WO2025237275A1 - Anti-lag-3 antibody or antigen-binding moiety thereof for treating tumor - Google Patents

Abstract

Provided is a use of an anti-LAG-3 antibody in the preparation of a drug for treating tumors.

Description

Anti-LAG-3 antibodies or their antigen-binding fractions used to treat tumors

Cross-reference to related applications

This disclosure claims priority to PCT patent application PCT/CN2024/092934, filed on May 13, 2024, the entire contents of which are incorporated herein by reference.

Technical Field

This invention relates to the use of an anti-LAG-3 antibody or its antigen-binding portion in the treatment of tumors, and more particularly to the use of the anti-LAG-3 antibody or its antigen-binding portion, and its combination with other therapeutic agents, in the preparation of a medicament for the treatment of tumors.

Background Technology

LAG-3 (Lymphocyte Activation Gene 3) is a member of the immunoglobulin superfamily, also known as CD223, and is an inhibitory immune checkpoint expressed on the surface of T cells. The LAG-3 gene and the CD4 gene are located in the same region and share some homology. Studies have found that LAG-3 has four ligands in the tumor microenvironment, mainly including Major Histocompatibility Complex Class II (MHC II), Galectin-3 (Gal-3), Liver sinusoidal endothelial cell lectin (LSECtin), and Fibrinogen-like protein 1 (FGL1). MHC-II molecules are considered typical ligands of LAG-3, with significantly higher affinity than CD4. The binding of MHC-II and LAG-3 negatively regulates T cell activation, cytotoxicity, and cytokine production. Gal-3 inhibits activated antigen-directed CD8 T cells and suppresses the proliferation of plasmacytoid dendritic cells through LAG-3 expression in the tumor microenvironment, thereby forming an anti-tumor-specific immune response. FGL1, a novel functional ligand of LAG-3, exhibits immunosuppressive activity by binding to LAG-3 and inhibiting antigen-specific T cells, and is considered a novel immune evasion mechanism. Therefore, LAG-3 mainly regulates T cell immune responses through three mechanisms: first, by directly inhibiting T cell proliferation and activation through negative regulation of T cells; second, by promoting the suppressive function of regulatory T cells (Tregs), thereby indirectly inhibiting T cell responses; and third, by preventing T cell activation by regulating the function of antigen-presenting cells (APCs). LAG-3 is highly expressed in various tumor-infiltrating lymphocytes (TILs), especially CD4+ and CD8+ T cells, including Tregs, natural killer T cells, B cells, NK cells, plasmacytoid dendritic cells, and tumor-associated macrophages. Blocking LAG-3 can enhance TIL proliferation and cytokine secretion, thereby enhancing anti-tumor immunity. LAG-3 has become a novel target for tumor immunotherapy after CTLA-4/PD-1/PD-L1.

PD-(L)1, a member of the B7/CD28 family, is composed of PD-1 ligand and PD-L1 receptor. It is expressed on T cells, B cells, monocytes/macrophages, and natural killer (NK) cells, primarily inhibiting the activation of effector T cells, controlling reactive T cells, and promoting the production of Tregs. Studies have shown that LAG-3 and PD-1 are co-expressed in tumor-infiltrating lymphocytes of various tumor types. In a mouse autoantigen tolerance model, CD8+ T cells co-expressing PD-1 and LAG-3 exhibited severe impaired effector function. Furthermore, in a mouse model of primary squamous cell carcinoma bearing a KrasG12D mutation and Smad4 deletion, CD8+ tumor-infiltrating lymphocytes showed a phenotype of chronic activation and exhaustion, while PD-1 and LAG-3 co-expression was found; Treg expression was upregulated in CD4+ tumor-infiltrating lymphocytes; and dual blocking of the PD-1 and LAG-3 pathways inhibited the growth of squamous cell carcinoma. A study analyzing the expression of tumor-infiltrating lymphocytes (TILs) in patients with hepatocellular carcinoma used flow cytometry to examine the expression of PD-1 and LAG-3 on TILs. The results showed that the expression levels of PD-1 and LAG-3 on TILs in the tumor tissue of the same patient were significantly higher than those in adjacent tissue and peripheral blood. At the same time, blocking PD-1 and LAG-3 had a good tumor-suppressive effect.

Based on the above information, LAG-3 and PD-1 molecules have a significant synergistic effect in tumor diseases. By coordinating the inhibition of LAG-3 and PD-1, the immune response can be enhanced, and the best anti-tumor effect of combined drugs can be achieved, providing a clear approach for the combined treatment of tumors.

Currently, there are nearly 30 LAG-3 monoclonal antibody products in development globally, most of which are for combination therapy, such as combination with anti-PD-1/PD-L1 monoclonal antibodies, combination with anti-PD-1/PD-L1 monoclonal antibodies and chemotherapy, and combination with anti-PD-1/PD-L1 monoclonal antibodies and anti-angiogenic agents. Internationally, there are Bristol-Myers Squibb's (BMS) Relatlimab, Novartis' LAG525/IMP701, Merck's MK-4280, Regeneron's REGN3767, GSK's Tesaro's TSR-033, and F-Star's FS118. Domestically, there are Hengrui Medicine's SHR-1802, Innovent Biologics' IBI110, Zai Lab's MGD013, and Kelun Biotech's KL-A289. BMS's Relatlimab study has progressed the most, having completed Phase III clinical trials. On September 20, 2021, the FDA announced that it had granted Priority Review designation to the Biologics License Application (BLA) for Relatlimab in combination with the anti-PD-1 antibody Opdivo (nivolumab) for the treatment of adult and pediatric patients with unresectable/metastatic melanoma. On March 19, 2022, BMS announced that the FDA had approved Relatlimab, making it the first LAG-3 antibody approved by the FDA in the United States.

Summary of the Invention

This disclosure provides the use of antibodies that specifically bind to LAG-3 or their antigen-binding moiety in the treatment of tumors.

Therefore, in a first aspect, this disclosure relates to the use of an antibody or antigen-binding moiety thereof that specifically binds to LAG-3 in the preparation of a medicament for treating malignant tumors, said antibody or antigen-binding moiety comprising a heavy chain variable region and a light chain variable region, wherein:

The heavy chain variable region

(i) Contains HCDR1, HCDR2 and HCDR3 derived from the heavy chain variable region consisting of the amino acid sequence of SEQ ID NO:4;

(ii) Containing HCDR1, HCDR2, and HCDR3, which respectively contain or constitute the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, or respectively contain or constitute amino acid sequences having one, two, or three amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; or

(iii) Containing or consisting of the amino acid sequence of SEQ ID NO:4, preferably having all three CDRs of SEQ ID NO:4; or containing or consisting of an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:4, preferably having all three CDRs of SEQ ID NO:4; or containing or consisting of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5) amino acid modifications (preferably amino acid substitutions, more preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:4, preferably, these amino acid modifications do not occur in the CDR region, more preferably, these amino acid modifications occur in the FR region, such as FR1, FR2, FR3, or FR4 region;

The variable region of the light chain:

(i) LCDR1, LCDR2 and LCDR3 comprising a light chain variable region composed of an amino acid sequence derived from SEQ ID NO:8;

(ii) comprising LCDR1, LCDR2, and LCDR3, which respectively comprise or consist of the sequences shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, or respectively comprise or consist of amino acid sequences having one, two, or three amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7; or

(iii) Containing or consisting of the amino acid sequence of SEQ ID NO:8, or containing or consisting of an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:8, preferably having all three CDRs of SEQ ID NO:8; or containing or consisting of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5) amino acid modifications (preferably amino acid substitutions, more preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:8, preferably, these amino acid modifications do not occur in the CDR region, more preferably, these amino acid modifications occur in the FR region, such as FR1, FR2, FR3, or FR4 region.

In one embodiment, the antibody or its antigen-binding portion further comprises a heavy chain constant region and/or a light chain constant region, the heavy chain constant region comprising or consisting of the amino acid sequence shown in SEQ ID NO:9, and the light chain constant region comprising or consisting of the amino acid sequence shown in SEQ ID NO:10.

In one embodiment, the antibody or its antigen-binding portion comprises a heavy chain and a light chain, wherein: the heavy chain comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:11, preferably having all three CDRs of SEQ ID NO:4; and the light chain comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:12, preferably having all three CDRs of SEQ ID NO:8.

In one embodiment, the antigen-binding portion is selected from: (i) the Fab fragment; (ii) the F(ab')2 fragment; (iii) the Fd fragment; (iv) the Fv fragment; (v) the dAb fragment; and (vii) the single-chain Fv (scFv).

In one embodiment, the antibody or antigen-binding portion described in the uses covered by this disclosure is a mouse antibody, a human antibody, a chimeric antibody, or a humanized antibody.

In one embodiment, the antibody or the antigen-binding portion is an IgG1, IgG2, or IgG4 isotype.

In one embodiment, the malignant tumor is an advanced malignant tumor, preferably selected from nasopharyngeal carcinoma.

In one embodiment, the drug is formulated for administration to a subject suffering from the malignant tumor, the subject being a subject with relapsed and refractory advanced malignant tumors, a subject with recurrent or metastatic advanced malignant tumors who has not previously received systemic treatment, or a subject with unresectable or metastatic advanced malignant tumors; preferably, the subject is a subject with recurrent or metastatic advanced nasopharyngeal carcinoma who has not previously received systemic treatment.

In some other embodiments, the nasopharyngeal carcinoma subject is LAG-3 positive (LAG-3 ≥ 1%) and PD-L1 positive (PD-L1 ≥ 1%). In still other embodiments, the nasopharyngeal carcinoma subject is LAG-3 negative (LAG-3 < 1%) or PD-L1 negative (PD-L1 < 1%). In still other embodiments, the nasopharyngeal carcinoma subject is a patient with nasopharyngeal carcinoma who has not received anti-PD-(L)1 antibody therapy or who has progressed or is intolerant to anti-PD-(L)1 antibody therapy.

In one embodiment, the drug is formulated for administration to a subject at each dosing cycle or per administration, the drug comprising 0.05 mg/kg to 200 mg/kg of the antibody or its antigen-binding portion. In a preferred embodiment, the drug comprises 0.25 mg/kg to 10 mg/kg of the antibody or its antigen-binding portion. In a preferred embodiment, the drug comprises 1.0 mg/kg to 6 mg/kg of the antibody or its antigen-binding portion, and in a preferred embodiment, the drug comprises 6.0 mg/kg of the antibody or its antigen-binding portion, for example, 0.25 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 1.0 mg/kg, 1.2 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 8 mg/kg, or 10 mg/kg of the antibody or its antigen-binding portion.

In one embodiment, the drug is formulated for administration to a subject in each dosing cycle or per administration, the drug comprising 50 mg to 5000 mg of the antibody or its antigen-binding portion. In a preferred embodiment, the drug comprises 60 mg to 4000 mg of the antibody or its antigen-binding portion. In a preferred embodiment, the drug comprises 80 mg to 3750 mg of the antibody or its antigen-binding portion. In a preferred embodiment, the drug comprises 100 mg to 3500 mg of the antibody or its antigen-binding portion, for example, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1250 mg, 1500 mg, 1750 mg, 2000 mg, 2250 mg, 2500 mg, 2750 mg, 3000 mg, 3250 mg, or 3500 mg of the antibody or its antigen-binding portion.

In one embodiment, the drug is administered every 12 weeks, every 9 weeks, every 6 weeks, every 5 weeks, every 4 weeks, every 3 weeks, every 2 weeks, once a week, twice a week, or three times a week.

In one embodiment, the drug is formulated as an intravenous infusion or subcutaneous injection preparation.

In one embodiment, the drug is also administered in combination with one or more other therapeutic agents. In some preferred embodiments, the other therapeutic agents are immunostimulatory antibodies and/or chemotherapeutic agents.

In one embodiment, the immunostimulating antibody is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody. In some preferred embodiments, the immunostimulating antibody is an anti-PD-1 antibody. In a more preferred embodiment, the anti-PD-1 antibody is selected from tislelizumab and toripalimab.

In one embodiment, the immunostimulatory antibody is administered at intervals of 12 weeks, 9 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, once a week, twice a week, or three times a week.

In one embodiment, the immunostimulatory antibody is formulated as an intravenous infusion or subcutaneous injection preparation.

In one implementation, the immunostimulatory antibody is administered before, after, or simultaneously with the administration of the drug to the subject in each dosing cycle or each time.

In one embodiment, the immunostimulatory antibody is administered 0–5 hours before or after each dosing cycle or each time, for example, immediately, 15 minutes, 30 minutes, 45 minutes, 1 hour, 1.25 hours, 1.5 hours, 1.75 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, or 5 hours after each dosing cycle or each time. In some preferred embodiments, 100 mg to 600 mg of the immunostimulatory antibody is administered each time, for example, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, or 600 mg of tislelizumab or 100 mg, 200 mg, 240 mg, 300 mg, 400 mg, 500 mg, or 600 mg of toripalimab each time.

In one embodiment, the chemotherapeutic agent is gemcitabine and/or platinum-based drugs. In some preferred embodiments, the chemotherapeutic agent is gemcitabine hydrochloride and/or cisplatin.

In one embodiment, the chemotherapeutic agent is administered every 5 weeks, every 4 weeks, every 3 weeks, every 2 weeks, once a week, twice a week, or three times a week.

In one embodiment, the chemotherapeutic agent is formulated for intravenous infusion or oral administration.

In one embodiment, the chemotherapy agent is administered after each dosing cycle or each time the antidrug and the immunostimulatory antibody are administered to the subject.

In one embodiment, the chemotherapeutic agent is administered 0–24 hours after each dosing cycle or each time the drug and the immunostimulatory antibody are applied.

In a preferred embodiment, when the chemotherapeutic agent is administered sequentially with gemcitabine and the platinum-based agent, gemcitabine is administered 0–24 hours after each dosing cycle or each administration of the immunostimulatory antibody. For example, gemcitabine is administered immediately, at 15 minutes, 30 minutes, 45 minutes, 1 hour, 1.25 hours, 1.5 hours, 1.75 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours after each dosing cycle or each administration of the immunostimulatory antibody. Each administration does not exceed 1000 mg/ ^m² of gemcitabine, for example, 1000 mg/m² per administration. ² % gemcitabine hydrochloride; the platinum-based drugs are administered from 0 to 24 hours after gemcitabine administration, for example, at each dosing cycle or at each administration immediately, 15 minutes, 30 minutes, 45 minutes, 1 hour, 1.25 hours, 1.5 hours, 1.75 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours after gemcitabine administration, with each administration not exceeding 80 mg/ ^m² of the platinum-based drugs, for example, 80 mg/ ^m² of cisplatin per administration.

In one embodiment, tislelizumab is administered 1 h ± 30 min after the completion of administration of the anti-LAG-3 antibody or its antigen-binding portion, and chemotherapy drugs are administered 1 h ± 30 min after the completion of tislelizumab administration. The chemotherapy drugs are gemcitabine and cisplatin. Cisplatin is administered after the gemcitabine infusion. In one embodiment, the ORR improvement rate achieved by the anti-LAG-3 antibody combined with tislelizumab and gemcitabine plus cisplatin is higher than the ORR improvement rate achieved by anti-LAG-3 antibody alone. In one embodiment, the DCR improvement rate achieved by the anti-LAG-3 antibody combined with tislelizumab and gemcitabine plus cisplatin is higher than the DCR improvement rate achieved by anti-LAG-3 antibody alone. In one embodiment, the PFS improvement rate achieved by the anti-LAG-3 antibody combined with tislelizumab and gemcitabine plus cisplatin is higher than the PFS improvement rate achieved by anti-LAG-3 antibody alone.

In a second aspect, this disclosure relates to a medicament for treating malignant tumors, said medicament comprising the antibody or its antigen-binding portion as defined in the first aspect.

In a third aspect, this disclosure relates to a pharmaceutical combination for treating malignant tumors, comprising the antibody or its antigen-binding portion as defined in the first aspect, and administration of one or more other therapeutic agents as defined in the first aspect.

In some embodiments, the other therapeutic agents are immunostimulatory antibodies and/or chemotherapeutic agents.

In some preferred embodiments, the immunostimulating antibody is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody. In a more preferred embodiment, the immunostimulating antibody is an anti-PD-1 antibody. In the most preferred embodiment, the anti-PD-1 antibody is selected from tislelizumab and toripalimab.

In some preferred embodiments, the chemotherapeutic agent is a platinum-based substance. In a more preferred embodiment, the platinum-based substance is cisplatin.

In some embodiments, this disclosure relates to a drug or a combination of drugs as defined in the second aspect, wherein the malignant tumor is an advanced malignant tumor. In some preferred embodiments, the malignant tumor is selected from nasopharyngeal carcinoma.

In some embodiments, this disclosure relates to the drug or combination of drugs as defined in the second aspect, wherein the drug is administered to a subject with the malignant tumor, the subject being a subject with relapsed and refractory advanced malignant tumors, or a subject with recurrent or metastatic advanced malignant tumors who has not previously received systemic therapy, or a subject with unresectable or metastatic advanced malignant tumors. In some preferred embodiments, the subject is a subject with recurrent or metastatic advanced nasopharyngeal carcinoma who has not previously received systemic therapy. In other embodiments, the nasopharyngeal carcinoma subject is LAG-3 positive (LAG-3 ≥ 1%) and PD-L1 positive (PD-L1 ≥ 1%). In still other embodiments, the nasopharyngeal carcinoma subject is LAG-3 negative (LAG-3 < 1%) or PD-L1 negative (PD-L1 < 1%). In still other embodiments, the nasopharyngeal carcinoma subject is a patient with nasopharyngeal carcinoma who has not received anti-PD-(L)1 antibody therapy or who has progressed or is intolerant to anti-PD-(L)1 antibody therapy.

In a fourth aspect, this disclosure relates to a method for treating malignant tumors, the method comprising administering to a subject an antibody or antigen-binding moiety thereof that specifically binds to LAG-3, said antibody or antigen-binding moiety comprising a heavy chain variable region and a light chain variable region, wherein:

The heavy chain variable region

(i) Contains HCDR1, HCDR2 and HCDR3 derived from the heavy chain variable region consisting of the amino acid sequence of SEQ ID NO:4;

(ii) Containing HCDR1, HCDR2, and HCDR3, which respectively contain or constitute the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, or respectively contain or constitute amino acid sequences having one, two, or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; or

(iii) Containing or consisting of the amino acid sequence of SEQ ID NO:4, preferably having all three CDRs of SEQ ID NO:4; or containing or consisting of an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:4, preferably having all three CDRs of SEQ ID NO:4; or containing or consisting of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5) amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:4, wherein, in a preferred embodiment, these amino acid modifications do not occur in the CDR region, and in a more preferred embodiment, these amino acid modifications occur in the FR region, such as FR1, FR2, FR3, or FR4 region;

The variable region of the light chain:

(i) LCDR1, LCDR2 and LCDR3 comprising a light chain variable region composed of an amino acid sequence derived from SEQ ID NO:8;

(ii) comprising LCDR1, LCDR2, and LCDR3, which respectively comprise or consist of the sequences shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, or respectively comprise or consist of amino acid sequences having one, two, or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7; or

(iii) Containing or consisting of the amino acid sequence of SEQ ID NO:8, or containing or consisting of an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:8, preferably having all three CDRs of SEQ ID NO:8; or containing or consisting of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5) amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:8, wherein, in a preferred embodiment, these amino acid modifications do not occur in the CDR region, and in a more preferred embodiment, these amino acid modifications occur in the FR region, such as FR1, FR2, FR3, or FR4 region.

In one embodiment, the antibody or its antigen-binding portion further comprises a heavy chain constant region and/or a light chain constant region, the heavy chain constant region comprising or consisting of the amino acid sequence shown in SEQ ID NO:9, and the light chain constant region comprising or consisting of the amino acid sequence shown in SEQ ID NO:10.

In one embodiment, the antibody or its antigen-binding portion comprises a heavy chain and a light chain, wherein: the heavy chain comprises or is composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 11; and the light chain comprises or is composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 12.

In one embodiment, the antigen-binding portion is selected from: (i) the Fab fragment; (ii) the F(ab')2 fragment; (iii) the Fd fragment; (iv) the Fv fragment; (v) the dAb fragment; and (vii) the single-chain Fv (scFv).

In one embodiment, the antibody or the antigen-binding portion is a mouse antibody, a human antibody, a chimeric antibody, or a humanized antibody.

In one embodiment, the antibody or the antigen-binding portion is an IgG1, IgG2, or IgG4 isotype.

In one embodiment, the malignant tumor is an advanced malignant tumor. In some preferred embodiments, the malignant tumor is selected from nasopharyngeal carcinoma and melanoma.

In one embodiment, the antibody or its antigen-binding fragment is administered to a subject suffering from the malignant tumor, wherein the subject is a subject with relapsed and refractory advanced malignant tumors, a subject with recurrent or metastatic advanced malignant tumors who has not previously undergone systemic treatment, or a subject with unresectable or metastatic advanced malignant tumors. In some preferred embodiments, the subject is a subject with recurrent or metastatic advanced nasopharyngeal carcinoma who has not previously undergone systemic treatment.

In some other embodiments, the nasopharyngeal carcinoma subject is LAG-3 positive (LAG-3 ≥ 1%) and PD-L1 positive (PD-L1 ≥ 1%). In still other embodiments, the nasopharyngeal carcinoma subject is LAG-3 negative (LAG-3 < 1%) or PD-L1 negative (PD-L1 < 1%). In still other embodiments, the nasopharyngeal carcinoma subject is a patient with nasopharyngeal carcinoma who has not received anti-PD-(L)1 antibody therapy or who has progressed or is intolerant to anti-PD-(L)1 antibody therapy.

In one embodiment, the antibody or its antigen-binding portion is administered to the subject at a dose of 0.05 mg/kg to 200 mg/kg per dosing cycle or per administration. In some preferred embodiments, the antibody or its antigen-binding portion is 0.25 mg/kg to 10 mg/kg. In some preferred embodiments, the antibody or its antigen-binding portion is 1.0 mg/kg to 6 mg/kg. In some preferred embodiments, the antibody or its antigen-binding portion is 6.0 mg/kg. Examples of dosages include 0.25 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 1.0 mg/kg, 1.2 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 8 mg/kg, or 10 mg/kg.

In one embodiment, the antibody or its antigen-binding portion is administered to the subject at a rate of 50 mg to 5000 mg per dosing cycle or per administration. In some preferred embodiments, the antibody or its antigen-binding portion is administered at a rate of 60 mg to 4000 mg. In some preferred embodiments, the antibody or its antigen-binding portion is administered at a rate of 80 mg to 3750 mg. In some preferred embodiments, the antibody or its antigen-binding portion is administered at a rate of 100 mg to 3500 mg, for example, 200 mg, 240 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1250 mg, 1500 mg, 1750 mg, 2000 mg, 2250 mg, 2500 mg, 2750 mg, 3000 mg, 3250 mg, or 3500 mg.

In one embodiment, the antibody or its antigen-binding portion is administered at intervals of 12 weeks, 9 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, once a week, twice a week, or three times a week.

In one embodiment, the antibody or its antigen-binding portion is formulated as an intravenous infusion or subcutaneous injection preparation.

In one embodiment, the antibody or its antigen-binding portion described in the method of this disclosure is further administered in combination with one or more other therapeutic agents. In some preferred embodiments, the other therapeutic agents are selected from immunostimulatory antibodies and/or chemotherapeutic agents.

In one embodiment, the immunostimulating antibody is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody. In some preferred embodiments, the immunostimulating antibody is an anti-PD-1 antibody. In a more preferred embodiment, the anti-PD-1 antibody is selected from tislelizumab and toripalimab.

In one embodiment, the immunostimulatory antibody is administered at intervals of 12 weeks, 9 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, once a week, twice a week, or three times a week.

In one embodiment, the immunostimulatory antibody is formulated as an intravenous infusion or subcutaneous injection preparation.

In one implementation, the immunostimulatory antibody is administered to the subject before, after, or simultaneously with each dosing cycle or each administration of the antibody or its antigen-binding portion.

In one embodiment, the immunostimulatory antibody is administered 0–5 hours before or after each dosing cycle or each administration of the antibody or its antigen-binding portion, for example, immediately, 15 minutes, 30 minutes, 45 minutes, 1 hour, 1.25 hours, 1.5 hours, 1.75 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, or 5 hours after each dosing cycle or each administration of the antibody or its antigen-binding portion, or tislelizumab and/or toripalimab. In some preferred embodiments, 100 mg to 600 mg of the immunostimulatory antibody is administered each time, for example, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, or 600 mg of tislelizumab or 100 mg, 200 mg, 240 mg, 300 mg, 400 mg, 500 mg, or 600 mg of toripalimab each time.

In one embodiment, the chemotherapeutic agent is gemcitabine and/or platinum-based drugs. In some preferred embodiments, the chemotherapeutic agent is gemcitabine hydrochloride and/or cisplatin.

In one embodiment, the chemotherapeutic agent is administered every 5 weeks, every 4 weeks, every 3 weeks, every 2 weeks, once a week, twice a week, or three times a week.

In one embodiment, the chemotherapeutic agent is formulated for intravenous infusion or oral administration.

In one implementation, the chemotherapeutic agent is administered after each dosing cycle or each administration of the antibody or its antigen-binding portion to the subject, as well as the immunostimulatory antibody.

In one embodiment, the chemotherapeutic agent is administered 0–24 hours after each dosing cycle or each administration of the antibody or its antigen-binding portion and the immunostimulatory antibody.

In some preferred embodiments, when the chemotherapeutic agent is administered sequentially with gemcitabine and the platinum-based agent, gemcitabine is administered 0–24 hours after each dosing cycle or each administration of the immunostimulatory antibody. For example, gemcitabine is administered immediately, at 15 minutes, 30 minutes, 45 minutes, 1 hour, 1.25 hours, 1.5 hours, 1.75 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours after each dosing cycle or each administration of the immunostimulatory antibody. Each administration does not exceed 1000 mg/ ^m² of gemcitabine, for example, 1000 mg/m² per administration. ² % gemcitabine hydrochloride; the platinum-based drugs are administered from 0 to 24 hours after gemcitabine administration, for example, at each dosing cycle or at each administration immediately, 15 minutes, 30 minutes, 45 minutes, 1 hour, 1.25 hours, 1.5 hours, 1.75 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours after gemcitabine administration, with each administration not exceeding 80 mg/ _m² of the platinum-based drugs, for example, 80 mg/ ^m² of cisplatin per administration.

In a fifth aspect, this disclosure relates to an antibody or antigen-binding moiety thereof that specifically binds to LAG-3 for the treatment of malignant tumors, said antibody or antigen-binding moiety comprising a heavy chain variable region and a light chain variable region, wherein:

The heavy chain variable region

(i) Contains HCDR1, HCDR2 and HCDR3 derived from the heavy chain variable region consisting of the amino acid sequence of SEQ ID NO:4;

(ii) Containing HCDR1, HCDR2, and HCDR3, which respectively contain or constitute the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, or respectively contain or constitute amino acid sequences having one, two, or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; or

(iii) Containing or consisting of the amino acid sequence of SEQ ID NO:4, preferably having all three CDRs of SEQ ID NO:4; or containing or consisting of an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:4, preferably having all three CDRs of SEQ ID NO:4; or containing or consisting of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5) amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:4, wherein, in a preferred embodiment, these amino acid modifications do not occur in the CDR region, and in a more preferred embodiment, these amino acid modifications occur in the FR region, such as FR1, FR2, FR3, or FR4 region;

The variable region of the light chain:

(i) LCDR1, LCDR2 and LCDR3 comprising a light chain variable region composed of an amino acid sequence derived from SEQ ID NO:8;

(ii) comprising LCDR1, LCDR2, and LCDR3, which respectively comprise or consist of the sequences shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, or respectively comprise or consist of amino acid sequences having one, two, or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7; or

(iii) Containing or consisting of the amino acid sequence of SEQ ID NO:8, or containing or consisting of an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:8, preferably having all three CDRs of SEQ ID NO:8; or containing or consisting of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5) amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:8, wherein, in a preferred embodiment, these amino acid modifications do not occur in the CDR region, and in a more preferred embodiment, these amino acid modifications occur in the FR region, such as FR1, FR2, FR3, or FR4 region.

In one embodiment, the antibody or its antigen-binding portion further comprises a heavy chain constant region and/or a light chain constant region, the heavy chain constant region comprising or consisting of the amino acid sequence shown in SEQ ID NO:9, and the light chain constant region comprising or consisting of the amino acid sequence shown in SEQ ID NO:10.

In one embodiment, the antibody or its antigen-binding portion comprises a heavy chain and a light chain, wherein: the heavy chain comprises or is composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 11; and the light chain comprises or is composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 12.

In one embodiment, the antigen-binding portion is selected from: (i) the Fab fragment; (ii) the F(ab')2 fragment; (iii) the Fd fragment; (iv) the Fv fragment; (v) the dAb fragment; and (vii) the single-chain Fv (scFv).

In one embodiment, the antibody or the antigen-binding portion is a mouse antibody, a human antibody, a chimeric antibody, or a humanized antibody.

In one embodiment, the antibody or the antigen-binding portion is an IgG1, IgG2, or IgG4 isotype.

In one embodiment, the malignant tumor is an advanced malignant tumor. In some preferred embodiments, the malignant tumor is selected from nasopharyngeal carcinoma and melanoma.

In one embodiment, the antibody or the antigen-binding portion is administered to a subject suffering from the malignant tumor, wherein the subject is a subject with relapsed and refractory advanced malignant tumors, a subject with recurrent or metastatic advanced malignant tumors who has not previously undergone systemic treatment, or a subject with unresectable or metastatic advanced malignant tumors. In some preferred embodiments, the subject is a subject with recurrent or metastatic advanced nasopharyngeal carcinoma who has not previously undergone systemic treatment.

In some other embodiments, the nasopharyngeal carcinoma subject is LAG-3 positive (LAG-3 ≥ 1%) and PD-L1 positive (PD-L1 ≥ 1%). In still other embodiments, the nasopharyngeal carcinoma subject is LAG-3 negative (LAG-3 < 1%) or PD-L1 negative (PD-L1 < 1%). In still other embodiments, the nasopharyngeal carcinoma subject is a patient with nasopharyngeal carcinoma who has not received anti-PD-(L)1 antibody therapy or who has progressed or is intolerant to anti-PD-(L)1 antibody therapy.

In one embodiment, the antibody or its antigen-binding portion is administered to the subject at a dose of 0.05 mg/kg to 200 mg/kg per dosing cycle or per administration. In some preferred embodiments, the antibody or its antigen-binding portion is 0.25 mg/kg to 10 mg/kg. In some preferred embodiments, the antibody or its antigen-binding portion is 1.0 mg/kg to 6 mg/kg. In some preferred embodiments, the antibody or its antigen-binding portion is 6.0 mg/kg. Examples of dosages include 0.25 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 1.0 mg/kg, 1.2 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 8 mg/kg, or 10 mg/kg.

In one embodiment, the antibody or its antigen-binding portion is administered to the subject at a rate of 50 mg to 5000 mg per dosing cycle or per administration. In some preferred embodiments, the antibody or its antigen-binding portion is administered at a rate of 60 mg to 4000 mg. In some preferred embodiments, the antibody or its antigen-binding portion is administered at a rate of 80 mg to 3750 mg. In some preferred embodiments, the antibody or its antigen-binding portion is administered at a rate of 100 mg to 3500 mg, for example, 200 mg, 240 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1250 mg, 1500 mg, 1750 mg, 2000 mg, 2250 mg, 2500 mg, 2750 mg, 3000 mg, 3250 mg, or 3500 mg.

In one embodiment, the antibody or its antigen-binding portion is administered at intervals of 12 weeks, 9 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, once a week, twice a week, or three times a week.

In one embodiment, the antibody or its antigen-binding portion is formulated as an intravenous infusion or subcutaneous injection preparation.

In one embodiment, the antibody or its antigen-binding portion is further administered in combination with one or more other therapeutic agents. In a preferred embodiment, the other therapeutic agents are selected from immunostimulatory antibodies and/or chemotherapeutic agents.

In one embodiment, the immunostimulating antibody is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody. In a preferred embodiment, the immunostimulating antibody is an anti-PD-1 antibody. In a more preferred embodiment, the anti-PD-1 antibody is selected from tislelizumab and toripalimab.

In one embodiment, the immunostimulatory antibody is administered at intervals of 12 weeks, 9 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, once a week, twice a week, or three times a week.

In one embodiment, the immunostimulatory antibody is formulated as an intravenous infusion or subcutaneous injection preparation.

In one implementation, the immunostimulatory antibody is administered to the subject before, after, or simultaneously with each dosing cycle or each administration of the antibody or its antigen-binding portion.

In one embodiment, the immunostimulatory antibody is administered 0–5 hours before or after each dosing cycle or each administration of the antibody or its antigen-binding portion, for example, immediately, 15 minutes, 30 minutes, 45 minutes, 1 hour, 1.25 hours, 1.5 hours, 1.75 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, or 5 hours after each dosing cycle or each administration of the antibody or its antigen-binding portion, or tislelizumab and/or toripalimab. In some preferred embodiments, 100 mg to 600 mg of the immunostimulatory antibody is administered each time, for example, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, or 600 mg of tislelizumab or 100 mg, 200 mg, 240 mg, 300 mg, 400 mg, 500 mg, or 600 mg of toripalimab each time.

In one embodiment, the chemotherapeutic agent is gemcitabine and/or platinum-based drugs. In some preferred embodiments, the chemotherapeutic agent is gemcitabine hydrochloride and/or cisplatin.

In one embodiment, the chemotherapeutic agent is administered every 5 weeks, every 4 weeks, every 3 weeks, every 2 weeks, once a week, twice a week, or three times a week.

In one embodiment, the chemotherapeutic agent is formulated for intravenous infusion or oral administration.

In one implementation, the chemotherapeutic agent is administered after each dosing cycle or each administration of the antibody or its antigen-binding portion to the subject, as well as the immunostimulatory antibody.

In one embodiment, the chemotherapeutic agent is administered 0–24 hours after each dosing cycle or each administration of the antibody or its antigen-binding portion and the immunostimulatory antibody.

In some preferred embodiments, when the chemotherapeutic agent is administered sequentially with gemcitabine and the platinum-based agent, gemcitabine is administered 0–24 hours after each dosing cycle or each administration of the immunostimulatory antibody. For example, gemcitabine is administered immediately, at 15 minutes, 30 minutes, 45 minutes, 1 hour, 1.25 hours, 1.5 hours, 1.75 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours after each dosing cycle or each administration of the immunostimulatory antibody. Each administration does not exceed 1000 mg/ ^m² of gemcitabine, for example, 1000 mg/m² per administration. ² % gemcitabine hydrochloride; the platinum-based drugs are administered from 0 to 24 hours after gemcitabine administration, for example, at each dosing cycle or at each administration immediately, 15 minutes, 30 minutes, 45 minutes, 1 hour, 1.25 hours, 1.5 hours, 1.75 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours after gemcitabine administration, with each administration not exceeding 80 mg/ ^m² of the platinum-based drugs, for example, 80 mg/ ^m² of cisplatin per administration.

Attached Figure Description

Figure 1 shows the structure of anti-LAG3 antibody No. 2.

Figure 2 shows the pharmacokinetic curves after a single dose (Figure 2A) and multiple doses (Figure 2B) of anti-LAG3 antibody 2.

Detailed Implementation

To facilitate a clearer understanding of this disclosure, certain terms are first defined. Further definitions are set forth throughout the detailed description.

The term "LAG-3" refers to lymphocyte activation gene-3. The term "LAG-3" includes variants, isoforms, homologs, orthologs, and paralogs. For example, in some cases, antibodies specific to human LAG-3 protein may cross-react with LAG-3 proteins from species other than humans. In other embodiments, antibodies specific to human LAG-3 protein may be completely specific to human LAG-3 protein and not cross-reactive with other species or types, or may cross-react with LAG-3 from some but not all other species (e.g., cross-reactive with monkey LAG-3 but not with mouse LAG-3).

The term "human LAG-3" refers to the human LAG-3 sequence, such as the complete amino acid sequence of human LAG-3 with NP 002277 (SEQ ID NO: 13). The term "mouse LAG-3" refers to the mouse LAG-3 sequence, such as the complete amino acid sequence of mouse LAG-3 with NP_032505. LAG-3 is also referred to in the art, for example, as CD223. The human LAG-3 sequence may differ from the human LAG-3 with NP_002277 in that it has, for example, a conserved mutation or a mutation in a non-conserved region, and the LAG-3 and the human LAG-3 with NP_002277 have substantially the same biological function. For example, the biological function of human LAG-3 is to have an epitope in the extracellular domain of LAG-3 that is specifically bound by the antibody of this disclosure, or the biological function of human LAG-3 is to bind to MHC class II molecules.

The terms “LAG-3 positive” and “LAG-3 negative” refer to the evaluation of LAG-3 expression in tumor cells using the standardized SP464 assay (Ventana Medical Systems). The score is the percentage of positively stained immune cells in the surviving tumor area. A score of LAG-3 ≥ 1% is considered LAG-3 positive, and a score of LAG-3 < 1% is considered LAG-3 negative.

The terms "PD-L1 positive" and "PD-L1 negative" refer to the assessment of PD-L1 expression in tumor cells using the standardized SP263 assay (Ventana Medical Systems). The score is the percentage of positively stained immune cells within the surviving tumor region; a score of PD-L1 ≥ 1% indicates PD-L1 positive, and a score of PD-L1 < 1% indicates PD-L1 negative. The term "immune response" refers to the action of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by these cells or the liver, resulting in selective damage, destruction, or elimination from the body of invading pathogens, pathogen-infected cells or tissues, cancer cells, or, in cases of autoimmunity or pathological inflammation.

"Antigen-specific T cell response" refers to the T cell response induced by an antigen specifically targeted by the T cell. Non-limiting examples of T cell responses to antigen-specific stimulation include proliferation and cytokine production (e.g., IL-2 production).

As used herein, the term "antibody" includes both intact antibodies and any antigen-binding fragments (i.e., "antigen-binding portions") or single chains thereof. An intact antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (abbreviated _VH herein) and a heavy chain constant region (abbreviated _CH herein). The heavy chain constant region contains three domains: _CH1 , _CH2 , and _CH3 . Each light chain contains a light chain variable region (abbreviated _VL herein) and a light chain constant region (abbreviated _CL herein). The light chain constant region contains one domain, _CL . The _VH and _VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which alternate with more conserved regions called framework regions (FRs). Each _VH and _VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

As used herein, the term "antigen-binding portion" (or simply "antibody portion") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., the LAG-3 protein). It has been demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments covered within the term "antigen-binding portion" of an antibody include (i) Fab fragments, which are monovalent fragments consisting of _VL , _VH , _CL , and _CH1 domains; (ii) F(ab') ₂ fragments, which are bivalent fragments containing two Fab fragments connected by a disulfide bridge in the hinge region; (iii) Fd fragments consisting of a _VH domain and a _CH1 domain; (iv) Fv fragments consisting of a single-arm _VL domain and a _VH domain; (v) bi-Fv fragments consisting of two Fv fragments; (vi) dAb fragments consisting of a _VH domain (Ward et al., (1989) Nature 341:544-546); (vii) separated complementarity-determining regions (CDRs); and (viii) nanobodies, which are heavy chain variable regions containing a single variable domain and two constant domains. Furthermore, although the two domains _VL and _VH of the Fv fragment are encoded by separate genes, they can be linked using recombination methods via synthetic adapters that allow them to be made into a single protein chain, wherein the _VL and _VH regions pair to form a monovalent molecule (referred to as a single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). These single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art and are screened for utility fragments in the same manner as intact antibodies.

The terms “Fc domain”, “Fc region”, or “Fc fragment” are used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region. This term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin “Fc domain” contains two or three constant domains: a CH2 domain, a CH3 domain, and optionally a CH4 domain. For example, in native antibodies, an immunoglobulin Fc domain contains the second and third constant domains (CH2 and CH3 domains) of two heavy chains derived from IgG, IgA, and IgD antibodies; or it contains the second, third, and fourth constant domains (CH2, CH3, and CH4 domains) of two heavy chains derived from IgM and IgE antibodies. Unless otherwise stated herein, amino acid residues in the Fc region or heavy chain constant region are numbered according to the EU numbering system (also known as the EU index) as described in Edelman, G.M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) (https://pubmed.ncbi.nlm.nih.gov/5257969/), and also see http://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html. In this document, the terms “Fc domain” or “Fc region” or “Fc fragment” do not include the heavy chain variable region VH and light chain variable region VL of immunoglobulins, nor the heavy chain constant region CH1 (hingeless region) and light chain constant region CL, but in some cases may include the hinge region at the N-terminus of the heavy chain constant region. In some embodiments, the heavy chain constant region Fc suitable for use in this invention is derived from the antibody heavy chain constant region, such as the constant region of human IgG1, IgG2, IgG3, or IgG4, preferably from the constant region of IgG1. In some embodiments, the Fc region comprises the amino acid sequence shown in SEQ ID NO:93, or comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence. In some embodiments of this invention, the Fc fragment dimerizes to form an Fc dimer. In some embodiments, the Fc fragment heterodimerizes to form an Fc heterodimer. In the case of the Fc fragment heterodimerizing to form a heterodimer, the Fc fragment may contain a mutation for heterodimerization, such as a knock-in-hole mutation. In some embodiments, the C-terminal lysine residue of the Fc region may be replaced with alanine. In some embodiments, Fc comprises the amino acid sequence shown in SEQ ID NO:15, or comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

The term "CH1 region" refers to the portion of the antibody heavy chain polypeptide extending from EU position 118 to EU position 216 (EU numbering system). In some cases, such as during Fab construction, the CH1 region may also include a partial hinge region, such as EPKSC. Therefore, in some embodiments, the term "CH1" region refers to the portion of the antibody heavy chain polypeptide extending from EU position 118 to EU position 220 (EU numbering system). In some embodiments, CH1 comprises the amino acid sequence shown in SEQ ID NO: 14, or comprises, or consists of, an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with, said amino acid sequence.

As used herein, "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to the LAG-3 protein is substantially free of antibodies that specifically bind to antigens other than the LAG-3 protein). However, isolated antibodies that specifically bind to the human LAG-3 protein may be cross-reactive to other antigens, such as LAG-3 proteins from other species. Furthermore, isolated antibodies may be substantially free of other cellular material and/or chemicals.

As used herein, the terms "monoclonal antibody" or "monoclonal antibody composition" refer to formulations of antibody molecules having a single molecular composition. Monoclonal antibody compositions exhibit single binding specificity and affinity for a specific epitope.

As used herein, the term "human antibody" is intended to include antibodies having variable regions, wherein both the framework region and the CDR region are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, then that constant region is also derived from a human germline immunoglobulin sequence. Human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced in vitro by random or site-specific mutagenesis or in vivo by somatic mutations). However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as a mouse, has been grafted onto a human framework sequence.

The term "human monoclonal antibody" refers to an antibody exhibiting single-binding specificity with a variable region, wherein both the framework region and the CDR region are derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibody is generated by a hybridoma comprising B cells obtained from a transgenic non-human animal, such as a transgenic mouse, having a genome containing both human heavy-chain and light-chain transgenes, fused with immortalized cells.

As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as (a) antibodies isolated from transgenic or transchromosomal animals (e.g., mice) of the human immunoglobulin gene or hybridomas prepared therefrom (described further below); (b) antibodies isolated from host cells transformed to express human antibodies, for example from transfected tumors; (c) antibodies isolated from recombinant combined human antibody libraries; and (d) antibodies prepared, expressed, produced, or isolated by any other means involving splicing a human immunoglobulin gene sequence into other DNA sequences. Such recombinant human antibodies have variable regions in which the frame region and CDR region are derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies can be mutagenized in vitro (or in vivo somatic cell mutagenized when using transgenic animals with human Ig sequences), and thus the amino acid sequences of the _VL and _VH regions of the recombinant antibody are sequences that, although derived from and associated with human germline _VL and _VH sequences, may not naturally exist in the in vivo human antibody germline library.

The term "Kabat numbering system" in this article usually refers to the immunoglobulin matching and numbering system proposed by Elvin A. Kabat (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

The term “Chothia numbering system” in this article usually refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classic rule for identifying the boundaries of CDR regions based on the location of structural loop regions (see, for example, Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883).

The term "IMGT numbering system" in this article usually refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classic rule for identifying the boundaries of CDR regions based on the location of structural loop regions (see, for example, Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al., (1989) Nature, 342: 878-883).

The Kabat numbering system is used to identify the CDR region in this paper, but the Chothia numbering system, the IMGT numbering system, or other immunoglobulin numbering systems are also applicable. Antibodies and their antigen-binding portions corresponding to the CDR regions identified by currently known numbering systems based on the heavy chain variable region or light chain variable region sequence of this invention are also within the scope of this invention.

The term "specific binding" refers to binding that is selective for antigens and can be distinguished from unwanted or nonspecific interactions. The ability of an antigen-binding region to bind to a specific antigenic determinant can be determined by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) techniques (e.g., analysis on a BIAcore instrument).

Unless otherwise stated, "binding affinity" as used herein refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., monoclonal antibody and antigen, antigen-binding region and antigen, or receptor and its ligand). Affinity can be determined by methods known in the art, including those described herein.

The term "isotype" refers to an antibody class (e.g., IgM or IgG1) encoded by a gene in the heavy chain constant region.

The phrases “antibody that recognizes an antigen” and “antibody that is specific to an antigen” are used interchangeably with the term “antibody that binds specifically to an antigen” in this document.

The term "human antibody derivative" refers to any modified form of human antibody, such as an antibody conjugate with another drug or antibody.

The term "humanized antibody" is intended to refer to antibodies in which a CDR sequence derived from the germline of another mammalian species, such as a mouse, has been transplanted onto a human frame sequence. Further frame region modifications can be performed within the human frame sequence.

The term "chimeric antibody" is intended to refer to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

As used herein, an antibody that "specifically binds to human LAG-3" is intended to refer to an antibody that binds to the human LAG-3 protein (and possibly LAG-3 proteins from one or more non-human species), but substantially does not bind to non-LAG-3 proteins. Preferably, the antibody binds to the human LAG-3 protein with "high affinity," i.e., with a _KD of 1 × ^10⁻⁷ M or less, more preferably 1 × ^10⁻⁸ M or less, more preferably ⁵ × ^10⁻⁹ M or less, and even more preferably 1 × ^10⁻⁹ M or less.

As used herein, the term "substantially does not bind to proteins or cells" means that it does not bind to said proteins or cells or does not bind to said proteins or cells with high affinity, i.e., with a _KD of 1 ^{×10⁻⁶ M} or greater, more preferably 1× ^10⁻⁵ M or greater, more preferably 1× ^10⁻⁴ M or greater, more preferably 1×10⁻³ M or greater, and even more preferably 1× ^10⁻² M or greater.

As used herein, the terms “K _-association ” or “ _Ka ” are intended to refer to the association rate of a particular antibody-antigen interaction, while the terms “K _{-dissociation} ” or “ _Kd ” are intended to refer to the dissociation rate of a particular antibody-antigen interaction. The term “ _KD ” is intended to refer to the dissociation constant, which is obtained from the ratio of _Kd to _Ka (i.e., _Kd / _Ka ) and is expressed as a molar concentration (M). The _KD value of an antibody can be determined using methods recognized in the art. A preferred method for determining the _KD of an antibody is by using surface plasmon resonance, preferably using a biosensor system such as the Biacore ^™ system.

The term "high affinity" for IgG antibodies refers to antibodies with a KD of 1× ^10⁻⁶ M or less, more preferably 5× ^10⁻⁸ M or less, even more preferably 1× ^10⁻⁸ M or less, even more preferably 5× ^10⁻⁹ M or less, and even more preferably 1× ^10⁻⁹ M or less for the _target antigen. However, "high affinity" binding may differ for other antibody isotypes. For example, "high affinity" binding for IgM isotypes refers to antibodies with a _KD of ^10⁻⁶ M or less, more preferably ^10⁻⁷ M or less, and even more preferably ^10⁻⁸ M or less.

The term “EC ₅₀ ”, also known as the half-maximum effective concentration, refers to the antibody concentration that induces half the response between baseline and maximum after a specified exposure time.

The term " _IC50 ," also known as the half-maximal inhibitory concentration, refers to the antibody concentration that inhibits a specific biological or biochemical function by 50% relative to the absence of the antibody.

The term “subject” includes any human or non-human animal. The term “non-human animal” includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cattle, horses, chickens, amphibians, and reptiles, although mammals are preferred, such as non-human primates, sheep, dogs, cats, cattle, and horses.

The term "therapeutic effective amount" refers to the amount of the disclosed antibody sufficient to prevent or improve symptoms associated with a disease or condition (such as cancer) and/or reduce the severity of the disease or condition. Therapeutic effective amount is understood to be relevant to the condition being treated, where the actual effective amount is readily discernible to those skilled in the art.

As used herein, the term "therapeutic agent" encompasses any substance that is effective in preventing or treating tumors (such as cancer) or infections or autoimmune diseases, including chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies (e.g., antibodies against immune checkpoint molecules), anti-infective agents, immunomodulators, and small molecule drugs.

The term "chemotherapeutic agents" includes compounds used to treat cancer.

The term "anti-infective agent" includes any molecule that specifically inhibits or eliminates the growth of microorganisms (such as viruses, bacteria, fungi, or protozoa, such as parasites) at the applied concentration and at the applied interval, but is not lethal to the host.

As used herein, the term anti-infective agent includes antibiotics, antibacterial agents, antiviral agents, antifungal agents, and antigenic agents. In one specific aspect, an anti-infective agent is non-toxic to the host at the given concentration and at the given interval.

Immunomodulators include immune checkpoint molecule inhibitors and co-stimulatory molecule activators.

The term "small molecule drug" refers to a low molecular weight organic compound capable of modulating biological processes. "Small molecule" is defined as a molecule with a molecular weight typically less than 2 kDa, preferably less than 1 kDa, and more preferably about 0.5 kDa or smaller. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimics, and antibody mimics.

The terms “cancer” and “cancerous” refer to or describe physiological diseases in mammals that are typically characterized by unregulated cell growth.

The term “tumor” refers to all proliferative cell growth and proliferation (whether malignant or benign), as well as all precancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “proliferative disorder,” “tumor” are not mutually exclusive when used in this document.

The term "infection" refers to a disease caused by a pathogen, including, for example, viral infections, bacterial infections, fungal infections, or protozoan infections such as parasitic infections.

The term "tumor immune escape" refers to a tumor evading immune recognition and clearance. Therefore, as a therapeutic concept, when this escape is reduced, tumor immunity is "treated," and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

The terms "drug combination" or "drug combination product" refer to non-fixed combination products or fixed combination products. The term "non-fixed combination" means that the active ingredients (e.g., (i) the antibodies of the present invention, and platinum and/or podosides) are administered to a patient simultaneously, without a specific time limit, or sequentially at the same or different time intervals, in separate entities, wherein such administration to the patient provides a preventive or therapeutically effective level of the two active agents; an exemplary non-fixed combination product is a kit. In some embodiments, the antibodies of the present invention and platinum and/or podosides used in the drug combination are administered at levels not exceeding those achieved when used alone. The term "fixed combination" means that the two active agents are administered to a patient simultaneously in the form of a single entity. Preferably, the dosage and/or time interval of the two active agents are selected so that the combined use of the components produces an effect greater than that achieved by using either component alone in treating a disease or condition. The components may be in separate formulations, which may be the same or different, and the components may also be used as a pharmaceutical composition.

The term "pharmaceutical composition" refers to a mixture of one or more active ingredients and pharmaceutically acceptable excipients.

The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiation therapy or surgery) to treat the disease described herein. Such administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule containing active ingredients in a fixed proportion. Alternatively, such administration includes the co-administration of individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and/or liquids may be reconstituted or diluted to the desired dose prior to administration. Furthermore, such administration includes the sequential administration of each type of therapeutic agent at substantially the same time or at different times. In either case, the treatment regimen will provide the beneficial effect of the combination of drugs in treating the condition or symptom described herein.

"Subject/Patient/Individual Sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample can be solid tissue, such as fresh, frozen, and/or preserved organ or tissue samples, biopsy samples, or puncture samples; blood or any blood component; body fluids, such as tears, vitreous fluid, cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; or cells from any stage of pregnancy or development in the subject. In some embodiments, the tissue sample is tumor tissue. The tissue sample may contain compounds that are naturally occurring and do not contaminate with tissues, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

The term "cycle" refers to a specific period of time, expressed in days or weeks, repeated according to a regular schedule. For example, each treatment cycle (or prevention cycle) of the drug combination of the present invention is 14 to 30 days, for example 14 to 28 days, preferably two weeks (i.e., 14 days), three weeks (i.e., 21 days), or four weeks (i.e., 28 days). The components of the drug combination of the present invention may be administered on the same day or different days of the cycle, that is, (i) and (ii) of the drug combination of the present invention may be administered separately, simultaneously, or sequentially within the cycle.

The term "administration" refers to the physical introduction of the active ingredients of the medicament or combination of medicines of the present invention into an individual using any of a variety of methods and delivery systems known to those skilled in the art. Routes of administration for the active ingredients of the medicament or combination of medicines of the present invention include oral, intravenous (e.g., infusion (also known as drip) or injection), intramuscular, subcutaneous, intraperitoneal, spinal, local, or other parenteral administration routes. The phrase "parenteral administration" as used herein refers to administration methods other than gastrointestinal and local administration, typically via intravenous, and non-limitingly includes intramuscular, intra-arterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as intracorporeal electroporation. Accordingly, the active ingredients of the medicament or combination of medicines of the present invention can be formulated into capsules, tablets, injections (including infusions or solutions), syrups, sprays, lozenges, liposomes, or suppositories, etc.

The term "continuous administration" refers to daily administration. In the case of continuous administration, the drug may be administered once or multiple times daily, for example, once daily, twice daily, or three times daily, preferably once daily.

The term "dosage" refers to the amount of a drug that produces a therapeutic effect. Unless otherwise stated, dosage relates to the amount of the drug in its free form. If the drug is in the form of a pharmaceutically acceptable salt, the amount of the drug is increased proportionally to the amount of the drug in its free form. For example, the dosage will be stated on the product packaging or product information sheet.

The term "pharmaceutical-acceptable salt" includes, but is not limited to, acid addition salts or base addition salts, such as: acid addition salts formed by compounds of formula (I) with inorganic acids, such as hydrochlorides, hydrobroms, carbonates, bicarbonates, phosphates, sulfates, sulfites, nitrates, etc.; and acid addition salts formed by compounds of formula (I) with organic acids, such as formates, acetates, malates, maleates, fumarates, tartrates, succinates, citrates, lactates, methanesulfonates, p-toluenesulfonates, 2-hydroxyethanesulfonates, benzoates, salicylates, stearates, and salts formed with alkyl dicarboxylic acids of formula HOOC-( _CH₂ ) _n -COOH (where n is 0-4), etc. "Pharmaceutically acceptable salt" also includes base addition salts formed by compounds of formula (I) with acidic groups and pharmaceutically acceptable cations such as sodium, potassium, calcium, aluminum, lithium, and ammonium.

The term "pharmaceutical-grade" refers to compounds, materials, compositions, and/or dosage forms that are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit/risk ratio.

The term "adverse event" (AE) is any unfavorable and generally unexpected or unwanted symptom (including abnormal laboratory findings), condition, or illness associated with the use of a medical treatment. For example, an adverse event may be associated with activation of the immune system in response to treatment or expansion of immune system cells (e.g., T cells) in response to treatment. Medical treatments may have one or more associated AEs, and these AEs may have the same or different levels of severity.

The term "overall survival" or "OS" refers to the time from when a patient first uses the investigated drug until they die from any cause.

The term “progression-free survival” or “PFS” refers to the time from when a patient first uses the investigational drug until disease progression or death from any cause.

The term "accumulation factor" (AF) is an indicator used to measure the degree of drug accumulation in the body. It reflects the relationship between the accumulation of drug in the body after multiple doses and the amount of drug in the body after a single dose.

The term "receptor occupancy" (RO) refers to the degree to which proteins such as antibodies occupy cell surface targets.

All numerical ranges herein should be understood as disclosing every numerical value and subset thereof within that range, regardless of whether they are specifically disclosed otherwise. For example, reference to any numerical range should be considered as reference to every numerical value within that range, such as every integer within that range. This invention relates to all values falling into these ranges, all smaller ranges, and upper or lower limits of numerical ranges.

The term "therapeuticly effective amount" is an amount sufficient to provide therapeutic benefit in treating or managing cancer, or to delay or minimize one or more symptoms associated with cancer. A therapeutically effective amount of a compound refers to a specific amount of a therapeutic agent, alone or in combination with other therapeutic agents, that provides therapeutic benefit in treating or managing cancer. The term "therapeuticly effective amount" can encompass amounts that improve overall cancer therapy, reduce or prevent symptoms or causes of cancer, or enhance the therapeutic efficacy of another therapeutic agent. Examples of "effective amounts" are amounts sufficient to help treat, prevent, or reduce one or more symptoms of a disease, which can also be referred to as "therapeuticly effective amounts." "Reduction" of symptoms means a decrease in the severity or frequency of one or more symptoms, or the elimination of one or more symptoms. The exact amount of the composition (including the “therapeutic effective amount”) will depend on the purpose of treatment and can be determined by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (Vols. 1–3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, edited by Gennaro, Lippincott, Williams & Wilkins).

The term "AUC" (area under the curve) refers to the total amount of drug absorbed or exposed to a subject, or it can refer to a portion of the AUC over a specific time interval. AUC can be obtained based on a curve showing the change in drug concentration in a subject over time.

The term "first-line treatment" refers to the first treatment given for a condition or disease. It is usually part of a standard treatment set, such as chemotherapy, radiation therapy, and/or immunotherapy following surgery, also known as initial treatment. Generally, surgical resection of malignant tumors is not considered a first-line treatment. First-line treatment alone is recognized as the best therapy for the condition or disease. If it fails to cure the condition or disease (e.g., treatment fails, stops working) or causes serious side effects, other treatments may be added or used.

Correspondingly, the term "second-line treatment" refers to treatment given when first-line treatment is unsuccessful, the term "third-line treatment" refers to treatment or treatment regimens given when both first-line and subsequent second-line treatments are unsuccessful, and the term "multi-line treatment" refers to second-line and higher-level treatments.

Various aspects of the invention are described in more detail in the following sections.

I. Antibody

Antibodies suitable for use in this invention include, for example, the antibodies disclosed in PCT/CN2018/095528.

Anti-LAG-3 antibodies with binding affinity to human LAG-3

The exemplary antibodies of this disclosure, or their antigen-binding portions, specifically bind to human LAG-3. The exemplary antibodies of this disclosure, or their antigen-binding portions, have the ability to stimulate an immune response, such as an antigen-specific T-cell response. This can be tested, for example, by assessing the antibody's ability to stimulate the production of interleukin-2 (IL-2) in an antigen-specific T-cell response. In some embodiments, the antibody binds to human LAG-3 and stimulates an antigen-specific T-cell response. In other embodiments, the antibody binds to human LAG-3 but does not stimulate an antigen-specific T-cell response.

The preferred antibody of the present invention is a human monoclonal antibody. Alternatively or additionally, the antibody may be, for example, a chimeric or humanized monoclonal antibody.

In one aspect, the present invention relates to an anti-LAG-3 antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising three heavy chain variable regions HCDR: HCDR1, HCDR2 and HCDR3; and three light chain variable regions LCDR: LCDR1, LCDR2 and LCDR3.

In another aspect, the present invention relates to an anti-LAG-3 antibody or an antigen-binding fragment thereof, the antibody or the antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region.

In one implementation, the heavy chain variable region comprises three heavy chain variable regions HCDR: HCDR1, HCDR2 and HCDR3; the light chain variable region comprises three light chain variable regions LCDR: LCDR1, LCDR2 and LCDR3.

In one implementation, the three heavy chain variable regions HCDR1, HCDR2, and HCDR3 are:

(i) HCDR1, HCDR2, and HCDR3 derived from the heavy chain variable region VH, wherein the VH contains or is composed of the amino acid sequence of SEQ ID NO:4, or

(ii) HCDR1, HCDR2 and HCDR3 of (i) together contain at least one and no more than five amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the three HCDRs of (i).

In one implementation, the three light chain variable regions LCDR: LCDR1, LCDR2, and LCDR3 are:

(i) LCDR1, LCDR2, and LCDR3 derived from the light chain variable region VL, wherein the VL contains or is composed of the amino acid sequence of SEQ ID NO:8, or

(ii) The LCDR1, LCDR2 and LCDR3 of (i) together contain at least one and no more than five amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the three LCDRs of (i).

In one embodiment, the present invention relates to an anti-LAG-3 antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising:

The three heavy chain variable regions HCDR1, HCDR2 and HCDR3 derived from the heavy chain variable region VH, wherein the VH is composed of the amino acid sequence of SEQ ID NO:4; and the three light chain variable regions LCDR1, LCDR2 and LCDR3 derived from the light chain variable region VL, wherein the VL is composed of the amino acid sequence of SEQ ID NO:8.

In one embodiment, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:1, or HCDR1 comprises or consists of an amino acid sequence having one, two, or three amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:1.

In one embodiment, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:2, or HCDR2 comprises or consists of an amino acid sequence having one, two, or three amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:2.

In one embodiment, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:3, or HCDR3 comprises or consists of an amino acid sequence having one, two, or three amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:3.

In one embodiment, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:5, or LCDR1 comprises or consists of an amino acid sequence having one, two, or three amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:5.

In one embodiment, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:6, or LCDR2 comprises or consists of an amino acid sequence having one, two, or three amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:6.

In one embodiment, LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:7, or LCDR3 comprises or consists of an amino acid sequence having one, two, or three amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:7.

In one embodiment, the three heavy chain variable regions HCDR1, HCDR2 and HCDR3 are composed of the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, or may also contain at least one and no more than five amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions).

In one embodiment, the three light chain variable regions LCDR1, LCDR2 and LCDR3 are composed of the amino acid sequences of SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively, or may also contain at least one and no more than five amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions).

In another embodiment, the present invention relates to an anti-LAG-3 antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising HCDR1, HCDR2 and HCDR3 composed of the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively, and LCDR1, LCDR2 and LCDR3 composed of the amino acid sequences of SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 respectively.

In one implementation, the heavy chain variable region includes

(i) an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:4, preferably having all three CDRs of SEQ ID NO:4; or

(ii) Containing or consisting of the amino acid sequence of SEQ ID NO:4; or

(iii) An amino acid sequence comprising or consisting of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5) amino acid modifications (preferably amino acid substitutions, more preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:4, preferably, such amino acid modifications do not occur in the CDR region, more preferably, such amino acid modifications occur in the FR region, such as FR1, FR2, FR3 or FR4 region.

In another implementation, the light chain variable region

(i) an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:8, preferably having all three CDRs of SEQ ID NO:8; or

(ii) Containing or consisting of the amino acid sequence of SEQ ID NO:8; or

(iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 10, more preferably no more than 5) amino acid modifications (preferably amino acid substitutions, more preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:8, preferably, such amino acid modifications do not occur in the CDR region, more preferably, such amino acid modifications occur in the FR region, such as FR1, FR2, FR3 or FR4 region.

In one implementation, the variable region can be modified to improve antibody purification. For example, at the end of the heavy chain variable region, an amino acid can be mutated from S to G to obtain an antibody that can be prepared with high purity.

In another embodiment, the variable region may be modified to improve the stability of the antibody or antigen-binding moiety (e.g., scFv). For example, mutations may be made to form disulfide bonds between the heavy chain variable region and the light chain variable region. For example, the mutation in the heavy chain variable region is G44C (Eu number) and/or the mutation in the light chain variable region is T104C (Eu number).

In another embodiment, the present invention relates to an anti-LAG-3 antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising:

Heavy chain variable region, which contains or is composed of the amino acid sequence of SEQ ID NO:4, and/or

The light chain variable region contains or is composed of the amino acid sequence of SEQ ID NO:8.

On the other hand, anti-LAG-3 antibodies or their antigen-binding fragments also contain heavy chain constant regions and/or light chain constant regions.

In one embodiment, the heavy chain constant region is or is derived from the human IgG constant region, such as the IgG1, IgG2, IgG3 or IgG4 constant region, preferably the IgG1 constant region, the IgG2 constant region or the IgG4 constant region.

In one implementation, the constant regions of the antibody can be mutated to improve antibody preparation and purification. For example, the IgG4 constant region can have the S228P (EU number) mutation. The IgG1 constant region can have mutations of L234A, L235A, D265A, and P329A (EU number).

In another implementation, the heavy chain constant region

(i) comprising or consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 9; or

(ii) Containing or consisting of the amino acid sequence of SEQ ID NO:9; or

(iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 10, more preferably no more than 5) amino acid modifications (preferably amino acid substitutions, more preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:9.

In one implementation, the light chain constant region is or is derived from the κ or λ light chain constant region, such as the human κ or λ light chain constant region, such as the human λ light chain constant region.

In another implementation, the light chain constant region

(i) comprising or consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 10; or

(ii) Containing or consisting of the amino acid sequence of SEQ ID NO:10; or

(iii) An amino acid sequence comprising or consisting of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5) amino acid modifications (preferably amino acid substitutions, more preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:10.

In one embodiment, the antigen-binding fragment of the antibody disclosed herein is an scFv fragment. Specifically, the scFv fragment comprises a light chain variable region and a heavy chain variable region linked by a linker. In one embodiment, the linker is a flexible linker, such as a linker having individual glycine and/or serine residues or combinations thereof. In one embodiment, the linker comprises the amino acid sequence (Gly4Ser)n or (GlySer4)n, where n is a positive integer equal to or greater than 1, for example, n is a positive integer from 1 to 7, for example, n is 2, 3, 4, 5, or 6. In one embodiment, n is 1, 2, 3, or 4.

Conservative modification

In another embodiment, the anti-LAG-3 antibody or its antigen-binding portion thereof disclosed herein includes one or more conserved modifications that differ from those antibodies or their antigen-binding portions described above. It should be understood in the art that certain conserved sequence modifications can be made that do not remove antigen binding. See, for example, Brummell et al. (1993) Biochem 32:1180-8; de Wildt et al. (1997) Prot. Eng. 10:835-41; Komissarov et al. (1997) J. Biol. Chem. 272:26864-26870; Hall et al. (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al. (1998) Int. Immunol. 10:341-6; and Beers et al. (2000) Clin. Can. Res. 6:2835-43.

Therefore, in one embodiment, the anti-LAG-3 antibody or its antigen-binding portion of the present disclosure comprises heavy chain variable region sequences and/or light chain variable region sequences that differ from those described above in that they have one or more conserved modifications, preferably not occurring in the CDR, and preferably occurring in the FR.

In some implementations, modifications may be introduced into the heavy chain variable region and/or the light chain variable region to form disulfide bonds with each other, thereby improving the stability of the scFv from which antibodies can be constructed.

In some implementations, modifications are substitutions, additions, and/or omissions.

In some implementations, the substitution is a conservative substitution. A conservative substitution means that one amino acid is replaced by another amino acid of the same class, such as an acidic amino acid being replaced by another acidic amino acid, a basic amino acid being replaced by another basic amino acid, or a neutral amino acid being replaced by another neutral amino acid. Exemplary substitutions are shown in Table 1 below:

Table 1. Exemplary amino acid substitutions

In various implementation schemes, the antibody may be, for example, a mouse antibody, a human antibody, a humanized antibody, or a chimeric antibody.

As used herein, the term "conserved sequence modification" is intended to refer to amino acid modifications that do not significantly affect or alter the binding properties of antibodies containing the said amino acid sequence. Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present invention using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conserved amino acid substitution is an amino acid substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues within the CDR region of the antibody of the present invention can be substituted with other amino acid residues from the same side chain family, and the retained function (i.e., the aforementioned function) of the altered antibody can be tested using the functional assays described herein.

Engineered and modified antibodies

The modified antibody can be engineered to prepare the antibody of the present invention using one or more of the _VH / _VL sequences of the anti-LAG-3 antibody of the present invention as starting material. The antibody can be engineered by modifying one or more residues within one or two variable regions (i.e., _VH and/or _VL ), such as within one or more CDR regions and/or one or more frame regions. Alternatively or additionally, the antibody can be engineered by modifying residues within one or more constant regions, for example, to alter one or more effector functions of the antibody.

In some implementations, CDR transplantation can be used to engineer the variable regions of an antibody. Antibodies primarily interact with target antigens through amino acid residues located in the six heavy chain complementarity-determining regions (CDRs) and light chain complementarity-determining regions. For this reason, the amino acid sequence within the CDRs is more diverse than the sequence outside the CDRs among individual antibodies. Since CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the characteristics of naturally occurring antibodies with specific properties by constructing expression vectors comprising CDR sequences from naturally occurring antibodies with different properties grafted onto frame sequences from said specific antibodies (see, for example, Riechmann et al. (1998) Nature 332:323-327; Jones et al. (1986) Nature 321:522-525; Queen et al. (1989) Proc. Natl. Acad. See. U.S.A. 86:10029-10033; U.S. Patent Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

Therefore, another embodiment of the present invention relates to an isolated monoclonal antibody or its antigen-binding portion comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO: 1, 2, and 3; and/or a light chain variable region comprising CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO: 5, 6, and 7. Although these antibodies contain the same _VH and _VL CDR sequences, they may contain different frame sequences.

Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences or from publicly available references. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the “VBase” human germline sequence database (available online at www.mrc-cpe.cam.ac.uk/vbase), as well as Kabat et al. (1991) (cited above); Tomlinson et al _{. (1992), “The Repertoire of Human Germline V H Sequences Reveals about Fifty Groups of V H Segments with} Different Hypervariable Loops”, J.Mol.Biol.227:776-798; and Cox et al _. (1994), “A Directory of Human Germ-line V _H Segments Reveals a Strong Bias in their Usage”. The catalog of _H- segments reveals a strong preference for their use,” Eur. J. Immunol. 24:827-836, each of which is explicitly incorporated herein by reference. As another example, germline DNA sequences of human heavy and light chain variable region genes can be found in gene bank databases. For example, the following heavy chain germline sequences found in HCo7 HuMAb mice can be obtained with the attached gene bank accession numbers: 1-69 (NG _-- 0010109, NT _-- 024637 and BC070333), 3-33 (NG _-- 0010109 and NT _-- 024637) and 3-7 (NG _-- 0010109 and NT _-- 024637). As another example, the following heavy chain germline sequences found in HCo12 HuMAb mice can be obtained from the attached gene bank accession numbers: 1-69 (NG _-- 0010109, NT _-- 024637 and BC070333), 5-51 (NG _-- 0010109 and NT _-- 024637), 4-34 (NG _-- 0010109 and NT _-- 024637), 3-30.3 (CAJ556644) and 3-23 (AJ406678).

Using one of the sequence similarity search methods known to those skilled in the art, known as Gapped BLAST, the antibody protein sequence was compared with a compiled protein sequence database (Altschul et al. (1997) (ibid.)).

Preferred frame sequences in the antibodies used in this invention are those frame sequences that are structurally similar to the frame sequences used in the antibodies of this invention. The _{V H} CDR1, CDR2, and CDR3 sequences can be transplanted into frame regions having sequences identical to those present in the germline immunoglobulin genes from which the frame sequences originate, or the CDR sequences can be transplanted into frame regions containing one or more mutations compared to the germline sequences. For example, it has been found that in certain circumstances, it is advantageous to mutate residues within the frame regions to maintain or enhance the antigen-binding capacity of the antibody (see, for example, U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

Another type of variable region modification involves mutating amino acid residues within the _VH and/or _VL CDR1, CDR2, and/or CDR3 regions to improve one or more binding properties (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce one or more mutations, and the effect on antibody binding or other functional properties of interest can be evaluated in in vitro or in vivo assays as described herein and in the examples. Preferably, conserved modifications (as known in the art) are introduced. The mutations can be amino acid substitutions, additions, or deletions, but substitution is preferred. Furthermore, typically no more than one, two, three, four, or five residues within the CDR regions are altered.

Therefore, in another embodiment, the present invention provides an isolated anti-LAG-3 monoclonal antibody or its antigen-binding moiety comprising a heavy chain variable region, said heavy chain variable region comprising: (a) a V H CDR1 region comprising SEQ ID NO:1 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions compared to SEQ ID NO:1; (b) a V _H CDR2 region comprising SEQ ID NO:2 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions compared to SEQ ID NO:2; (c) a V _H CDR3 region comprising SEQ ID NO:3 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions compared to SEQ ID NO:3; (d) a V _L CDR1 region comprising SEQ ID NO:5 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions compared to SEQ ID NO:5; (e) a V _H CDR1 region comprising SEQ ID NO:6 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions compared to SEQ ID NO:5; NO:6 compared to the V _L CDR2 region having an amino acid sequence with 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions; and (f) the V L CDR3 region containing SEQ ID NO:7 or having an amino acid sequence with 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions compared to _SEQ ID NO:7.

The engineered antibodies of this invention include those in which framework residues within _VH and/or _VL have been modified, for example, to improve antibody properties. Typically, such framework modifications are performed to reduce the immunogenicity of the antibody. For example, one method is to “reverse mutate” one or more framework residues to the corresponding germline sequence. More specifically, antibodies that have undergone somatic mutations may contain framework residues different from the germline sequence from which said antibody originates. Such residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody originates.

Another type of framework modification involves mutating one or more residues within the framework region or even one or more CDR regions to remove T-cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also known as "deimmunization" and is described in more detail in U.S. Patent Publication No. 20030153043.

In addition to modifications within the frame region or CDR region, or as an alternative to modifications within the frame region or CDR region, the antibodies of the present invention can be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cytotoxicity. Furthermore, the antibodies of the present invention can be chemically modified (e.g., one or more chemical moieties can be linked to the antibody), or modified to alter its glycosylation, thereby again altering one or more functional properties of the antibody. Each of these embodiments is described in more detail below. The residues in the Fc region are numbered according to the Kabat EU index.

In a preferred embodiment, the antibody is an IgG4 isotype antibody containing a serine-proline mutation at position 241 in the hinge region of the heavy chain constant region, corresponding to position 241 as described in Angal et al. (1993) Mol. Immunol. 30:105-108. This mutation reportedly eliminates the heterogeneity of inter-heavy chain disulfide bridges in the hinge region (Angal et al. (ibid.); position 241 is based on the Kabat numbering system).

In one embodiment, the hinge region of CH1 is modified to alter the number of cysteine residues in the hinge region, for example, by increasing or decreasing them. This method is further described in U.S. Patent No. 5,677,425. Changing the number of cysteine residues in the hinge region of CH1 can, for example, promote the assembly of light and heavy chains or increase or decrease antibody stability.

In another embodiment, the Fc hinge region of the antibody is mutated to shorten the antibody's biological half-life. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc hinge fragment to such that the antibody has impaired binding to staphylococcal protein A (SpA) relative to the native Fc hinge domain SpA binding. This method is described in more detail in U.S. Patent No. 6,165,745.

In yet another embodiment, the glycosylation of the antibody is modified. For example, glycosylated-free antibodies (i.e., antibodies lacking glycosylation) can be prepared. Glycosylation can be altered to, for example, increase the antibody's affinity for the antigen. Such carbohydrate modification can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be performed, which cause the elimination of one or more variable region framework glycosylation sites, thereby eliminating the glycosylation at that site. Such glycosylation can increase the antibody's affinity for the antigen. See, for example, U.S. Patent Nos. 5,714,350 and 6,350,861.

Alternatively or additionally, antibodies with altered glycosylation profiles can be prepared, such as low-fucosylated antibodies with reduced fucosylation residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation profiles have been shown to increase the ADCC ability of antibodies. Such carbohydrate modification can be achieved, for example, by expressing the antibody in host cells with altered glycosylation mechanisms. Cells with altered glycosylation mechanisms have been described in the art and can be used as host cells for expressing the recombinant antibodies of the present invention, thereby producing antibodies with altered glycosylation. For example, cell lines Ms704, Ms705, and Ms709 lack the fucosylation gene FUT8 (α(1,6)-fucosylation), and therefore antibodies expressed in Ms704, Ms705, and Ms709 cell lines lack fucosylation on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8 ^-/- cell lines were generated by targeting and disrupting the FUT8 gene in CHO/DG44 cells using two alternative vectors (see U.S. Patent Publication No. 20040110704 and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene (which encodes fucosyltransferase), and thus antibodies expressed in such cell lines exhibit low fucosylation by reducing or eliminating α-1,6 bond-associated enzymes. EP 1,176,195 also describes cell lines with low or no enzymatic activity for adding fucose to the Fc region of an antibody, such as the rat myeloma cell line YB2/0 (ATCC CRL 1662). PCT Publication WO03/035835 describes a variant CHO cell line, Lec13, with a reduced ability to link fucose to Asn(297)-linked carbohydrates, thereby also causing hypofucosylation of antibodies expressed in this host cell (see also Shields et al. (2002) J. Biol. Chem. 277:26733-26740). Antibodies with modified glycosylation profiles can also be produced in eggs, as described in PCT Publication WO 06/089231. Alternatively, antibodies with modified glycosylation profiles can be produced in plant cells, such as duckweed. A method for producing antibodies in plant systems is disclosed in U.S. Patent Application No. 040989/314911, filed August 11, 2006, by Attorney General for Alston & Bird LLP. PCT publication WO 99/54342 describes cell lines engineered to express glycosyltransferases (e.g., β(1,4)-N-acetylglucosamine transferase III (GnTIII)) that modify glycoproteins. Antibodies expressed in these engineered cell lines exhibit an increased bifurcated GlcNac structure, which leads to increased ADCC activity (see also Umana et al. (1999) Nat. Biotech. 17:176-180). Alternatively, fucosidases can be used to cleave the fucose residues from the antibody; for example, fucosidase α-L-fucosidase removes fucose residues from the antibody (Tarentino et al. (1975) Biochem. 14:5516-23).

Another modification of the antibodies described herein that is considered in this disclosure is polyethylene glycol (PEG) oxidation. Antibodies can be PEGylated to, for example, extend their biological (e.g., serum) half-life. To PEGylate an antibody, the antibody or a fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups are attached to the antibody or antibody fragment. Preferably, PEGylation is performed via an acylation or alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). The term “polyethylene glycol” as used herein is intended to cover any of the forms of PEG already used to derivatize other proteins, such as mono(C1-C10)alkoxy polyethylene glycol, aryloxy polyethylene glycol, or polyethylene glycol-cis-butenediamide. In some embodiments, the antibody to be PEGylated is a glycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of this invention. See, for example, EPO 154 316 and EP 0 401 384.

II. Antibody Physical Properties

The antibodies of the present invention can be characterized by their various physical properties in order to detect and/or distinguish their different categories.

For example, antibodies may contain one or more glycosylation sites in the variable regions of the light chain or heavy chain. Such glycosylation sites may lead to increased immunogenicity or altered pK of the antibody due to altered antigen binding (Marshall et al. (1972) Annu Rev Biochem 41:673-702; Gala and Morrison (2004) J Immunol 172:5489-94; Wallick et al. (1988) J Exp Med 168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al. (1985) Nature 316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706). Glycosylation is known to occur at motifs containing N-X-S/T sequences. In some cases, it is preferable to have anti-LAG-3 antibodies that do not contain glycosylation in the variable region. This can be achieved by selecting antibodies that do not contain the glycosylation motif in the variable region or by mutating residues within the glycosylation region.

In a preferred embodiment, the antibody does not contain an asparagine isomer site. Deamidation of asparagine may occur at the N-G or D-G sequence and result in the formation of isoaspartic residues, which introduce bends into the polypeptide chain and reduce its stability (isoaspartic effect).

Each antibody will have a unique isoelectric point (pI), which generally falls within the pH range of 6 to 9.5. The pI value of IgG1 antibodies typically falls within the pH range of 7–9.5, and the pI value of IgG4 antibodies typically falls within the pH range of 6–8. It is presumed that antibodies with pI values outside the normal range may exhibit some unfolding and instability under in vivo conditions. Therefore, anti-LAG-3 antibodies with pI values falling within the normal range are preferred. This can be achieved by selecting antibodies with pI values within the normal range or by mutating the charged surface residues.

III. Pharmaceutical Composition

In another aspect, this disclosure provides pharmaceutical compositions comprising the anti-AG-3 antibody of this disclosure formulated with pharmaceutically acceptable excipients and optionally containing one or more other therapeutic agents. In some embodiments, the other therapeutic agents are immunostimulatory antibodies and/or chemotherapeutic agents.

The pharmaceutical composition may contain any number of excipients. Excipients that may be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersants or suspending agents, solubilizers, colorants, flavoring agents, coating agents, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof. The selection and use of suitable excipients are taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th edition (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

In some embodiments, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound may be encapsulated in a material to protect it from the effects of acids and other natural conditions that may inactivate it. The phrase "parenteral administration" as used herein refers to a method of administration other than enteral and local administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal injections and infusions. Optionally, the antibodies of the present invention may be administered via non-parenteral routes, such as local, epidermal, or mucosal administration routes, for example, intranasal, oral, vaginal, rectal, sublingual, or local administration. Preferably, the antibodies are formulated as intravenous infusion or subcutaneous injection preparations.

Pharmaceutical compositions can be in the form of sterile aqueous solutions or dispersions. They can also be formulated into microemulsions, liposomes, or other ordered structures suitable for high drug concentrations.

The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the specific method of administration, and will generally be the amount of the composition that produces the therapeutic effect. Generally, in 100 percent, when combined with a pharmaceutically acceptable carrier, this amount will be in the range of about 0.01% to about 99% of the active ingredient, preferably about 0.1% to about 70%, and most preferably about 1% to about 30% of the active ingredient.

The pharmaceutical composition may be a controlled-release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene-vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. See, for example, *Sustained and Controlled Release Drug Delivery Systems*, ed. J.R. Robinson, Marcel Dekker, Inc., New York, 1978.

Therapeutic drugs can be administered via medical devices such as (1) needle-free subcutaneous injection devices (e.g., U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 and 4,596,556); (2) microinfusion pumps (U.S. Patent No. 4,487,603); (3) transdermal devices (U.S. Patent No. 4,486,194); (4) infusion devices (U.S. Patent Nos. 4,447,233 and 4,447,224); and (5) permeation devices (U.S. Patent Nos. 4,439,196 and 4,475,196); the disclosures of these documents are incorporated herein by reference.

In some embodiments, the pharmaceutical composition comprises (i) an anti-LAG-3 antibody of the present disclosure; (ii) an immunostimulatory antibody, such as an anti-PD-1 antibody (e.g., tislelizumab, toripalimab), an anti-PD-L1 antibody, or an anti-CTLA-4 antibody; (iii) gemcitabine, such as gemcitabine hydrochloride; and (iv) a platinum-based antibody, such as carboplatin or cisplatin.

IV. Combination therapy and drug combinations

On the other hand, this disclosure provides combination therapies in which the anti-LAG-3 antibody of this disclosure is administered in combination with one or more other therapeutic agents (i.e., drug combinations) and/or treatment modalities that effectively alleviate cancer, infection or autoimmune disease in a subject, such as other antibodies or chemotherapeutic agents.

In some embodiments, this disclosure provides a combination therapy for treating a subject’s cancer disease, the therapy comprising administering the antibody of the present invention to the subject and one or more other therapies (e.g., standard disease treatment, such as standard cancer treatment), such as treatment modalities and/or other therapeutic agents.

In some implementations, the treatment methods include surgery, radiation, cryosurgery, and/or thermotherapy.

In some embodiments, the additional therapeutic agent is, for example, another antibody, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, and/or an anti-CTLA-4 antibody.

In some embodiments, the therapeutic agent is selected from chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infective agents, small molecule drugs, or immunomodulators.

In one aspect, the present invention provides a pharmaceutical combination comprising the anti-LAG-3 antibody disclosed herein and one or more other therapeutic agents.

In some implementations, the methods described herein and the anti-LAG-3 antibody are administered in combination with one or more of the following: other antibody molecules, chemotherapy, other anticancer therapies (e.g., targeted anticancer therapies or oncolytic agents), cytotoxic agents, immune-based therapies (e.g., cytokines), surgery, and/or radiation.

In exemplary embodiments, the anti-LAG-3 antibody of this disclosure can be combined with other cancer treatments. For example, the antibody of this disclosure can be combined with immunostimulatory antibody therapy. For instance, the anti-LAG-3 antibody of this disclosure can be effectively combined with anti-PD-1 antibody (such as tislelizumab, toripalimab) treatment regimens. For instance, the anti-LAG-3 antibody of this disclosure can be effectively combined with standard cancer chemotherapy regimens. In these cases, the dosage of the administered chemotherapy agent can be reduced (Mokyr, M. et al., (1998) Cancer Research 58:5301-5304).

In some embodiments, the anti-LAG-3 antibody, immunostimulatory antibody, and standard cancer treatment chemotherapy agents of this disclosure are used in combination. The immunostimulatory antibody includes, but is not limited to, anti-PD-1 antibodies, anti-PD-L1 antibodies, or anti-CTLA-4 antibodies, such as anti-PD-1 antibodies like tislelizumab or toripalimab. The standard cancer treatment chemotherapy agents include, but are not limited to, platinum-based drugs and gemcitabine; more preferably, the platinum-based drug is carboplatin or cisplatin, and the gemcitabine is gemcitabine hydrochloride. In some embodiments, the drug combination comprises the anti-LAG-3 antibody of this invention; an immunostimulatory antibody, such as tislelizumab or toripalimab; gemcitabine, such as gemcitabine hydrochloride; and a platinum-based drug, such as carboplatin or cisplatin.

In some implementations, the disclosed anti-LAG-3 antibody is combined with tislelizumab, gemcitabine hydrochloride, and cisplatin.

In some embodiments, the anti-LAG-3 antibody and one or more other therapeutic agents described in this disclosure are administered to a patient simultaneously, without a specific time limit, or sequentially at the same or different time intervals, in separate entities, wherein such administration to the patient provides a preventive or therapeutically effective level of the two active agents. Exemplarily, the anti-LAG-3 antibody and one or more other therapeutic agents of this disclosure are a kit.

In some specific embodiments, the anti-LAG-3 antibody described in the pharmaceutical combination of this disclosure is simultaneously administered to a patient in the form of a single entity with one or more other therapeutic agents. Preferably, the dosage and/or time interval between the anti-LAG-3 antibody of this disclosure and one or more other therapeutic agents are selected so that the combined use of the components produces an effect greater than that achieved by using any one component alone in treating the disease or condition. Each component may be in a separate formulation, and their formulations may be the same or different. The anti-LAG-3 antibody or its antigen-binding portion and/or other therapeutic agents in the pharmaceutical combination of this invention may be a drug dosing unit, such as a single drug dosing unit.

In other embodiments, the anti-LAG-3 antibody or its antigen-binding portion described in the pharmaceutical compositions of this disclosure is present in the same pharmaceutical composition as one or more other therapeutic agents.

In one aspect, the invention also relates to a pillbox containing the drug combination of the invention, preferably in the form of a drug dosage unit. This allows the dosage unit to be provided according to a dosing regimen or drug administration interval.

In one embodiment, the complete medicine box of the present invention comprises, within the same package:

- A first container containing the anti-LAG-3 antibody or pharmaceutical composition disclosed herein;

- A second container containing one or more other therapeutic agents, such as immunostimulatory antibodies and/or chemotherapeutic agents, such as anti-PD-1 antibodies and/or standard cancer treatment chemotherapeutic agents. In some embodiments, the anti-LAG-3 antibody of this disclosure and other therapeutic agents, such as immunostimulatory antibodies and/or chemotherapeutic agents, in the pharmaceutical combination of this invention may each be in a separate dosage form, for example, any dosage form known to those skilled in the art, such as tablets, capsules, granules, syrups, powders, lozenges, capsules, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments, creams, and injections, etc., and their dosage forms may be different or the same. In some embodiments, the anti-LAG-3 antibody of this disclosure or its antigen-binding portion is preferably formulated as an intravenous infusion or subcutaneous injection formulation. In some embodiments, the immunostimulatory antibody of this invention is formulated as an intravenous infusion or subcutaneous injection formulation. In some embodiments, the chemotherapeutic agent of this invention is formulated as an intravenous infusion or oral formulation for administration. In some embodiments, in the drug combination of the present invention, the anti-LAG-3 antibody or its antigen-binding portion thereof disclosed herein is administered intravenously, the immunostimulatory antibody is administered intravenously, and the chemotherapeutic agent is also administered intravenously.

V. Uses/Treatment Methods

The anti-LAG-3 antibody or its antigen-binding portion disclosed herein has numerous in vitro and in vivo uses relating to the treatment of, for example, cancer, infections, and autoimmune diseases. The anti-LAG-3 antibody or its antigen-binding portion disclosed herein can be administered to human subjects, e.g., in vivo, to treat these diseases. In some embodiments, the treatment methods of this disclosure include administering the anti-LAG-3 antibody or its antigen-binding portion disclosed herein to a subject. In other embodiments, the treatment methods of this disclosure include administering the anti-LAG-3 antibody or its antigen-binding portion disclosed herein to a subject and one or more other therapies (e.g., standard disease treatment, such as standard cancer treatment), such as modalities of treatment and/or other therapeutic agents.

In one aspect, the present invention provides a method for modulating the immune response of a subject.

In some embodiments, the present invention provides a method for antitumor activity by enhancing immune responses (e.g., T cell-mediated immune responses).

In some embodiments, the present invention provides a method for blocking LAG-3.

In some embodiments, the above methods include administering the anti-LAG-3 antibody of this disclosure or a pharmaceutical combination/combination therapy containing the antibody. Specifically, the above methods include administering the anti-LAG-3 antibody of this disclosure, a pharmaceutical composition or formulation containing the anti-LAG-3 antibody of this disclosure, a combination product, or a nucleic acid encoding the anti-LAG-3 antibody of this disclosure or its antigen-binding portion.

In another aspect, the present invention provides a method for treating a tumor in a subject, the method comprising administering an anti-LAG-3 antibody of the present disclosure or a pharmaceutical composition or combination thereof containing the present disclosure.

In some implementations, the tumor is a malignant tumor, such as cancer.

In some implementations, cancer includes solid cancer, non-solid cancer, and metastatic lesions. In one implementation, an example of solid cancer includes malignant tumors. Cancer can be early, intermediate, late, or metastatic.

In some embodiments, the malignant tumor is an advanced malignant tumor, preferably selected from nasopharyngeal carcinoma and melanoma.

In some implementations, cancer treatment will benefit from: (a) binding to human LAG-3; (b) binding to monkey LAG-3; (c) lack of binding to mouse LAG-3; (d) binding to LAG-3 at a domain that binds to MHC class II molecules; (e) inhibiting the binding of LAG-3 to major MHC class II molecules; (f) inhibiting the binding of LAG-3 to LSECtin; (g) stimulating an immune response; (h) stimulating an antigen-specific T cell response; and (i) any one or a combination of the above.

In some implementations, treatments using the anti-LAG-3 antibody or its antigen-binding portion disclosed herein can be used as first-line, second-line, or multiple-line treatments for malignant tumors or cancers.

In some specific implementations, the drug is administered to a subject suffering from the malignant tumor, the subject being a subject with relapsed and refractory advanced malignant tumors, a subject with recurrent or metastatic advanced malignant tumors who has not previously received systemic treatment, or a subject with unresectable or metastatic advanced malignant tumors; preferably, the subject is a subject with recurrent or metastatic advanced nasopharyngeal carcinoma who has not previously received systemic treatment.

In some other embodiments, the nasopharyngeal carcinoma subject is LAG-3 positive (LAG-3 ≥ 1%) and PD-L1 positive (PD-L1 ≥ 1%). In still other embodiments, the nasopharyngeal carcinoma subject is LAG-3 negative (LAG-3 < 1%) or PD-L1 negative (PD-L1 < 1%). In still other embodiments, the nasopharyngeal carcinoma subject is a patient with nasopharyngeal carcinoma who has not received anti-PD-(L)1 antibody therapy or who has progressed or is intolerant to anti-PD-(L)1 antibody therapy.

VI. Dosing regimen

In the treatment methods of the present invention, the anti-LAG-3 antibody or its antigen-binding portion thereof disclosed herein is administered to a human subject at a therapeutically effective dose.

The therapeutically effective dose of the anti-LAG-3 antibody or its antigen-binding portion thereof of this disclosure preferably causes a reduction in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods, or prevention of impairment or disability caused by disease distress. For example, in treating a subject carrying a tumor, the therapeutically effective dose preferably inhibits tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more preferably at least about 80%, compared to an untreated subject. The therapeutically effective dose of the therapeutic compound can reduce tumor size or otherwise improve symptoms in a subject who is typically human or may be another mammal.

The dosing regimen can be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus injection may be administered, several fractionated doses may be administered over time, or the dose may be proportionally reduced or increased, as indicated by the urgency of the treatment situation. Particularly advantageous is to formulate the parenteral composition into unit dosage forms for ease of administration and to maintain uniform dosage. As used herein, unit dosage forms refer to physically discrete units suitable for use as unit doses in the subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with the desired drug carrier. Alternatively, the antibody may be administered as a sustained-release formulation, in which case less frequent administration is required.

The dosing regimens of the anti-LAG-3 antibody described below are applicable to both treatment methods that administer the anti-LAG-3 antibody of this disclosure alone and to drug combinations/combination therapies of the present invention.

In the drug combination/combination therapy of the present invention, the dosing cycles of the anti-LAG-3 antibody and one or more other therapeutic agents disclosed herein may be the same or different.

In the dosing regimen of this invention, the anti-LAG-3 antibody disclosed herein can be administered to individuals in need at one or more doses.

In some embodiments, approximately 50 mg to 5000 mg of the anti-LAG-3 antibody, preferably 60 mg to 4000 mg, more preferably 80 mg to 3750 mg, and most preferably 100 mg to 3500 mg of the anti-LAG-3 antibody, such as 200 mg, 400 mg, 600 mg, 800 mg, 1000 mg, 1250 mg, 1500 mg, 1750 mg, 2000 mg, 2250 mg, 2500 mg, 2750 mg, 3000 mg, 3250 mg, and 3500 mg of the anti-LAG-3 antibody, are administered to the subject during each dosing cycle or each administration.

In some embodiments, approximately 0.05 mg/kg to 200 mg/kg of the anti-LAG-3 antibody or its antigen-binding portion is administered to the subject per dosing cycle or per administration, preferably 0.25 mg/kg to 10 mg/kg of the anti-LAG-3 antibody or its antigen-binding portion, more preferably 1.0 mg/kg to 6 mg/kg of the anti-LAG-3 antibody or its antigen-binding portion, and most preferably 6.0 mg/kg of the anti-LAG-3 antibody or its antigen-binding portion. In some embodiments, the anti-LAG-3 antibody or its antigen-binding portion is administered to the subject at a dose of 0.25 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 1.0 mg/kg, 1.2 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 8 mg/kg, or 10 mg/kg per dosing cycle or per administration.

In some implementations, the administration of the anti-LAG-3 antibody can be completed within 30-120 minutes, for example, more than 45 minutes, more than 60 minutes, more than 75 minutes, more than 90 minutes, or more than 105 minutes.

In some embodiments, when multiple doses are administered, the next dose is administered approximately 2 to 84 days after the previous dose. For example, each administration cycle of the anti-LAG-3 antibody of this disclosure is approximately 2 to 84 days, for example, administration cycles are every 12 weeks, every 9 weeks, every 6 weeks, every 5 weeks, every 4 weeks, every 3 weeks, every 2 weeks, once a week, twice a week, or three times a week. In some embodiments, the anti-LAG-3 antibody of this disclosure is administered to the subject for, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 administration cycles, preferably, the anti-LAG-3 antibody administration time does not exceed two years.

Another therapeutic agent in the pharmaceutical combination of the present invention, such as an immunostimulating antibody, may be administered approximately once a day, once every two days, once every three days, or once every four days. In some embodiments, the immunostimulating antibody may be administered for 1-5 days, for example, for 1 day or 3 days.

Another therapeutic agent, such as a chemotherapeutic agent, in the drug combination of the present invention may be administered approximately once a day, once every two days, once every three days, or once every four days. In some embodiments, the chemotherapeutic agent may be administered for 1-8 days, for example, for 1 day, 3 days, 5 days, or 8 days.

In some embodiments, the immunostimulatory antibody in the drug combination/combination therapy of the present invention is an anti-PD-1 antibody (e.g., tislelizumab or toripalimab), wherein 100 mg to 600 mg of the immunostimulatory antibody is administered to the subject in each dosing cycle or per administration, for example, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg or 600 mg of tislelizumab per administration or 100 mg, 200 mg, 240 mg, 300 mg, 400 mg, 500 mg or 600 mg of toripalimab per administration.

In some embodiments, the chemotherapeutic agents in the drug combination/combination therapy of the present invention are gemcitabine and platinum, such as gemcitabine hydrochloride and cisplatin, wherein: gemcitabine is administered at a rate not exceeding 1000 mg/ ^m² per dosing cycle or per administration, for example, gemcitabine hydrochloride at a rate of 1000 mg/ ^m² per administration; and the platinum is administered at a rate not exceeding 80 mg/ ^m² per dosing cycle or per administration, for example, cisplatin at a rate of 80 mg/ ^m² per administration.

The anti-LAG-3 antibody or drug combination of this disclosure may be administered before, simultaneously with, or after the administration of other therapeutic agents. When the anti-LAG-3 antibody of this disclosure is administered "before" the administration of other therapeutic agents, the interval between the administration of the anti-LAG-3 antibody of this disclosure and the administration of other therapeutic agents may be greater than 150 hours, about 150 hours, about 100 hours, about 72 hours, about 60 hours, about 48 hours, about 36 hours, about 24 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1.5 hours, about 1 hour, or about 30 minutes, about 15 minutes, or about 10 minutes, or no interval. When administered after the initiation of other therapeutic agents, the interval between administration of the anti-LAG-3 antibody of this disclosure and other therapeutic agents may be no interval, approximately 10 minutes, approximately 15 minutes, approximately 30 minutes, approximately 1 hour, approximately 1.5 hours, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 8 hours, approximately 10 hours, approximately 12 hours, approximately 24 hours, approximately 36 hours, approximately 48 hours, approximately 60 hours, approximately 72 hours, or more than 72 hours. "Simultaneous" administration with the initiation of other therapeutic agents means that the anti-LAG-3 antibody of this disclosure is administered to the individual within less than 10 minutes (before, after, or simultaneously) of the initiation of other therapeutic agents. Preferably, the anti-LAG-3 antibody or the anti-LAG-3 antibody in the drug combination of the present disclosure is administered before the administration of other therapeutic agents. For example, the interval between the administration of the anti-LAG-3 antibody of the present disclosure and other therapeutic agents may be no interval, about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 6 hours, about 12 hours, about 15 hours, or about 24 hours.

In some implementations, the drug combination/combination therapy is administered in cycles, for example, each administration cycle is at least 14-35 days, such as 2, 3, 4 or 5 weeks, preferably 3 weeks.

The drug combination/combination therapy of the present invention can be administered for at least one cycle, for example, 2-6 or more treatment cycles. The number of administration cycles for the anti-LAG-3 antibody and other therapeutic agents in the drug combination/combination therapy can differ; for example, the number of administration cycles for antibody therapeutic agents is generally no more than two years, while the number of administration cycles for other therapeutic agents (e.g., chemotherapeutic agents) is 2-6 cycles, preferably 4-6 cycles.

Preferably, the anti-LAG-3 antibody of this disclosure is administered once or twice in each cycle, or the anti-LAG-3 antibody of this disclosure is administered in each cycle; and/or

Administer additional treatments at least on days 1 through 5 of each cycle, such as on day 1 or day 1 through 3, and then discontinue treatment until the end of the cycle.

The drug combination/combination therapy of the present invention can be administered every 5 weeks, every 4 weeks, every 3 weeks, every 2 weeks or weekly.

In some embodiments, the drug combination/combination therapy comprises

(i) The anti-LAG-3 antibody or its antigen-binding portion disclosed herein;

(ii) Anti-PD-1 antibodies, such as tislelizumab;

(iii) Gemcitabine, such as gemcitabine hydrochloride; and

(iv) Platinum compounds, such as cisplatin.

In some embodiments of the drug combination/combination therapy,

(i) The single-dose administration is approximately 0.05 mg/kg to 200 mg/kg, preferably 0.25 mg/kg to 10 mg/kg, more preferably 1.0 mg/kg to 6.0 mg/kg, most preferably 6.0 mg/kg, for example 0.25 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 1.0 mg/kg, 1.2 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 8 mg/kg or 10 mg/kg; or 50 mg to 5000 mg, preferably 60 mg to 4000 mg, more preferably 80 mg to 3750 mg, most preferably 100 mg to 3500 mg, for example 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1250 mg, 1500 mg, 1750 mg, 2000 mg, 2250 mg, 2500 mg, 2750 mg, 3000 mg, 3250 mg, 3500 mg;

(ii) A single dose is approximately 100 mg to 600 mg, such as 100 mg, 200 mg, 300 mg, 400 mg, 500 mg or 600 mg;

(iii) A single administration dose not exceeding 1000 mg/ ^m² , for example, approximately 1000 mg/ ^m² ; and/or

(iv) The single dose is not higher than 80 mg/ ^m² , for example, about 80 mg/ ^m² .

In a specific implementation plan, the anti-LAG-3 antibody disclosed herein is used in combination with tislelizumab, gemcitabine hydrochloride, and cisplatin to treat patients with nasopharyngeal carcinoma who are LAG-3 positive (LAG-3 ≥ 1%) and PD-L1 positive (PD-L1 ≥ 1%), or who are LAG-3 negative (LAG-3 < 1%) or PD-L1 negative (PD-L1 < 1%).

In some embodiments, the drug combination/combination therapy comprises

(i) the anti-LAG-3 antibody or its antigen-binding portion disclosed herein; and

(ii) Anti-PD-1 antibodies, such as toripalimab.

In some embodiments of the drug combination/combination therapy,

(i) The single-dose administration is approximately 0.05 mg/kg to 200 mg/kg, preferably 0.25 mg/kg to 10 mg/kg, more preferably 1.0 mg/kg to 6.0 mg/kg, most preferably 6.0 mg/kg, for example 0.25 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 1.0 mg/kg, 1.2 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 8 mg/kg or 10 mg/kg; or 50 mg to 5000 mg, preferably 60 mg to 4000 mg, more preferably 80 mg to 3750 mg, most preferably 100 mg to 3500 mg, for example 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1250 mg, 1500 mg, 1750 mg, 2000 mg, 2250 mg, 2500 mg, 2750 mg, 3000 mg, 3250 mg, 3500 mg;

(ii) A single dose is approximately 100 mg to 600 mg, such as 100 mg, 200 mg, 240 mg, 300 mg, 400 mg, 500 mg or 600 mg; or 1 mg/kg to 10 mg/kg, such as 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 8 mg/kg or 10 mg/kg.

In a specific implementation plan, the combination of the anti-LAG-3 antibody and toripalimab disclosed herein is used to treat nasopharyngeal carcinoma patients who have not received anti-PD-(L)1 antibody therapy or who have progressed or are intolerant to anti-PD-(L)1 antibody therapy.

Other therapeutic agents in the drug combination/combination therapy of the present invention, such as immunostimulatory antibodies, are administered approximately once a day, once every two days, once every three days, or once every four days. In some embodiments, the immunostimulatory antibodies are administered for 1-5 days, for example, for 1 day or 3 days.

Other therapeutic agents, such as chemotherapeutic agents, in the drug combination/combination therapy of the present invention are administered approximately once a day, once every two days, once every three days, or once every four days. In some embodiments, the chemotherapeutic agent is administered for 1-8 days, for example, for 1 day, 3 days, 5 days, or 8 days.

In some embodiments of the drug combination, the drug combination is administered in a three-week cycle, wherein each cycle is administered once (i), once (ii), once (iii), and once (iv), preferably, the first administration is administered after (i) (e.g., immediately or about 15, 30, 60 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, or 3 hours later), the first administration is administered after (ii) (e.g., immediately or about 15, 30, 60 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, or 3 hours later), and the first administration is administered after (iii) (e.g., immediately or about 15, 30, 60 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, or 3 hours later), and the first administration is administered after (iii) (e.g., immediately or about 15, 30, 60 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, or 3 hours later), and the first administration is administered after (iii) (e.g., immediately or about 15, 30, 60 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, or 3 hours later), and the first administration is administered after (iv). In some embodiments, (i) a single dose is 5-20 mg/kg, for example about 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mg/kg; (ii) a single dose is 100 mg/ ^m² (body surface area), preferably gemcitabine with a single dose of 100 mg/ ^m² , preferably administered once daily for 3 days; (iii) a single dose is 100 mg/ ^m² , or AUC = 5 mg/mL/min, ^preferably cisplatin or carboplatin with an AUC = 5 mg/mL/min.

In a preferred embodiment, the drug combination is administered for 4 to 6 cycles.

In one embodiment, after the completion of the entire dosing cycle of the drug combination, the disclosed anti-LAG-3 antibody or its antigen-binding portion is continuously administered, for example, every three weeks, every four weeks, every five weeks, and every six weeks, with each single dose being approximately 0.05 mg/kg to 200 mg/kg, preferably 0.25 mg/kg to 10 mg/kg, more preferably 1.0 mg/kg to 6.0 mg/kg, most preferably 6.0 mg/kg, for example 0.25 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 1.0 mg/kg, 1.2 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1 0.8 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 8 mg/kg or 10 mg/kg; or 50 mg to 5000 mg, preferably 60 mg to 4000 mg, more preferably 80 mg to 3750 mg, most preferably 100 mg to 3500 mg, for example 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1250 mg, 1500 mg, 1750 mg, 2000 mg, 2250 mg, 2500 mg, 2750 mg, 3000 mg, 3250 mg, 3500 mg.

In one specific embodiment, after the completion of all dosing cycles of the drug combination, the disclosed anti-LAG-3 antibody or its antigen-binding portion is continuously administered, for example, once every three weeks, four weeks, five weeks, or six weeks, for dosing cycles of, for example, 1, 3, 5, 7, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 cycles. Preferably, the total duration of drug combination administration and administration of the anti-LAG-3 antibody or its antigen-binding portion alone does not exceed two years.

In a preferred embodiment, the anti-LAG-3 antibody of this disclosure is used in combination with tislelizumab, gemcitabine hydrochloride, and cisplatin, with each dosing cycle lasting 3 weeks. On day 1 of each dosing cycle, 400 mg or 600 mg of anti-LAG-3 antibody is administered intravenously; tislelizumab is administered intravenously at a dose of 200 mg; gemcitabine hydrochloride is administered intravenously at a dose of 1000 mg/ ^m² or less; and cisplatin is administered intravenously at a dose of 80 mg/ ^m² or less. The dosing sequence is as follows: anti-LAG-3 antibody is administered first, followed by tislelizumab after the anti-LAG-3 antibody infusion is completed, followed by gemcitabine hydrochloride after the tislelizumab administration is completed, and cisplatin is administered after the gemcitabine hydrochloride administration is completed. On day 8, gemcitabine is administered intravenously alone at a daily dose of 1000 mg/ ^m² or less. The chemotherapy agent is administered for a maximum of 6 cycles. In a preferred embodiment, the anti-LAG-3 antibody of this disclosure is used in combination with toripalimab, with each dosing cycle being 3 weeks. On day 1 of each dosing cycle, 400 mg of anti-LAG-3 antibody is administered intravenously, followed by 240 mg of toripalimab intravenously. The order of administration is as follows: anti-LAG-3 antibody is administered first, and toripalimab is administered after the anti-LAG-3 antibody infusion is completed.

In some embodiments, administration of the disclosed anti-LAG-3 antibody or its antigen-binding portion as monotherapy or in combination with other therapeutic agents results in an increased, preferably synergistic, increase in progression-free survival (PFS) or overall survival (OS) in patients. In some embodiments, administration of at least one cycle of the disclosed anti-LAG-3 antibody or its antigen-binding portion or combination of drugs results in an increase in PFS of at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 2 years, or longer in patients. In some embodiments, administration of at least one cycle of the disclosed anti-LAG-3 antibody or its antigen-binding portion or combination of drugs results in an increase in OS of at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 2 years, or longer in patients.

The drug combination of the anti-LAG-3 antibody or its antigen-binding portion disclosed herein results in an increased, preferably synergistic, increase in the inhibitory effect on tumor growth. In some embodiments, the anti-LAG-3 antibody or its antigen-binding portion or drug combination of the present invention results in an inhibition of tumor growth of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%. In some embodiments, administration of the drug combination of the present invention results in increased tumor regression, tumor shrinkage, and/or disappearance.

In some embodiments, the drug combination of the anti-LAG-3 antibody or its antigen-binding portion disclosed herein prevents tumor recurrence and/or increases survival duration in an individual, for example, by increasing survival duration by more than 15 days, more than 1 month, more than 3 months, more than 6 months, more than 12 months, more than 18 months, more than 24 months, more than 36 months, or more than 48 months. In some embodiments, the drug combination of the present invention may increase progression-free survival or overall survival.

In some embodiments, administration of the disclosed anti-LAG-3 antibody or its antigen-binding portion or drug combination to an individual with cancer results in the complete disappearance of the tumor (“complete response”). In some embodiments, administration of the disclosed anti-LAG-3 antibody or its antigen-binding portion or drug combination to an individual with cancer results in a reduction of tumor cells or tumor size of at least 30% or more (“partial response”). The reduction in tumor size can be measured by any method known in the art, such as X-ray, positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), cytological, histological, or molecular genetic analysis.

In some embodiments, the anti-LAG-3 antibody of this disclosure or its antigen-binding portion or drug combination may reduce adverse events resulting from the administration of known therapies, such as hematological toxicities, non-hematological toxicities, or other toxicities, such as pneumonia, diarrhea, enterocolitis, renal insufficiency, rash, hepatitis, endocrine disorders, peripheral or central neuritis, and abnormal liver function.

Example

The anti-LAG-3 antibody used in this disclosure is the "anti-LAG3 antibody No. 2" constructed and expressed as described in PCT/CN2018/095528, hereinafter referred to as "antibody No. 2" (heavy chain amino acid sequence as shown in SEQ ID NO:11, light chain amino acid sequence as shown in SEQ ID NO:12).

Example 1: Preclinical Toxicology Study

Toxicity studies and toxicokinetics were conducted on cynomolgus monkeys administered antibody 2 intravenously for 4 weeks and during the recovery period.

This study evaluated the toxicity and toxicokinetics of cynomolgus monkeys after a 4-week recovery period following intravenous infusion of antibody 2. The nature, severity, dose-response relationship, time-response relationship, and reversibility of potential toxic reactions were analyzed. Target organs or tissues were identified, and the immunogenicity and immunotoxicity of the test product were investigated to provide reference information for clinical research. The study consisted of four groups: a control group (0.9% sodium chloride) and antibody 2 dose groups of 10, 30, and 100 mg/kg. Each group contained 10 cynomolgus monkeys, half male and half female (age: 3–5 years; weight: female 2.40–3.55 kg, male 2.30–4.85 kg). The drug was administered once weekly for 5 consecutive weeks, followed by a 4-week recovery period. During the experiment, clinical observations and tests were conducted on the animals, including monitoring of food intake, body weight, body temperature, lead II electrocardiogram, blood pressure, respiratory rate, hematology, blood biochemistry, complement (C3, C4), immunoglobulins (IgG, IgA, IgM), circulating immune complexes (CIC), lymphocyte subsets (CD3+, CD3+CD4+, CD3+CD8+, CD16+CD56+, CD3+CD4+/CD3+CD8+), cytokines (IL-2, IL-6, IL-10, tumor necrosis factor α (TNF-α), IFN-γ), LAG-3 receptor occupancy (RO), anti-drug antibodies, ophthalmological examination, urine analysis, bone marrow smears, gross anatomical observation, organ weight measurement, and histopathological examination. Blood samples were collected from monkeys in each group before the first and last administration of antibody 2, 5 minutes after the start of administration, 5 minutes after the end of administration, and at 6, 24, 72, 120 and 168 hours.

The results showed that no animals in any group died or were near death. No significant abnormalities were observed in the general condition, weight, food intake, body temperature, blood pressure, lead II electrocardiogram, respiratory rate, urine examination, ophthalmological examination, complement levels, immune complexes, immunoglobulins, lymphocyte subsets, cytokines, hormones, bone marrow smears, gross anatomical observation, or histopathological examination. Under the conditions of this experiment, no adverse reactions were observed with antibody 2, and the recommended dosage is 100 mg/kg/day.

At 1–2, 24–25, and 168–169 hours after the first administration, and at 24–25 and 168–169 hours after the last administration, LAG-3RO levels increased in both male and female monkeys in each administration group. No significant dose-related increase in LAG-3RO was observed at any of the dose levels (10, 30, and 100 mg/kg).

Anti-antibody 2 was not detected in any of the animals in the treatment groups on days 17 and 24 of the administration period and on days 2, 9 and 22 of the recovery period.

Within the dosage range of 10–100 mg/kg, after the first administration, the exposure of antibody 2 in the serum of monkeys in each group was generally positively correlated with the infused dose, and there was no sex difference in exposure in monkeys. After five consecutive administrations, the accumulation factor of antibody 2 in monkeys ranged from 2.02 to 0.61, indicating slight accumulation.

Example 2: Clinical Pharmacology Study

Antibody 2 was administered intravenously, with a median time to peak concentration ( _tmax ) ranging from 1 to 9 hours across all dose groups. For most patients, peak plasma concentration was reached at the end of administration. Comparing the different dose groups of Antibody 2, as the dose increased from 0.05 mg/kg to 1 mg/kg, the t1 _/2 of Antibody 2 tended to increase with increasing dose; clearance (CL) tended to decrease with increasing dose, but after reaching 3 mg/kg, CL no longer changed with dose. There was no significant dose-dependent trend in _tmax , terminal volume of distribution ( _Vz ), and steady-state volume of distribution ( _Vss ) among the dose groups. The mean t1/2 after a single intravenous infusion of Antibody 2 ranged from 1.98 days to 7.38 days. Both _Cmax and AUC _0-t increased with increasing dose, indicating that in vivo drug exposure increased proportionally to the dose.

The pharmacokinetic characteristics after multiple administrations were similar to those after a single administration, with _tmax ranging from 1 to 9 hours for most subjects in each dose group. After multiple intravenous infusions of antibody 2 at doses of 0.05 mg/kg, 0.25 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg, and 10 mg/kg every 2 weeks, _Cmax and AUC _0-t increased with increasing dose. No significant accumulation was observed with consecutive administrations of 0.05–3 mg/kg, but serum levels in the 6–10 mg/kg dose groups showed an increasing trend with increasing dosing frequency, suggesting a possible small amount of accumulation.

The mean serum concentration-time curves after single and multiple administrations are shown in Figures 2A and 2B.

Example 3: Clinical study of the safety, tolerability, and preliminary efficacy of antibody 2 in subjects with advanced malignant tumors.

1. Drug name and specifications:

Antibody No. 2 Injection Solution :

Specifications: 85mg/5.0mL/vial;

Prescription composition: Antibody No. 2, glacial acetic acid, anhydrous sodium acetate, trehalose dihydrate and polysorbate 80;

Storage conditions: Store at 2–8℃ away from light;

Tislelizumab injection (abbreviated as tislelizumab or PD-1 monoclonal antibody or...) ) :

Specification: 10mL:100mg;

The prescription consists of: tislelizumab, sodium citrate dihydrate, citrate monohydrate, L-histidine hydrochloride monohydrate, L-histidine, trehalose dihydrate, polysorbate 20, and water for injection;

Storage conditions: Store at 2–8℃ away from light;

2. Safety evaluation in subjects with advanced malignant tumors

Safety was assessed in the subjects and found to be within manageable limits.

3. Validity Evaluation

As of January 15, 2022, 22 patients were enrolled and received monotherapy with antibody 2. Among them, 18 patients with solid tumors were evaluable for efficacy (2 in the 0.05 mg/kg group, 1 in the 0.25 mg/kg group, 3 in the 1 mg/kg group, 3 in the 3 mg/kg group, 3 in the 6 mg/kg group, and 6 in the 10 mg/kg group). Results showed an objective response rate (ORR) of 5.6% and a disease control rate (DCR) of 55.6% (1 partial response (PR) and 9 stable disease cases).

Example 4: A multicenter phase Ib/II trial evaluating the safety, tolerability, and efficacy of antibody 2 in combination with tislelizumab for the treatment of malignant tumors. Clinical research

1. Purpose

The main objectives of this embodiment include (1) evaluating the safety and tolerability of antibody 2 combined with tislelizumab in the treatment of malignant tumors, and providing a basis for dose selection in subsequent clinical studies; (Phase Ib); (2) evaluating the efficacy of antibody 2 combined with other drugs in the treatment of malignant tumors based on investigator-assessed objective response rate (ORR); (Phase II);

Other objectives include evaluating the pharmacokinetic (PK) characteristics of antibody 2 in combination with tislelizumab for the treatment of malignant tumors; evaluating the immunogenicity of antibody 2 in combination therapy for malignant tumors; evaluating the safety of antibody 2 in combination therapy for malignant tumors; assessing the efficacy of antibody 2 in combination therapy for malignant tumors based on efficacy endpoints; assessing the expression of programmed cell death protein ligand-1 (PD-L1), investigational drug-specific protein and/or ligand, tumor cell surface receptor occupancy, gene expression profile, tumor mutational burden/microsatellite instability/gene mutation profile, and paired whole blood controls, as well as the level of tumor-infiltrating immune cells, in tumor tissues before and/or after treatment; and evaluating the efficacy of tumor treatment according to the iRECIST criteria for evaluating the efficacy of immunotherapy for solid tumors.

2. Study endpoint

The primary endpoints included: (1) Safety and tolerability (Part A): Determining the maximum tolerated dose (MTD) and the recommended phase II dose (RP2D) based on dose-limiting toxicity (DLT), all adverse events (AE), and serious adverse events (SAE); (Phase Ib) (2) Efficacy (Part B–Part H): Investigator-assessed ORR (assessed according to RECIST 1.1).

Other endpoints include: (1) PK (Pharmacokinetics) indicators, including peak concentration (Cmax), time to peak (Tmax), area under the serum concentration-time curve from 0 to the last time point (AUC0-last), and if data permits, area under the serum concentration-time curve from 0 to infinity (AUC0-inf), clearance (CL), half-life (t _1/2 ), apparent volume of distribution at the terminal phase (Vz), elimination rate constant (Kel), steady-state peak concentration (Cmax, ss), steady-state trough concentration (Cmin, ss), Tmax, AUC0-tau, peak concentration accumulation factor (Rac_Cmax), etc.; (2) Immunogenicity evaluation indicators are the incidence of anti-drug antibodies (ADA) and the incidence of neutralizing antibodies (NAbs) in the subjects (if applicable). If applicable, the impact of ADAs on the PK (if applicable) characteristics, efficacy, and safety of the investigational drug will be further evaluated; (3) Safety (Part Part B to Part H monitors and records the occurrence of adverse events (AEs), as well as changes in laboratory tests (including complete blood count, blood biochemistry, urinalysis, coagulation function, thyroid function, etc.), vital signs, 12-lead electrocardiogram (12-ECG), physical examination, ECOG score, etc., as specified in the protocol; (4) ORR (limited to Part A), duration of response (DOR), disease control rate (DCR), time to response (TTR), progression-free survival (PFS), and overall survival (OFS) as assessed according to RECIST 1.1. Survival (OS); (5) assess the expression of programmed cell death protein ligand-1 (PD-L1), investigational drug-specific protein and/or ligand, tumor cell surface receptor occupancy, gene expression profile, tumor mutation burden/microsatellite instability/gene mutation profile and paired whole blood controls, and the level of tumor-infiltrating immune cells in tumor tissues before and/or after treatment; (6) assess ORR, DCR, DOR, TTP and PFS according to the iRECIST criteria for evaluating the efficacy of immunotherapy in solid tumors.

3. Research Design

This is an open-label, multicenter phase Ib/II clinical trial designed to evaluate the safety, tolerability, pharmacokinetic (PK) characteristics, immunogenicity, and efficacy of antibody 2 in combination with tislelizumab for the treatment of malignant tumors. The trial comprises the following components: Phase Ib: dose escalation and PK expansion; Phase II: nasopharyngeal carcinoma cohort. Details are as follows:

The nasopharyngeal carcinoma cohort program plans to enroll patients who have never received systemic anti-tumor therapy. Participants will receive antibody 2 in combination with tislelizumab and gemcitabine plus cisplatin (GP regimen). The study will have a safety introductory period, initially enrolling 6 evaluable participants for 21 days of safety monitoring. If the safety monitoring committee (SMC) assesses tolerability, the remaining participants will be enrolled. Antibody 2 will be administered at a dose of 600 mg every 3 weeks, and tislelizumab at a dose of 200 mg every 3 weeks.

Qualified subjects will receive treatment with antibody 2 in combination with tislelizumab. The dose of antibody 2 will be 600 mg every 3 weeks, and the dose of tislelizumab will be 200 mg every 3 weeks.

Dosage and administration

This study drug should be administered under the guidance of a physician experienced in cancer treatment. The initial intravenous infusion time for both antibody 2 and tislelizumab should be at least 60 minutes. If the first infusion is well-tolerated (e.g., no adverse reactions), subsequent intravenous infusion times may be shortened but should still be at least 30 minutes. Intravenous bolus injections or single rapid intravenous injections are prohibited. The intravenous access should be flushed at the end of the infusion. Concurrent administration of other medications to the same infusion set is prohibited.

The chemotherapy regimen and dosage for the nasopharyngeal carcinoma cohort are as follows:

The administration sequence was as follows: tislelizumab was administered 1 hour ± 30 minutes after the completion of antibody administration, and chemotherapy drugs were administered 1 hour ± 30 minutes after the completion of tislelizumab administration.

Cisplatin is administered after gemcitabine infusion.

Gemcitabine hydrochloride for injection: One treatment cycle is 3 weeks. Administer intravenously on days 1 and 8 of each cycle at a dose of 1000 mg/ ^m² over at least 30 minutes, for 4–6 cycles. In addition, all patients should undergo pretreatment according to guidelines or research center protocols.

Cisplatin for injection: One treatment cycle is 3 weeks, administered intravenously on day 1 of each cycle at a dose of 80 mg/ ^m² , over at least 4 hours (or as determined by the investigator based on local clinical guidelines or clinical practice), for 4–6 cycles. All patients should receive adequate hydration (including pre-treatment hydration) and diuretics. Urine output must be maintained above 2000 mL within 24 hours of infusion.

Primary inclusion criteria for subjects

(1) Patients with recurrent or metastatic advanced malignant tumors diagnosed by histology and/or cytology and who have failed previous standard treatment, have no standard treatment, or are not currently eligible for standard treatment; (2) Nasopharyngeal carcinoma cohort: Patients with recurrent or metastatic advanced nasopharyngeal carcinoma diagnosed by histology and/or cytology who have never received systemic antitumor therapy.

(2) The Eastern Oncology Collaborative Group's physical condition assessment (ECOG) score is 0-1.

(3) Expected survival time is at least 12 weeks;

(4) The subject has at least one measurable tumor lesion.

Dosage adjustment

For antibody 2 and tislelizumab, dose increases or decreases of the investigational drug are not permitted in this trial. If a subject experiences a grade ≥3 investigational drug-related adverse event (AE), a recurring grade 2 investigational drug-related AE, a grade ≥3 infusion adverse event, or a life-threatening adverse event, the investigator should refer to the "Toxicity-Based Dose Adjustment" recommendations in the protocol and consider the subject's specific circumstances to determine whether to pause or permanently discontinue the investigational drug. Subjects whose treatment is delayed by more than 12 weeks should be considered for permanent discontinuation of the investigational drug. If the investigator determines that the subject will continue to benefit from treatment, a decision on whether to resume treatment will be made jointly with the sponsor. Adjustments or dose delays for gemcitabine combined with cisplatin should be made in accordance with the drug's package insert and clinical practice. If one or two chemotherapy drugs in the combination therapy regimen are discontinued, the remaining combination therapy can continue. For patients requiring delayed treatment, study evaluation should not be delayed. The reasons for dose adjustments, treatment delays, and supportive measures should be documented in the medical record and CRF.

Duration of drug treatment

Antibody 2 should be used in combination therapy and continued until intolerable toxicity, disease progression or death, voluntary withdrawal, loss to follow-up, or continued treatment for more than 2 years or study termination (whichever occurs first). When combined with gemcitabine and cisplatin, chemotherapy should last for a maximum of 6 cycles.

Safety assessment

During the trial, participants were required to undergo safety checks at specified times, including laboratory tests (complete blood count, urinalysis, blood biochemistry, coagulation function, and thyroid function, etc.), vital signs, physical examination, and a 12-lead electrocardiogram (12-ECG). The clinical characteristics, severity, onset time, end time, duration, treatment, and outcome of any adverse events were recorded, and their correlation with antibody 2 was determined. Adverse events were classified using the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) V 5.0. The correlation between adverse events and antibody 2 was assessed by the investigator according to the attribution assessment criteria specified in the protocol.

Validity evaluation

The efficacy of antitumor therapy was evaluated according to the revised criteria for evaluating the efficacy of treatment in solid tumors (RECIST version 1.1) and the iRECIST criteria for evaluating the efficacy of immunotherapy in solid tumors (iRECIST). The efficacy evaluation indicators included objective response rate (ORR), duration of response (DOR), disease control rate (DCR), progression-free survival (PFS), 6-month overall survival (6M-OS), and overall survival (OS).

Objective response rate (ORR): Includes partial response (PR) and complete response (CR), defined as the proportion of subjects achieving a complete response or a partial response. For subjects with solid tumors, antitumor efficacy is evaluated according to RECIST 1.1. For subjects initially evaluated as PR or CR, efficacy must be confirmed at least 4 weeks later.

Duration of remission (DOR) is defined as the time from the first occurrence of a confirmed objective remission to the onset of disease progression or death from any cause (whichever comes first).

Disease control rate (DCR): defined as the proportion of subjects who achieve remission (PR+CR) and stable disease (SD) (lasting at least 12 weeks) after treatment.

Progression-free survival (PFS) is defined as the time from the date of the subject's first dose of medication until the first assessment of confirmed disease progression or death from any cause.

Overall survival (OS) is defined as the time from enrollment of a subject to death from any cause.

Pharmacokinetic (PK) evaluation

The main PK parameters include peak concentration ( _Cmax ), time to peak concentration ( _Tmax ), elimination half-life (T1 _/2 ), clearance rate (CL), volume of distribution (Vd), area under the concentration-time curve (AUC0 _-t ) from 0 to t, area under the concentration-time curve (AUC0 _{- inf} ) from 0 to infinity, area under the steady-state concentration-time curve (AUCss), steady-state peak concentration (Cmax,ss), steady-state trough concentration ( _Cmin,ss ), steady-state time to peak concentration (Tmax _,ss ), and accumulation factor.

Immunogenicity evaluation

Immunogenicity assessment endpoints include the incidence of anti-drug antibodies (ADAs) and the incidence of neutralizing antibodies (if applicable) in subjects. If applicable, the impact of ADAs on the efficacy, pharmacokinetic characteristics, and safety of the investigational drug will be further evaluated.

Biomarker evaluation

The biomarker evaluation indicators are PD-L1 and LAG-3, which will be used to evaluate the expression levels of PD-L1 and LAG-3 in tumor tissues.

Statistical analysis

(1) 1. Sample size estimation

The Phase Ib cohort plans to enroll patients with malignant tumors who have no standard treatment, have failed previous standard treatment, or are not currently eligible for standard treatment, with an estimated enrollment of approximately 20–30 participants (the exact sample size is subject to change). The nasopharyngeal carcinoma cohort plans to enroll approximately 30–60 participants.

(2) Statistical Analysis

1. General Method

All statistical analyses will be performed using SAS 9.4 or later. Unless otherwise specified, all statistical tests will be performed using a two-tailed test with α = 0.05, and two-tailed 95% confidence intervals (CI) will be calculated.

2. Subject distribution

The subject distribution includes a summary of subject screening, grouping, dropout or exclusion, and dataset partitioning for each group, presented using a subject distribution flowchart.

3. Baseline statistical analysis

Descriptive statistics were used to perform descriptive analysis on baseline characteristics of each group, including demographics, medical history, treatment history, medication history, disease characteristics, etc.

4. Security Analysis

Adverse events will be coded using the Medical Dictionary of Regulatory Activities (MedDRA) version 25.0 or later.

5. Validity Analysis

Descriptive statistics were used to summarize the number and proportion of subjects with the best response as complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD) according to cancer type, and the objective response rate and its 95% confidence interval were calculated based on the Clopper-Pearson method.

We will use the Kaplan-Meier method to plot progression-free survival (PFS) curves for patients treated with the investigational drug, differentiating between different dosage groups and different cancer types. We will calculate the median, first quartile, and third quartile, and then use the Greenwood formula to calculate the two-sided 95% confidence intervals for these statistics. Similar statistical methods will be used to analyze and summarize other survival endpoints (DOR, TTR, OS, etc.).

6. Pharmacokinetic Analysis

Individual pharmacokinetic (PK) data are presented, and mean PK data within groups are descriptively summarized. Plasma concentration-time curves for antibody 2 are plotted. PK parameters are summarized using descriptive statistics. Multiple-dose PK studies will discuss PK parameters, drug accumulation, and time to steady state.

7. Immunogenicity analysis

For all subjects who received at least one dose of the investigational drug, the incidence of anti-antibody 2 and neutralizing antibodies (if applicable) will be summarized. If applicable, the impact of ADA on the pharmacokinetic, efficacy, and safety of anti-antibody 2 will be assessed.

8. Biomarker Analysis

Assess the expression levels of PD-L1 and LAG-3 in tumor tissue samples.

4. Treatment Results

As of March 31, 2025, 21 patients with stage Ib advanced malignant tumors were enrolled and received antibody 2 monotherapy. Of these, 20 patients were evaluable for efficacy. Results showed an objective response rate (ORR) of 23.8% and a disease control rate (DCR) of 61.9%.

As of March 31, 2025, 42 patients in the first-line treatment cohort for stage II nasopharyngeal carcinoma were enrolled and received antibody 2 in combination with tislelizumab and gemcitabine plus cisplatin. Of these, 41 patients were evaluable for efficacy, and the results are shown in Table 2 below.

Table 2. Efficacy of first-line treatment in cohort of stage II nasopharyngeal carcinoma


* LAG-3 and PD-L1 were tested in 37 and 39 patients, respectively.
**Data source: Yang Y, et al. Cancer Cell. 2023.

In first-line treatment of nasopharyngeal carcinoma, the combination of antibody 2 with tislelizumab and gemcitabine plus cisplatin showed significantly improved efficacy compared to the combination of tislelizumab and gemcitabine plus cisplatin (NCT03924986).

Example 5: A multicenter Ib/II study evaluating the safety, tolerability, and efficacy of antibody 2 in combination with toripalimab for the treatment of advanced malignant tumors. Phase II clinical trials

This example is a single-arm, open-label, multicenter phase Ib/II clinical trial designed to evaluate the safety, tolerability, pharmacokinetic characteristics, immunogenicity, and efficacy of antibody 2 in combination with toripalimab for the treatment of advanced malignant tumors. The trial was divided into a dose-escalation phase (phase Ib) and an expansion phase (phase II), as detailed below:

Phase Ib (Dosage escalation phase of combination therapy)

The Phase Ib study plans to enroll patients with advanced malignancies who have no standard treatment, have failed previous standard treatment, or are not currently eligible for standard treatment, including patients with advanced malignancies who have not received anti-PD-(L)1 antibody therapy or who have progressed or are intolerant to anti-PD-(L)1 antibody therapy.

Following a "rolling six design" dose escalation method, the tolerability of antibody 2 at 200 mg and 400 mg dose levels was evaluated. Each dose group included 3–6 subjects, with a total sample size of approximately 9–12 subjects in Phase Ib (subject to actual occurrence). The antibody 2 dosing regimen was once every 3 weeks (Q3W), administered intravenously; the toripalimab dosing regimen was 240 mg, Q3W, administered intravenously. During the trial, the dosing frequency or escalation group setup could be adjusted as necessary based on the obtained PK and safety data. Each dosing cycle in this study was 3 weeks, with a 3-week follow-up period for dose-limiting toxicity (DLT) assessment (performed in subjects enrolled during the dose escalation phase).

Phase II (Combination Therapy Expansion Phase)

Based on global R&D data for Antibody 2 and safety, tolerability, and pharmacokinetic (PK) data from Phase Ib clinical trials, the recommended dose (RP2D) from Phase II clinical trials was obtained to expand the target indications, including patients with malignant tumors such as nasopharyngeal carcinoma.

This study plans to enroll patients with advanced malignant tumors, including those who have not received anti-PD-(L)1 antibody therapy or whose advanced malignant tumors have progressed or are intolerant to anti-PD-(L)1 antibody therapy. During the screening period, subjects will undergo relevant examinations or observations, and those meeting the screening criteria will enter the treatment period. During the treatment period, all subjects will receive antibody 2 in combination with toripalimab until intolerable toxicity, disease progression, death, voluntary withdrawal, loss to follow-up, or continued administration for 2 years (whichever occurs first). The antibody 2 dosing regimen is 400 mg, Q3W, intravenously; the toripalimab dosing regimen is 240 mg, Q3W, intravenously. The antitumor efficacy of the subjects will be evaluated according to the RECIST 1.1 criteria for evaluating the efficacy of immunotherapy in solid tumors and the iRECIST criteria for evaluating the efficacy of immunotherapy in solid tumors. After the first dose, the frequency will be adjusted to once every 9 weeks after week 24. All subjects in this study were followed up for safety 30 days (+7 days) after the last dose or before starting a new anti-tumor therapy. After that, they were followed up for survival, and information such as whether they received a new anti-tumor therapy and their survival time was collected until the subject died, was lost to follow-up, or withdrew their informed consent.

In this trial, all subjects in the Ib/II phase underwent a population pharmacokinetic study with sparse blood collection; blood samples were collected from all subjects for the evaluation of the immunogenicity of antibody 2; tumor tissue samples were collected from all subjects for biomarker-related exploration, and blood samples were collected from some subjects for receptor occupancy-related exploration.

Treatment results:

For nasopharyngeal carcinoma patients who had not received anti-PD-(L)1 antibody therapy (n=12): the objective response rate (ORR) was 33.3%, the disease control rate (DCR) was 75%, and the median progression-free survival (PFS) was 10.8 months (95% CI, 1.3 to unestimated).

Patients with nasopharyngeal carcinoma who progressed or were intolerant to anti-PD-(L)1 antibody therapy (n=17): ORR was 11.8%, DCR was 64.7%, and median PFS was 2.7 months (95% CI, 1.4 to 4.9).

Table 3. Sequence Information

Claims (27)

Use of an antibody that specifically binds to LAG-3 or its antigen-binding moiety in the preparation of a medicament for treating malignant tumors, wherein the antibody or its antigen-binding moiety comprises a heavy chain variable region and a light chain variable region, wherein The heavy chain variable region (i) Contains HCDR1, HCDR2 and HCDR3 derived from the heavy chain variable region consisting of the amino acid sequence of SEQ ID NO:4; (ii) Containing HCDR1, HCDR2, and HCDR3, which respectively contain or constitute the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, or respectively contain or constitute amino acid sequences having one, two, or three amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; or (iii) Containing or consisting of the amino acid sequence of SEQ ID NO:4, or containing or consisting of an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:4, or containing or consisting of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5) amino acid modifications (preferably amino acid substitutions, more preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:4, preferably, these amino acid modifications do not occur in the CDR region, more preferably, these amino acid modifications occur in the FR region, such as the FR1, FR2, FR3, or FR4 region; The variable region of the light chain: (i) LCDR1, LCDR2 and LCDR3 comprising a light chain variable region composed of an amino acid sequence derived from SEQ ID NO:8; (ii) comprising LCDR1, LCDR2, and LCDR3, which respectively comprise or consist of the sequences shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, or respectively comprise or consist of amino acid sequences having one, two, or three amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7; or (iii) Containing or consisting of the amino acid sequence of SEQ ID NO:8, or containing or consisting of an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:8, or containing or consisting of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5) amino acid modifications (preferably amino acid substitutions, more preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:8, preferably, these amino acid modifications do not occur in the CDR region, more preferably, these amino acid modifications occur in the FR region, such as the FR1, FR2, FR3, or FR4 region. According to the use of claim 1, the antibody or its antigen-binding portion further comprises a heavy chain constant region and/or a light chain constant region, the heavy chain constant region comprising or consisting of the amino acid sequence shown in SEQ ID NO:9, and the light chain constant region comprising or consisting of the amino acid sequence shown in SEQ ID NO:10. According to claim 1 or 2, the antibody or its antigen-binding portion comprises a heavy chain and a light chain, wherein: the heavy chain comprises or is composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 11; and the light chain comprises or is composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 12. For use according to any one of claims 1-3, wherein the antigen-binding portion is selected from: (i) Fab fragment; (ii) F(ab')2 fragment; (iii) Fd fragment; (iv) Fv fragment; (v) dAb fragment; and (vii) single-chain Fv (scFv). The use according to any one of claims 1-3, wherein the antibody or the antigen-binding portion is a mouse antibody, a human antibody, a chimeric antibody, or a humanized antibody. The use according to any one of claims 1-3, wherein the antibody or the antigen-binding portion is an IgG1, IgG2 or IgG4 isotype. The use according to any one of claims 1-6, wherein the malignant tumor is an advanced malignant tumor, and preferably the malignant tumor is selected from nasopharyngeal carcinoma. According to the use of claim 7, the drug is formulated for administration to a subject suffering from the malignant tumor, the subject being a subject with relapsed and refractory advanced malignant tumors, a subject with recurrent or metastatic advanced malignant tumors who has not previously received systemic treatment, or a subject with unresectable or metastatic advanced malignant tumors. Preferably, the subject is a subject with recurrent or metastatic advanced nasopharyngeal carcinoma who has not previously received systemic treatment. The medicament is formulated for administration to a subject in each dosing cycle or per administration, according to any one of claims 1-8, wherein the medicament comprises 0.05 mg/kg to 200 mg/kg of the antibody or its antigen-binding portion; Preferably, the drug comprises 0.25 mg/kg to 10 mg/kg of the antibody or its antigen-binding portion; More preferably, the drug comprises 1.0 mg/kg to 6.0 mg/kg of the antibody or its antigen-binding portion; Most preferably, the drug comprises 6.0 mg/kg of the antibody or its antigen-binding portion, for example, 0.25 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 1.0 mg/kg, 1.2 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.8 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 8 mg/kg or 10 mg/kg of the antibody or its antigen-binding portion. The medicament is formulated for administration to a subject in each dosing cycle or per administration, according to any one of claims 1-8, wherein the medicament comprises 50 mg to 5000 mg of the antibody or its antigen-binding portion; Preferably, the drug comprises 60 mg to 4000 mg of the antibody or its antigen-binding portion; More preferably, the drug comprises 80 mg to 3750 mg of the antibody or its antigen-binding portion; Most preferably, the drug comprises 100 mg to 3500 mg of the antibody or its antigen-binding portion, for example, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1250 mg, 1500 mg, 1750 mg, 2000 mg, 2250 mg, 2500 mg, 2750 mg, 3000 mg, 3250 mg, or 3500 mg of the antibody or its antigen-binding portion. According to the use described in claim 9 or 10, the drug is administered every 12 weeks, every 9 weeks, every 6 weeks, every 5 weeks, every 4 weeks, every 3 weeks, every 2 weeks, once a week, twice a week, or three times a week. The drug is formulated as an intravenous infusion or subcutaneous injection preparation according to any one of claims 1-11. The use according to any one of claims 1-12, wherein the drug is further administered in combination with one or more other therapeutic agents; Preferably, the other therapeutic agents are immunostimulatory antibodies and/or chemotherapeutic agents. According to the use of claim 13, the immunostimulatory antibody is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody; Preferably, the immunostimulatory antibody is an anti-PD-1 antibody; More preferably, the anti-PD-1 antibody is selected from tislelizumab and toripalimab. According to the use of claim 14, the administration cycle of the immune-stimulating antibody is every 12 weeks, every 9 weeks, every 6 weeks, every 5 weeks, every 4 weeks, every 3 weeks, every 2 weeks, once a week, twice a week, or three times a week. According to the use described in claim 14 or 15, the immunostimulatory antibody is formulated as an intravenous infusion or subcutaneous injection preparation. The immunostimulatory antibody is administered before, after, or simultaneously with each administration of the drug to the subject in each dosing cycle or each time. According to the use of claim 17, the immunostimulatory antibody is administered 0 to 5 hours before or after each dosing cycle or each time the drug is administered, for example, immediately, 15 minutes, 30 minutes, 45 minutes, 1 hour, 1.25 hours, 1.5 hours, 1.75 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, or 5 hours after the drug is administered. Preferably, each administration is of 100 mg to 600 mg of the immunostimulatory antibody, such as 100 mg, 200 mg, 300 mg, 400 mg, 500 mg or 600 mg of tislelizumab or 100 mg, 200 mg, 240 mg, 300 mg, 400 mg, 500 mg or 600 mg of toripalimab. The use according to any one of claims 13-18, wherein the chemotherapeutic agent is gemcitabine and/or platinum-based; Preferably, the chemotherapeutic agent is gemcitabine hydrochloride and/or cisplatin. According to the use of claim 19, the chemotherapy agent is administered every 5 weeks, every 4 weeks, every 3 weeks, every 2 weeks, once a week, twice a week, or three times a week. According to the use described in claim 19 or 20, the chemotherapeutic agent is formulated for intravenous infusion or oral administration. The chemotherapeutic agent is administered in each dosing cycle or each time after the administration of the drug and the immunostimulatory antibody to the subject. According to claim 22, the chemotherapy agent is administered in each dosing cycle or each time 0 to 24 hours after the administration of the drug and the immunostimulatory antibody; Preferably, when the chemotherapeutic agent is administered sequentially as gemcitabine and the platinum-based agent, gemcitabine is administered 0–24 hours after each dosing cycle or each administration of the immunostimulatory antibody. For example, gemcitabine is administered immediately, at 15 minutes, 30 minutes, 45 minutes, 1 hour, 1.25 hours, 1.5 hours, 1.75 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours after each dosing cycle or each administration of the immunostimulatory antibody. Each administration does not exceed 1000 mg/ ^m² of gemcitabine, for example, 1000 mg/m² per administration. ² % gemcitabine hydrochloride; the platinum-based drugs are administered from 0 to 24 hours after gemcitabine administration, for example, at each dosing cycle or at each administration immediately, 15 minutes, 30 minutes, 45 minutes, 1 hour, 1.25 hours, 1.5 hours, 1.75 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours after gemcitabine administration, with each administration not exceeding 80 mg/ ^m² of the platinum-based drugs, for example, 80 mg/ ^m² of cisplatin per administration. A medicament for treating malignant tumors, the medicament comprising an antibody or its antigen-binding portion as described in any one of claims 1-6. A pharmaceutical combination for treating malignant tumors, comprising an antibody or antigen-binding portion thereof as defined in claims 1-6, and one or more other therapeutic agents as defined in claims 13-14 and 19, administered together. Preferably, the other therapeutic agents are immunostimulatory antibodies and/or chemotherapeutic agents; Preferably, the immunostimulatory antibody is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody; More preferably, the immunostimulatory antibody is an anti-PD-1 antibody; Most preferably, the anti-PD-1 antibody is selected from tislelizumab and toripalimab; Preferably, the chemotherapeutic agent is gemcitabine or a platinum-based drug; More preferably, the gemcitabine is gemcitabine hydrochloride; More preferably, the platinum group is cisplatin. The medicament according to claim 24 or the combination of medicaments according to claim 25, wherein the malignant tumor is an advanced malignant tumor, preferably the malignant tumor is nasopharyngeal carcinoma. The drug or combination of drugs according to claim 26, wherein the drug is administered to a subject suffering from the malignant tumor, the subject being a subject with relapsed and refractory advanced malignant tumor, a subject with recurrent or metastatic advanced malignant tumor who has not previously received systemic treatment, or a subject with unresectable or metastatic advanced malignant tumor. Preferably, the subject is a subject with recurrent or metastatic advanced nasopharyngeal carcinoma who has not previously received systemic treatment.