be about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15 conservative amino acid substitutions, or a range between any two of these values (inclusive) or any value therein. The number of amino acids of a "conservative amino acid substitution of a heavy or light chain" can be about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15, about 18, about 19, about 22, about 24, about 25, about 29, about 31, about 35, about 38, about 41, about 45 conservative amino acid substitutions, or a range between any two of these values (including the endpoints) or any value therein.

The term "encoding" as applied to a polynucleotide refers to a polynucleotide that is said to "encode" a polypeptide, which polypeptide and/or fragments thereof can be produced by transcription and/or translation in its native state or when manipulated by methods well known to those skilled in the art.

The antibodies, antigen binding fragments or derivatives disclosed herein include, but are not limited to, polyclonal, monoclonal, multispecific, fully human, humanized, primatized, chimeric antibodies, single chain antibodies, epitope binding fragments (e.g., Fab-like ', and F-like (ab') ₂ ) Single-chain-like Fvs (scFv).

The term "recombinant" refers to a polypeptide or polynucleotide, and means a form of a polypeptide or polynucleotide that does not occur in nature, and non-limiting examples may include combinations that produce polynucleotides or polypeptides that do not normally occur.

"identity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions in each sequence that can be aligned. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.

"at least 80% identity" is about 80% identity, about 81% identity, about 82% identity, about 83% identity, about 85% identity, about 86% identity, about 87% identity, about 88% identity, about 90% identity, about 91% identity, about 92% identity, about 94% identity, about 95% identity, about 98% identity, about 99% identity, or a range between any two of these values (including endpoints), or any value therein.

A polynucleotide or polynucleotide sequence (or polypeptide or antibody sequence) has a certain percentage (e.g., 90%, 95%, 98%, or 99%) of "identity" or "sequence identity" with another sequence, meaning that the percentage of bases (or amino acids) in the two sequences being compared are the same when the sequences are aligned. The percent identity or sequence identity of the alignment can be determined using visual inspection or software programs known in the art, such as the software program described in Current Protocols in Molecular Biology, Ausubel et al. Preferably, the alignment is performed using default parameters. One alignment program is BLAST using default parameters, such as BLASTN and BLASTP, both using the following default parameters: geneticcode ═ standard; filter is none; strand ═ booth; cutoff is 60; expect is 10; matrix ═ BLOSUM 62; descriptors is 50 sequences; sortby ═ HIGHSCORE; databases is non-redundant; GenBank + EMBL + DDBJ + PDB + GenBank CDStranslations + Swi ssProtein + Spupdate + PIR. A biologically equivalent polynucleotide is a polynucleotide having the above specified percentage of identity and encoding a polypeptide having the same or similar biological activity.

"antibody," "antigen-binding fragment," refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen. The antibody may be a whole antibody and any antigen binding fragment thereof or a single chain thereof. The term "antibody" thus includes any protein or peptide in a molecule that contains at least a portion of an immunoglobulin molecule having biological activity that binds to an antigen. Antibodies and antigen-binding fragments include, but are not limited to, Complementarity Determining Regions (CDRs), heavy chain variable regions (VH), light chain variable regions (VL), heavy chain constant regions (CH), light chain constant regions (CL), Framework Regions (FR), or any portion thereof of a heavy chain or light chain or ligand-binding portion thereof, or at least a portion of a binding protein. The CDR regions include the CDR region of the light chain (LCDR1-3) and the CDR region of the heavy chain (HCDR 1-3).

The term "antibody" includes a wide variety of polypeptides that can be biochemically distinguished. Those skilled in the art will appreciate that the class of heavy chains includes gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses (e.g., γ 1- γ 4). The nature of this chain determines the "class" of the antibody as IgG, IgM, IgA, IgG or IgE, respectively. Immunoglobulin subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgG5, and the like have been well characterized and the functional specificity conferred is also known. All immunoglobulin classes are within the scope of the present disclosure. In some embodiments, the immunoglobulin molecule is an IgG class.

Light chains can be classified as kappa (. kappa.) or lambda (. lamda.). Each heavy chain may be associated with a kappa or lambda light chain. In general, when immunoglobulins are produced by hybridomas, B cells or genetically engineered host cells, the light and heavy chains are joined by covalent bonds and the "tail" portions of the two heavy chains are joined by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus of the forked end of the Y configuration to the C-terminus of the bottom of each chain. The immunoglobulin kappa light chain variable region is Vkappa; immunoglobulin lambda light chain variable region is V _λ 。

Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used in accordance with function. The light chain variable region (VL) and the heavy chain variable region (VH) determine antigen recognition and specificity. The light chain constant region (CL) and the heavy chain constant region (CH) confer important biological properties such as secretion, transplacental movement, Fc receptor binding, complement fixation, etc. By convention, the numbering of constant regions increases as they become further away from the antigen binding site or amino terminus of the antibody. The N-terminal part is a variable region and the C-terminal part is a constant region; the CH3 and CL domains actually comprise the carboxy-termini of the heavy and light chains, respectively.

Where two or more definitions are provided for a term used and/or accepted in the art, the definition of the term as used herein includes all such meanings unless explicitly stated to the contrary. One specific example is the use of the term "complementarity determining regions" ("CDRs") to describe non-contiguous antigen binding sites found within the variable regions of heavy and light chain polypeptides. This particular region is described in Kabat et al, U.S. Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and Chothia et al, J.mol.biol.196:901-917 (1987), which are incorporated herein by reference in their entirety.

CDRs defined according to Kabat and Chothia include overlaps or subsets of amino acid residues when compared to each other. Nevertheless, it is within the scope of the invention to apply either definition to refer to the CDRs of an antibody or variant thereof. The exact residue number comprising a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can generally determine which specific residues a CDR contains based on the amino acid sequence of the variable region of an antibody.

Kabat et al also defines a numbering system for the variable region sequences applicable to any antibody. One of ordinary skill in the art can apply this "Kabat numbering" system to any variable region sequence without relying on other experimental data beyond the sequence itself. "Kabat numbering" refers to the numbering system proposed by Kabat et al, U.S. Dept. of Health and Human Services at "Sequence of proteins of Immunological Interest" (1983). Antibodies may also be used with the EU or Chothia numbering system.

"treatment" refers to both therapeutic treatment and prophylactic or preventative measures, with the object of preventing, slowing, ameliorating, and halting undesirable physiological changes or disorders, such as the progression of a disease, including, but not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment or elimination (whether partial or total), extending the expected life span when not treated, and the like, whether detectable or undetectable. Patients in need of treatment include patients already with a condition or disorder, patients susceptible to a condition or disorder, or patients in need of prevention of the condition or disorder, patients who may or are expected to benefit from administration of the antibodies or compositions disclosed herein for detection, diagnostic procedures, and/or treatment.

"patient" refers to any mammal in need of diagnosis, prognosis or treatment, including humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, etc. In some embodiments, the patient is a human.

"about" refers to the conventional error range for corresponding numerical values as would be readily understood by one of ordinary skill in the relevant art. In some embodiments, reference herein to "about" refers to the numerical values recited and ranges of ± 10%, ± 5%, or ± 1% thereof.

"effective amount" refers to an amount of an active compound or agent that elicits a biological or medical response in a tissue, system, animal, subject, or human; an effective amount is sought by a researcher, veterinarian, medical doctor or other clinician.

“IC ₅₀ "means the 50% inhibitory concentration, i.e., the concentration of drug or inhibitor required to inhibit half of a given biological process.

As used herein, the term "in need thereof refers to a patient having been identified as in need of a particular method or treatment. In some embodiments, identification may be by any diagnostic means. In any of the methods and treatments described herein, a patient may be in need thereof.

DNA encoding the antibody can be synthesized according to the amino acid sequence design of the antibody described herein by a conventional method, and placed into an expression vector, followed by transfection of host cells, and culture of the transfected host cells in a medium to produce monoclonal antibodies. In some embodiments, the expression antibody vector comprises at least one promoter element, an antibody coding sequence, a transcription termination signal, and a polyA tail. Other elements include enhancers, Kozak sequences and donor and acceptor sites for RNA splicing on both sides of the insert. High transcription efficiency can be achieved by the early and late promoters of SV40, long terminal repeats from retroviruses such as RSV, HTLV1, HIVI and the early promoters of cytomegalovirus, and other cellular promoters such as actin can also be used. Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or Plncx, pcDNA3.1(+/-), pcDNA/Zeo (+/-), pcDNA3.1/Hygro (+/-), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI, pCS2, and the like. Commonly used mammalian cells include HEK293 cells, Cos1 cells, Cos7 cells, CV1 cells, murine L cells, CHO cells, and the like.

Detailed Description

The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 preparation of antibodies

The heavy and light chain DNA sequences of the antibodies were optimized according to CHO codon preference. Modifying the 5 'end of the DN A sequence by using a PCR primer, adding a kozak sequence and a signal peptide DNA sequence at the 5' ends of the light chain and heavy chain DNA sequences, cloning the sequences into the existing expression vector, and verifying the correct construction of the recombinant plasmid through sequencing analysis. The recombinant plasmid was transfected into expression cells for expression, and the supernatant was collected and purified to obtain an antibody protein sample, which was used in the following examples.

Wherein, 1) in the preparation process of the antibody h10D8OF, the adopted expression vector is pCDNA3.1 ^TM (+) (Invitr ogen, cat # V79020) and the expression cells were CHO cells; 2) in the preparation process of the antibody h10D8OFKF, the adopted expression vector is pCDNA3.1 ^TM (+), the expressing cells were alpha- (1,6) -fucosyltransferase gene knock-out CHO cells tested for fucosylation levels of about 0; 3) in the preparation process of the reference antibody Tiragolumab, the adopted expression vector is pCDNA3.1 ^TM (+), the expression cells were CHO cells. The amino acid sequences of the antibodies h10D8OF and h10D8OFKF are shown in table 1 (the sequences of the antibodies h10D8OF and h10D8OFKF are identical), the amino acid sequence of the reference antibody Tiragolumab is shown in table 2, and the DNA sequences of the antibodies h10D8OF and h10D8OFKF are shown in table 3 (the sequences of the antibodies h10D8OF and h10D8OFKF are identical).

The DNA sequence added to the 5' -end of the light chain DNA sequence of antibodies h10D8OF and h10D8OFKF was

As shown in SEQ ID NO 19, the kozak sequence is underlined and the signal peptide is in italics; the DNA sequence added to the 5' end of the heavy chain DNA sequence is

As shown in SEQ ID NO:20, the kozak sequence is underlined and the signal peptide is in italics. The DNA sequence added to the 5' -end of the light chain DNA sequence of the reference antibody Tira golumab is

As shown in SEQ ID NO:21, the kozak sequence is underlined and the signal peptide is in italics; the DNA sequence added to the 5' end of the heavy chain DNA sequence is

As shown in SEQ ID NO:22, the ko zak sequence is underlined and the signal peptide is in italics.

Amino acid sequences of the antibodies h10D8OF and h10D8OFKF of Table 1

TABLE 2 amino acid sequence of the reference antibody Tiragolumab

TABLE 3 DNA sequences of antibodies h10D8OF and h10D8OFKF

Example 2 blocking TIGIT binding assay to ligand PVR

Flow cytometry was used to detect the binding of anti-TIGIT antibody blocking free PVR-Fc to TIGIT on the surface of TIGIT-Jurkat cells. The test steps are as follows: taking TIGIT-Jurkat cells with good activity (the cell activity is more than 90 percent), centrifuging, then resuspending the cells into the density of 1000 ten thousand/ml by PBS, adding the cells into a 96-hole sharp bottom plate, wherein each hole has 50 mu l, namely the number of the cells in each hole is 50 ten thousand; taking a proper amount of biotinylated PVR-Fc (namely PVR-Fc-bio), diluting with PBS (phosphate buffer solution) to prepare PVR-Fc-bio diluent with the final concentration of 12.5 nM; taking a proper amount of anti-TIGIT antibody or a reference antibody Tiragolumab, diluting with PVR-Fc-bio diluent, wherein the initial concentration of the antibody is 200 nM, diluting with 2-fold gradient to obtain 10 concentration gradients, and arranging 3 compound holes at each concentration point; incubating at 2-8 deg.C for 1 hr, washing with PBS for 2 times, adding diluted 1:1000 diluted fluorescent secondary antibody Streptavidin-PE (eBioscience, CAT #12-4317-87) diluent, incubating at 2-8 deg.C for 30min and with 100 μ l per well; PBS was then washed 2 times and the fluorescence intensity was measured using a flow analyzer (Mean PE-A).

The preparation method of the PVR-Fc-bio comprises the following steps: the nucleic acid sequence of the human PVR extracellular region is added with enzyme cutting sites (HindIII and EcoRI), and is fused with the nucleic acid sequence of the human IgG1 heavy chain constant region through a linker; the sequences after the fusion were inserted into pCDNA3.1 ^TM (+) vector, then transiently transfected into HEK293F cells; purifying the cultured cell supernatant through ProteinA affinity chromatography, and obtaining a fusion protein named as PVR-Fc through purification; taking a proper amount of PVR-Fc protein,biotin labeling kit (Biotin labeling kit) from Thermo scientific Co Ltd

HSulfo-NHS-LC-Biotinylationkit, cat #: 21435) The PVR-Fc was biotinylated according to the protocol described in the specification, and the labeled protein was named PVR-Fc-bio. Wherein, the amino acid sequence of the human PVR extracellular region is shown as SEQ ID NO. 13, the nucleotide sequence of the linker is shown as SEQ ID NO. 14, the amino acid sequence of the linker is shown as SEQ ID NO. 23, the amino acid sequence of the human IgG1 heavy chain constant region is shown as SEQ ID NO. 15, and the amino acid sequence of the PVR-Fc is shown as SEQ ID NO. 24 (see Table 4).

The preparation method of the TIGIT-Jurkat cell comprises the following steps: human full-length TIGIT gene was used to replace the objective gene on pCMV2-CFD-Flag (Shenzhou, Yinqiao, Cat.: HG10160-M-F) to obtain a recombinant plasmid, the recombinant plasmid was linearized with restriction enzyme ClaI (Bsu15I), and then transfected into Jurkat cell line (ATCC, Clone E6-1, TIB-152) ^TM ). Screening hygromycin, obtaining a positive cell strain, and then subcloning to obtain a TIGIT cell line capable of stably expressing human, namely: TIGIT-Jurkat cells. Wherein, the sequence of the human full-length TIGIT gene is shown as SEQ ID NO:16 (see Table 4).

1) Under the same conditions, the antibody h10D8OF and the antibody Tiragolumab can effectively block the combination of TIGIT and PVR-Fc, and the IC of the two antibodies ₅₀ Values were 0.4409nM and 2.820nM, respectively; the blocking ability of antibody h10D8OF was superior to that of antibody Tiragolumab.

2) Under the same conditions, the antibody h10D8OFKF and the antibody Tiragolumab can effectively block the combination of TIGIT and PVR-Fc, and the IC of the two antibodies ₅₀ Values were 0.742nM and 2.820nM, respectively; the blocking ability of the antibody h10D8OFKF is superior to that of the antibody Tiragolumab.

TABLE 4 related sequences

Example 3 in vivo drug efficacy test

Humanized mouse BALB/c-hPD1/hTIGIT (Jiangsu Jiejiao kang Biotech Co., Ltd.) was inoculated subcutaneously with CT26 colon cancer tumor cells; after inoculation of tumor cells, when the mean tumor volume of the mice was 79.65mm ³ At times, groups were made of 10 individuals each. Grouping the day is defined as D0 days, and the administration is carried out by intraperitoneal injection (I.P.) on days D0, D4, D7, D11, D14 and D18, and the administration dosage is 10mg/kg or 30 mg/kg. The dosing regimen is shown in table 5.

TABLE 5 dosing regimen

After cell inoculation, the effect of tumors on the normal behavior of animals was routinely monitored weekly; the specific indexes comprise: mobility of the mice, feeding and drinking conditions, weight gain or loss, eyes, hair and other abnormalities. Tumor volume (mm) ³ ) 0.5X (tumor major diameter. times. tumor minor diameter) ² ) Tumor volumes of the mice in each group are expressed as mean ± standard error (mean ± SEM). TGItw (tumor weight inhibition rate) is calculated as:

TGItw＝(1-(mean TW _{administration set} )/(mean TW _{Control group} ))×100％；Mean TW _{Administration set} : mean value of tumor weight at end-point treatment, Mean TW, in mice of the administration group _{Control group} : mean value of tumor weight at end-point treatment of control mice.

1) As shown in fig. 1A, both antibody h10D8OF and antibody Tiragolumab can inhibit the growth of CT26 colon cancer, and antibody h10D8OF has better effect in inhibiting tumor growth than antibody Tiragolumab; at the 30mg/kg dose, the relative tumor inhibition rate TGItw (%) at the test end-point (D20 days) was 92.89% for the former and 71.07 for the latter.

2) As shown in fig. 1B, both the antibody h10D8OFKF and the antibody Tiragolumab can inhibit the growth of CT26 colon cancer, and the effect of the antibody h10D8OFKF in inhibiting the growth of tumor was superior to that of the antibody Tiragolumab. The relative tumor inhibition TGItw (%) at the end of the experiment (D20 days) was 92.39%, 95.94% and 71.07% at the 10mg/kg and 30mg/kg doses of h10D8OFKF and at the 30mg/kg dose of Tiragolumab, respectively.

Example 4 pharmacokinetic and toxicology testing

4a) 18 cynomolgus monkeys (half female and half male) were used in this study and randomly divided into 3 groups of 3 female and male animals per group; animals in each group were not fasted and given 2, 12 and 24mg/kg antibody (antibody h10D8OF or h10D8OFKF) injections, respectively, by single intravenous infusion for 1 total, with intravenous infusion for 30min ± 10 s; blood samples were collected for 29 days after the end of dosing. By means of blood concentration data, Phoenix is applied

7.0 software used a non-compartmental model to calculate its main pharmacokinetic parameters.

As shown in table 6 and table 7, there was no significant sex difference in the systemic exposure of antibodies (antibodies h10D8OF and h10D8OFKF) in serum after single intravenous infusion administration to cynomolgus monkeys in the range of 2-24mg/kg administration dose, and the systemic exposure in serum was positively correlated with the administration dose.

TABLE 6 pharmacokinetic parameters of cynomolgus monkeys after administration of the antibody h10D8OF injection

TABLE 7 pharmacokinetic parameters after cynomolgus monkey administration of antibody h10D8OFKF injection

4b) And (4) observing the acute toxic reaction of the cynomolgus monkey after intravenous infusion administration of the anti-TIGIT antibody. The experiment was designed into 4 groups of 1 female each. The administration dosage is 0, 100, 200, 400mg/kg respectively, the intravenous infusion administration is carried out, the administration is carried out once, and the infusion time is 30min +/-10 s; observations were continued for 22 days after dosing.

After the injection of 100, 200 and 400mg/kg antibody (antibody h10D8OF or h10D8OFKF) is administered to the cynomolgus monkey by single intravenous infusion for 30min, the cynomolgus monkey can better tolerate the injection; compared with the control group at the same period, in all the dosage groups of the antibody h10D8OFKF injection, the consumption of food related to the antibody test article is reduced after the cynomolgus monkey is dosed, and then the cynomolgus monkey shows a recovery trend and lacks of relevance and is not considered to be harmful; the antibody h10D8OF dose groups have no obvious abnormal change of food consumption; no other survival rate, clinical observation, body weight, body temperature, electrocardiogram, clinical pathology and macroscopic anatomical changes related to the antibody test sample appear. Thus, under the conditions of this test, the Maximum Tolerated Dose (MTD) of an intravenous single administration of an antibody injection is greater than 400 mg/kg.

4c) Cynomolgus monkeys were administered an injection of antibody (antibody h10D8OF or h10D8OFKF) by intravenous infusion once a week for a total of five replicates to assess toxicity responses and their pharmacokinetics; after four weeks of recovery, the lesions were observed for reversible events or possible delayed toxic reactions. After the dosing was completed, 3 animals per sex were dissected per group, and the recovery period was completed and the remaining 2 animals per sex were dissected per group.

As shown in tables 8 and 9, the exposure (in AUC) of the antibodies (antibodies h10D8OF or h10D8OFK) in the serum after the first and 4 th administrations _(0-t) Meter) increases with increasing dose. During the test period, no abnormal change in the body weight, food consumption, stimulation of administration site, ophthalmology, respiration, hematology, serum biochemistry, blood coagulation, urine, cytokine, lymphocyte phenotype, organ weight, etc. associated with the administration of the antibody (antibody h10D8OF or h10D8OFK) was observed in any of the test animals.

After intravenous administration of 10, 30, 100mg/kg of the antibody h10D8OF or h10D8OFKF injection solution 5 times, the antibody test article did not cause significant or severe toxic reaction, and it was assumed that the dose level (NOAEL) at which no damaging effect was observed with the antibody h10D8OF and h10D8OFKF injection solutions was 100 mg/kg.

The immunogenicity results show that 1) after the cynomolgus monkey is given 10, 30 and 100mg/kg of antibody h10D8OF in the repeated administration toxicity test, the positive rates of the anti-drug antibody (ADA) of 10 and 30mg/kg dose groups are respectively 50 percent and 25 percent, and the ADA is not detected in the high dose group; 2) after 10, 30 and 100mg/kg of antibody h10D8OFKF is administered to the cynomolgus monkey in the repeated administration toxicity test, the positive rates of the anti-drug antibody in the 10, 30 and 100mg/kg dose groups are 70%, 33% and 50%, respectively.

TABLE 8 toxicity kinetics parameters for antibody h10D8OF in cynomolgus monkey Long-term toxicity test

TABLE 9 toxicity kinetics parameters of h10D8OFKF in cynomolgus monkey Long-term toxicity test

4d) Hemolytic assays were performed using New Zealand rabbit red blood cells. When the injection (25mg/mL) of the antibody (antibody h10D8OF or h10D8OFKF) was diluted more than 10-fold, it did not cause hemolysis or agglutination of rabbit erythrocytes.

Example 5 clinical Studies of antibodies h10D8OF and h10D8OFKF

The study is two multicenter, open, dose escalating phase I clinical trials evaluating the safety, tolerability, Pharmacokinetic (PK) profile and preliminary clinical efficacy of antibody h10D8OF and h10D8OFKF injections in patients with advanced malignant solid tumors, respectively; the Maximum Tolerated Dose (MTD) or Maximum Administered Dose (MAD) was explored.

The clinical study of antibody h10D8OF was divided into two phases, the first phase was based on an accelerated titration method and a "3 + 3" dose escalation rule to explore safe dose ranges, which were mainly divided into: dose escalation studies were performed in the 30mg (initial dose) and 100mg groups using the accelerated titration method; carrying out dose increasing research on the 300mg group, the 600mg group and the 900mg group according to the standard 3+3 rule; and in the second stage, dose extension research is carried out, and the safety and clinical effectiveness of the antibody injection are further researched.

The clinical study of the antibody h10D8OFKF was divided into two phases, the first phase was based on the accelerated titration method and the dose escalation rule of "3 + 3" to explore the safe dose range, which was mainly divided into: the 10mg (initial dose) group, the 30mg group and the 100mg group were subjected to dose escalation studies using an accelerated titration method; carrying out dose increasing research on the 300mg group, the 600mg group and the 900mg group according to the standard 3+3 rule; and in the second stage, dose extension research is carried out, and the safety and clinical effectiveness of the antibody injection are further researched.

Three groups used a dose escalation protocol with accelerated titration, i.e., cohort size 1. The first 1 subject was included and if no DLT event was observed during the DLT assessment period, the next group could be entered directly for dose escalation, by analogy with this rule ("1 + 1"), to accelerate the escalation to the (300mg) dose group. During the accelerated dose escalation process, if grade 2 or greater toxic responses associated with study drug were observed in a certain group, the accelerated dose escalation was stopped and the standard "3 + 3" dose escalation (i.e., cohort size 3) rule was followed.

The medicine is administered by intravenous infusion for 1 time every 3 weeks for 1 cycle (treatment course), and the infusion time can be more than or equal to 60 minutes. If the patient has infusion-related reactions and is able to continue treatment, a prophylactic administration of diphenhydramine or acetaminophen can be used based on clinical practice. Study drug treatment should continue until appearance: disease progression, or withdrawal from intolerable toxicity, or acceptance of new anti-tumor therapy for lack of therapeutic benefit, or withdrawal of informed consent and other reasons for active withdrawal, or a maximum of 17 cycles (about 1 year), whichever comes first. DLT evaluation period: treatment cycle 1 (from first dose to 21 days post dose).

The tolerance evaluation index relates to: dose-limiting toxicity (DLT) events and their incidence; the safety evaluation index relates to: vital signs and physical examination, laboratory examination (hematology, hematobiochemistry, thyroid function, clotting routine, urine routine, stool routine, pregnancy test), ECOG score, electrocardiogram, adverse events (including immune related adverse events), and the like.

Pharmacodynamic Receptor Occupancy (RO) study: the receptor occupancy studies of antibody injections were performed by detecting T cell surface TIGIT receptor binding in peripheral blood. Subjects in all dose groups required blood sample collection at a specific time point during treatment. Pharmacodynamic receptor occupancy studies were performed only in dose-escalating subjects. 2mL blood samples were collected at each time point scheduled for 1 cycle pre-dose, end of dose, 168h, 336h intensive sampling, and 2 to 6 cycle pre-dose sampling, respectively.

The immunogenicity evaluation index involves: the sample positive rate and individual positive rate of the anti-drug antibody (ADA), the titer of the ADA positive sample, and whether the ADA positive sample will be tested for neutralizing antibody (nab) will continue.

Subjects in all dose groups required blood sample collection at the prescribed time point during the treatment period (first 3 treatment cycles). Plasma concentrations (C) were monitored for 60 min prior to dosing _trough ). Blood samples of 2mL were planned for each time point, serum drug concentration levels were tested, and Pharmacokinetic (PK) profiles were studied. The parameters involved in PK are: in a single administration, the parameters include C _max 、T _max 、T _1/2 、CL、Vd、Ke、MRT、AUC _(0-τ) 、AUC _(0-∞) (ii) a When administered multiple times, the parameters include C _max,ss 、C _avg,ss 、C _min,ss 、AUC _(0-τ) ,ss、AU _C(0-∞) ,ss、Tmax,ss、T _1/2 Ss, CL, Vss, Ke, MRT, accumulation index (Rac), and fluctuation index DF.

And (3) evaluating clinical effectiveness: objective Remission Rate (ORR), duration of remission (DOR), Disease Control Rate (DCR), Progression Free Survival (PFS), and Overall Survival (OS).

Objective Response Rate (ORR): proportion of subjects in Complete Remission (CR) and Partial Remission (PR); duration of mitigation (DOR): the duration of ORR is reflected in the time from the first assessment of Objective Remission (OR) to the first assessment of PD (progressive disease) OR death of any cause prior to PD; Progression-Free Survival (Progression-Free Survival: PFS): the time from first administration to the appearance of objective progression or all-cause death of the tumor (whichever occurred first). The "MTD" is the highest dose level of DLT explored in subjects with a dose group observing ≦ 1/6 during the DLT assessment period.

"dose-limiting toxicity (DLT)" is an Adverse Event (AE) that occurs during the DLT observation period and is considered to be at least likely related to the study drug, as follows:

grade 5 toxicity;

■ hematological toxicity:

grade 4 hematological toxicity (note: grade 3-4 lymphopenia did not include DLT);

grade 4 thrombocytopenia for any duration;

grade 3 thrombocytopenia with bleeding tendency or requiring platelet infusion;

grade 4 neutropenia for any duration;

grade 3 neutropenia with infection or duration ≥ 7 days or grade 3 neutropenia with fever;

■ non-hematologic toxicity:

grade 3, 4 non-hematologic toxicities (note: except 3 grade nausea, vomiting, rash; except 3 grade diarrhea which can be controlled within 3 days after clinical intervention; except fatigue/hypodynamia within 7 days);

grade 3, 4 non-hematological toxicity, found by clinical laboratory examination, meeting any of the following conditions: firstly, clinical intervention is needed; ② hospitalization is induced; duration is more than or equal to 7 days; all resulting in cessation of treatment in cycle 1; any treatment-related toxicity that results in a delay of more than 2 weeks for cycle 2 administration;

not less than grade 2 cerebral lesions;

eye toxicity to be treated systemically (note: any patient with vision changes or eye toxicity of grade 2 or higher will be evaluated by an ophthalmologist);

in the determination of DLT, some obvious and undisputed AEs due to disease progression or causes other than DLT should not be counted in DLT. Subjects who withdraw due to non-DLT causes (such as adverse events clearly associated with underlying disease, disease progression, concomitant medication or concomitant disease) during the 21-day DLT observation period will be considered unevaluable and will be replaced. If the subject is actively withdrawn from the study, the last visit should be completed as soon as possible and as required. Subjects who were replaced were included in the SS pool when counted, but were not included in the cohort population when the percentage of DLT events occurred was calculated. After agreement between the sponsor and the investigator, repeated screening of a subset of subjects was allowed, requiring reassignment of screening numbers.

Sequence listing

<110> Baiotai biopharmaceutical GmbH

Application of <120> anti-TIGIT antibody in treatment of tumor or cancer

<150> PCT/CN2021/078724

<151> 2021-03-02

<160> 24

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Ser Tyr Gly Met Ser

1 5

<210> 2

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Thr Ile Asn Ser Asn Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val Lys

1 5 10 15

Gly

<210> 3

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Leu Gly Thr Gly Thr Leu Gly Phe Ala Tyr

1 5 10

<210> 4

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Lys Ala Ser Gln Asp Val Lys Thr Ala Val Ser

1 5 10

<210> 5

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Trp Ala Ser Thr Arg Ala Thr

1 5

<210> 6

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Gln Gln His Tyr Ser Thr Pro Trp Thr

1 5

<210> 7

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Leu Val

35 40 45

Ala Thr Ile Asn Ser Asn Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Gly Thr Gly Thr Leu Gly Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 8

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Asp Val Lys Thr Ala

20 25 30

Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 9

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Leu Val

35 40 45

Ala Thr Ile Asn Ser Asn Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Gly Thr Gly Thr Leu Gly Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 10

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Asp Val Lys Thr Ala

20 25 30

Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 11

<211> 456

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Lys Thr Tyr Tyr Arg Phe Lys Trp Tyr Ser Asp Tyr Ala

50 55 60

Val Ser Val Lys Gly Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Phe Tyr Cys Thr Arg Glu Ser Thr Thr Tyr Asp Leu Leu Ala Gly Pro

100 105 110

Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

115 120 125

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

130 135 140

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

145 150 155 160

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

165 170 175

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

180 185 190

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

195 200 205

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val

210 215 220

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

225 230 235 240

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

245 250 255

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

260 265 270

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

275 280 285

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

290 295 300

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

305 310 315 320

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

325 330 335

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

340 345 350

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

355 360 365

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

370 375 380

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

385 390 395 400

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

405 410 415

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

420 425 430

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

435 440 445

Ser Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 12

<211> 220

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Thr Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Lys Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Asn Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ser Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 13

<211> 343

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Met Ala Arg Ala Met Ala Ala Ala Trp Pro Leu Leu Leu Val Ala Leu

1 5 10 15

Leu Val Leu Ser Trp Pro Pro Pro Gly Thr Gly Asp Val Val Val Gln

20 25 30

Ala Pro Thr Gln Val Pro Gly Phe Leu Gly Asp Ser Val Thr Leu Pro

35 40 45

Cys Tyr Leu Gln Val Pro Asn Met Glu Val Thr His Val Ser Gln Leu

50 55 60

Thr Trp Ala Arg His Gly Glu Ser Gly Ser Met Ala Val Phe His Gln

65 70 75 80

Thr Gln Gly Pro Ser Tyr Ser Glu Ser Lys Arg Leu Glu Phe Val Ala

85 90 95

Ala Arg Leu Gly Ala Glu Leu Arg Asn Ala Ser Leu Arg Met Phe Gly

100 105 110

Leu Arg Val Glu Asp Glu Gly Asn Tyr Thr Cys Leu Phe Val Thr Phe

115 120 125

Pro Gln Gly Ser Arg Ser Val Asp Ile Trp Leu Arg Val Leu Ala Lys

130 135 140

Pro Gln Asn Thr Ala Glu Val Gln Lys Val Gln Leu Thr Gly Glu Pro

145 150 155 160

Val Pro Met Ala Arg Cys Val Ser Thr Gly Gly Arg Pro Pro Ala Gln

165 170 175

Ile Thr Trp His Ser Asp Leu Gly Gly Met Pro Asn Thr Ser Gln Val

180 185 190

Pro Gly Phe Leu Ser Gly Thr Val Thr Val Thr Ser Leu Trp Ile Leu

195 200 205

Val Pro Ser Ser Gln Val Asp Gly Lys Asn Val Thr Cys Lys Val Glu

210 215 220

His Glu Ser Phe Glu Lys Pro Gln Leu Leu Thr Val Asn Leu Thr Val

225 230 235 240

Tyr Tyr Pro Pro Glu Val Ser Ile Ser Gly Tyr Asp Asn Asn Trp Tyr

245 250 255

Leu Gly Gln Asn Glu Ala Thr Leu Thr Cys Asp Ala Arg Ser Asn Pro

260 265 270

Glu Pro Thr Gly Tyr Asn Trp Ser Thr Thr Met Gly Pro Leu Pro Pro

275 280 285

Phe Ala Val Ala Gln Gly Ala Gln Leu Leu Ile Arg Pro Val Asp Lys

290 295 300

Pro Ile Asn Thr Thr Leu Ile Cys Asn Val Thr Asn Ala Leu Gly Ala

305 310 315 320

Arg Gln Ala Glu Leu Thr Val Gln Val Lys Glu Gly Pro Pro Ser Glu

325 330 335

His Ser Gly Ile Ser Arg Asn

340

<210> 14

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Ala Thr Thr Gly Ala Ala Gly Gly Thr Ala Gly Ala Ala Thr Gly Gly

1 5 10 15

Ala Thr

<210> 15

<211> 330

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 16

<211> 735

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

atgcgctggt gtctgctgct gatttgggcc cagggactga gacaggctcc tctggcttca 60

ggaatgatga ccggcaccat cgagaccacc ggaaacatca gcgccgagaa gggaggaagc 120

atcatcctcc agtgccacct gagtagcaca accgcacagg tcacccaggt caattgggag 180

cagcaggacc agctgctggc catttgcaac gccgatctgg gttggcacat ctctcctagc 240

ttcaaggaca gagtggcccc aggaccagga ctgggactga cactgcagag tctgaccgtg 300

aacgacaccg gcgagtactt ctgcatctac cacacctacc cagacggcac ctacacagga 360

cggatcttcc tggaggtgct ggagtctagc gtggcagagc acggagccag attccagatc 420

cctctgctgg gagctatggc agctacactg gtcgtgatct gcaccgcagt gatcgtggtc 480

gtggctctga cacggaagaa gaaggccctg agaatccaca gcgtggaggg agacctgaga 540

agaaagagcg ccggacagga ggagtggtct cctagcgctc cttctcctcc aggctcttgt 600

gtgcaggcag aagcagctcc agcaggtctc tgcggagaac agagaggaga ggattgcgcc 660

gagctgcacg actacttcaa cgtgctgagc taccggagcc tgggcaattg cagcttcttc 720

accgagaccg gatga 735

<210> 17

<211> 1350

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

caggtgcagc tggtggagtc cggcggcgga gtggtgcagc ctggaaggtc cctgagactg 60

gactgtaagg ccagcggctt cacctttagc agctacggca tgagctgggt gagacaggcc 120

cctggcaagg gcctggagct ggtggctacc atcaatagca atggcggcag cacctactac 180

cccgacagcg tgaagggcag attcactatc agcagagaca actccaagaa taccctgttc 240

ctgcagatga atagcctgag agccgaggac accgccgtgt actactgcgc caggctgggc 300

accggcaccc tgggatttgc ctactggggc cagggtaccc tggttaccgt tagcagcgcg 360

agcaccaaag gcccgagcgt gtttccgctg gccccgagca gcaaaagcac cagcggtggc 420

accgcagcgc tgggttgcct ggtgaaagat tatttcccgg aaccggtgac ggtgtcgtgg 480

aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 540

ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 600

atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt tgagcccaaa 660

tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 720

tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 780

gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 840

gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 900

acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 960

tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1020

gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggatgagctg 1080

accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1140

gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1200

gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag 1260

caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1320

aagagcctct ccctgtctcc gggtaaatga 1350

<210> 18

<211> 645

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gagatcgtga tgacccagag ccccgccacc ctgtccctga gcccaggaga gagagccacc 60

ctgagctgca aggcctccca ggacgtgaag accgccgtga gctggtatca acagaagcct 120

ggccaggccc ccagactgct gatctactgg gcctccacca gggccaccgg catccctgct 180

agattcagcg gctccggctc cggcaccgat tacaccctga ccatcagcag cctggagcct 240

gaggatttcg ccgtgtacta ctgtcagcag cactactcca ccccttggac cttcggccag 300

ggcaccaagg tggagatcaa gcgtacggtg gctgcaccat ctgtcttcat cttcccgcca 360

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttga 645

<210> 19

<211> 75

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gccgccacca tggactttca ggtgcagatc atctccttcc tgctgatcag cgcctccgtg 60

atcatgtcca ggggc 75

<210> 20

<211> 66

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

gccgccacca tgggctggag cctgatcctg ctgttcctgg tggccgtggc caccagagtg 60

ctgtcc 66

<210> 21

<211> 75

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gccgccacca tggacatgag ggtgctggcc cagctgctgg gactgctgct gctgtgcttc 60

ccaggcgcca gatgc 75

<210> 22

<211> 66

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

gccgccacca tggagtttgg gctgagctgg gttttccttg ttgctatatt aaaaggtgtc 60

cagtgt 66

<210> 23

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

Ile Glu Gly Arg Met Asp

1 5

<210> 24

<211> 659

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

Trp Pro Pro Pro Gly Thr Gly Asp Val Val Val Gln Ala Pro Thr Gln

1 5 10 15

Val Pro Gly Phe Leu Gly Asp Ser Val Thr Leu Pro Cys Tyr Leu Gln

20 25 30

Val Pro Asn Met Glu Val Thr His Val Ser Gln Leu Thr Trp Ala Arg

35 40 45

His Gly Glu Ser Gly Ser Met Ala Val Phe His Gln Thr Gln Gly Pro

50 55 60

Ser Tyr Ser Glu Ser Lys Arg Leu Glu Phe Val Ala Ala Arg Leu Gly

65 70 75 80

Ala Glu Leu Arg Asn Ala Ser Leu Arg Met Phe Gly Leu Arg Val Glu

85 90 95

Asp Glu Gly Asn Tyr Thr Cys Leu Phe Val Thr Phe Pro Gln Gly Ser

100 105 110

Arg Ser Val Asp Ile Trp Leu Arg Val Leu Ala Lys Pro Gln Asn Thr

115 120 125

Ala Glu Val Gln Lys Val Gln Leu Thr Gly Glu Pro Val Pro Met Ala

130 135 140

Arg Cys Val Ser Thr Gly Gly Arg Pro Pro Ala Gln Ile Thr Trp His

145 150 155 160

Ser Asp Leu Gly Gly Met Pro Asn Thr Ser Gln Val Pro Gly Phe Leu

165 170 175

Ser Gly Thr Val Thr Val Thr Ser Leu Trp Ile Leu Val Pro Ser Ser

180 185 190

Gln Val Asp Gly Lys Asn Val Thr Cys Lys Val Glu His Glu Ser Phe

195 200 205

Glu Lys Pro Gln Leu Leu Thr Val Asn Leu Thr Val Tyr Tyr Pro Pro

210 215 220

Glu Val Ser Ile Ser Gly Tyr Asp Asn Asn Trp Tyr Leu Gly Gln Asn

225 230 235 240

Glu Ala Thr Leu Thr Cys Asp Ala Arg Ser Asn Pro Glu Pro Thr Gly

245 250 255

Tyr Asn Trp Ser Thr Thr Met Gly Pro Leu Pro Pro Phe Ala Val Ala

260 265 270

Gln Gly Ala Gln Leu Leu Ile Arg Pro Val Asp Lys Pro Ile Asn Thr

275 280 285

Thr Leu Ile Cys Asn Val Thr Asn Ala Leu Gly Ala Arg Gln Ala Glu

290 295 300

Leu Thr Val Gln Val Lys Glu Gly Pro Pro Ser Glu His Ser Gly Ile

305 310 315 320

Ser Arg Asn Ile Glu Gly Arg Met Asp Ala Ser Thr Lys Gly Pro Ser

325 330 335

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

340 345 350

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

355 360 365

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

370 375 380

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

385 390 395 400

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

405 410 415

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

420 425 430

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

435 440 445

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

450 455 460

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

465 470 475 480

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

485 490 495

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

500 505 510

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

515 520 525

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

530 535 540

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

545 550 555 560

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

565 570 575

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

580 585 590

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

595 600 605

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

610 615 620

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

625 630 635 640

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

645 650 655

Pro Gly Lys

Claims (10)

1. A method for treating a tumor or cancer, comprising: administering to a patient in need thereof an effective amount of an anti-TIGIT antibody or antigen-binding fragment, the effective amount being 9mg to 1200mg per treatment cycle;

the anti-TIGIT antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 2, HCDR3 shown in SEQ ID NO. 3, LCDR1 shown in SEQ ID NO. 4, LCDR2 shown in SEQ ID NO. 5, and LCDR3 shown in SEQ ID NO. 6.

2. The method of claim 1, wherein the anti-TIGIT antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein:

the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 7, or an amino acid sequence with at least 80% of identity compared with the sequence shown in SEQ ID NO. 7, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 7; and/or

The light chain variable region comprises the sequence shown in SEQ ID NO. 8, or an amino acid sequence with at least 80% identity compared with the sequence shown in SEQ ID NO. 8, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 8.

3. The method of claim 1 or 2, the heavy chain of the anti-TIGIT antibody comprises the amino acid sequence set forth in SEQ ID No. 9, or an amino acid sequence having at least 80% identity compared to the sequence set forth in SEQ ID No. 9, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence set forth in SEQ ID No. 9; and/or

The light chain of the anti-TIGIT antibody comprises the amino acid sequence shown in SEQ ID NO. 10, or an amino acid sequence with at least 80% of identity compared with the sequence shown in SEQ ID NO. 10, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 10.

4. The method of any one of claims 1-3, wherein the anti-TIGIT antibody or antigen binding fragment has a level of fucosylation of 0-10%.

5. The method of any one of claims 1-4, wherein the anti-TIGIT antibody or antigen-binding fragment is expressed by an α - (1,6) -fucosyltransferase gene knockout cell; alternatively, the anti-TIGIT antibody or antigen binding fragment is expressed by α - (1,6) -fucosyltransferase gene knock-out CHO cells.

6. The method of any one of claims 1-5, wherein the tumor or cancer is selected from the group consisting of acute lymphocytic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, myeloproliferative diseases/tumors, Hodgkin's lymphoma, indolent and aggressive non-Hodgkin's lymphoma, Burkitt's lymphoma, follicular lymphoma, multiple myeloma, giant cell myeloma, heavy chain myeloma, light chain or Bense-Jones myeloma, breast cancer, ovarian cancer, pancreatic cancer, prostate cancer, melanoma, colorectal cancer, colon cancer, lung cancer, head and neck cancer, bladder cancer, esophageal cancer, liver cancer, and kidney cancer.

7. The method of any one of claims 1-6, wherein one treatment cycle is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, or 7 weeks.

8. The method of any one of claims 1-7, wherein the single dose amount of the anti-TIGIT antibody or antigen-binding fragment is from 0.01mg/kg to 26 mg/kg.

9. The method of any one of claims 1-8, wherein the administration is intravenous or subcutaneous.

10. A kit comprising an anti-TIGIT antibody or antigen-binding fragment and instructions for directing administration of the anti-TIGIT antibody or antigen-binding fragment to a patient in need thereof;

the anti-TIGIT antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 2, HCDR3 shown in SEQ ID NO. 3, LCDR1 shown in SEQ ID NO. 4, LCDR2 shown in SEQ ID NO. 5, and LCDR3 shown in SEQ ID NO. 6.

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