Anti-PD-l1 and Anti-4BB bispecific antibodies for treating tumors

Abstract

Provided is the use of anti-4BB and anti-PD-L1 bispecific antibodies and combinations with chemotherapeutic agents in the preparation of a medicament for the treatment of tumors.

Description

Anti-PD-L1 and anti-4-1BB bispecific antibodies for treating tumors Technical Field

[0001] The present invention relates to the use of an anti-4-1BB and anti-PD-L1 bispecific antibody in treating tumors, and more specifically, to the use of anti-PD-L1 and anti-4-1BB, and a combination thereof with a chemotherapeutic agent, in preparing a medicament for treating tumors. Background Art

[0002] Tumor immunotherapy has made significant progress over the past decade and has become a widely used and effective clinical treatment, following surgery, radiotherapy, chemotherapy, and targeted therapy. Tumor immunotherapy can trigger a tumor immune response in the patient's body by blocking the immunosuppressive pathways activated by cancer cells and activating the patient's own immune system to kill the tumor. It has the advantages of high specificity, long-lasting effects, and minimal side effects.

[0003] Programmed cell death protein 1 (PD-1) is an immunosuppressive receptor primarily expressed on the surfaces of T cells, B cells, monocytes, and natural killer cells. Its corresponding ligands are programmed death ligand-1 (PD-L1) and programmed death ligand-2 (PD-L2), respectively. PD-L1 is widely expressed on various tumor cells and immune cells, and its expression is upregulated in the tumor microenvironment by various cytokines, such as interferon (IFN)-γ. Binding of PD-L1 to PD-1 on the surfaces of tumor cells and antigen-presenting cells continuously activates the PD-1 / PD-L1 pathway, inhibiting the activation of tumor antigen-specific T cells and weakening T cell anti-tumor activity. Antibodies to PD-1 / PD-L1 disrupt the immunosuppressive effects of this pathway by interfering with the binding of PD-1 to PD-L1, thereby restoring T cell anti-tumor immune responses. Immune checkpoint inhibitors are currently a research hotspot in the field of tumor immunotherapy, and several anti-PD-(L)1 antibodies have been marketed both domestically and internationally. Although PD-(L)1 inhibitors have an efficacy of 40-70% in tumors such as melanoma, Merkel cell carcinoma, Hodgkin lymphoma, and microsatellite instability-high tumors (MSI-H tumors), in many tumors for which immune checkpoint inhibitors have been approved, such as advanced small cell lung cancer and gastric cancer, the efficacy is only 10-25% (refer to the product marketing instructions). In addition, existing immune checkpoint inhibitors may have problems such as primary and acquired drug resistance or may induce immune-related adverse events. Therefore, it is necessary to find other immune checkpoints in the tumor microenvironment to reduce the occurrence of drug resistance and improve anti-tumor efficacy.

[0004] 4-1BB (CD137) is a potential target in tumor immunotherapy. 4-1BB belongs to the tumor necrosis factor (TNF) receptor family and is primarily expressed in activated T cells, natural killer cells, regulatory T cells, dendritic cells, and mast cells. Its ligand, 4-1BBL, is primarily expressed in activated antigen-presenting cells (APCs), including dendritic cells, macrophages, and B cells. 4-1BB is a T cell-specific co-stimulatory molecule that plays a key role in fully activating T cells and other immune cells in immunotherapy. The primary physiological function of 4-1BB on T cells is as follows: 4-1BB binds to 4-1BBL and recruits TNF receptor-associated factors 1 and 2 (TRAF1 and TRAF2) to form a heterotrimer, thereby activating downstream signaling pathways led by nuclear factor-κ-light-chain-enhancer of activated B cells (NF-κB), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK). These activated signaling pathways generate co-stimulatory signals, promoting the production and secretion of cytokines (such as interleukin [IL]-2, IL-4, and IFN-γ) and the activation of CD8+ and CD4+ T lymphocytes. NF-κB activation increases the survival rate of CD8+ T lymphocytes by increasing the expression of the anti-apoptotic genes bcl-XL and bfl-1. In addition to activating the immune system through T cells, 4-1BB can also activate or regulate the immune system through natural killer cells, macrophages, and B cells, thereby inhibiting tumor proliferation.

[0005] In addition, combination therapies of anti-4-1BB antibodies and other antibodies (such as anti-PD-1 antibodies) are also undergoing clinical trials. For example, there is a need for bispecific antibodies that can simultaneously target PD-L1 and 4-1BB to block the PD-1 / PD-L1 pathway and provide co-stimulatory signals through 4-1BB when PD-L1-expressing tumor cells undergo PD-L1 cross-linking. However, the bispecific antibodies described in patent US_2017_0198050_A1 can activate or induce 4-1BB signaling without cross-linking with target cells, raising concerns about the potential toxicity induced by 4-1BB at high concentrations. There are already several bispecific antibodies targeting PD-L1 / 4-1BB targets in clinical research and development at home and abroad. GEN1046 jointly developed by BioNTech and Genmab, FS222 developed by F-star Therapeutics in the UK, and PM1003 developed by Promis Biotechnology (Zhuhai) Co., Ltd. are currently in clinical phase I / II. At present, there are no bispecific antibodies targeting the PD-L1 / 4-1BB signaling pathway on the market at home and abroad.

[0006] Summary of the Invention

[0007] The applicant has developed a bispecific antibody that specifically binds to 4-1BB and PD-L1 based on its 4-1BB antibody platform "X-Body". The present disclosure provides the use of this bispecific antibody in treating tumors.

[0008] Therefore, in a first aspect, the present disclosure relates to use of a bispecific antibody in the preparation of a medicament for treating a malignant tumor, wherein the bispecific antibody comprises a first antigen-binding region that specifically binds to 4-1BB and a second antigen-binding region that specifically binds to PD-L1.

[0009] In one embodiment, the present disclosure relates to a use in which the first antigen-binding region comprises a first heavy chain variable region and / or a first light chain variable region, wherein:

[0010] The first heavy chain variable region:

[0011] (i) comprising HCDR1, HCDR2, and HCDR3 derived from the heavy chain variable region consisting of the sequence shown in SEQ ID NO: 7 or SEQ ID NO: 9;

[0012] (ii) comprises HCDR1, HCDR2 and HCDR3, which comprise or consist of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, or comprise or consist of an amino acid sequence 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, respectively; or

[0013] (iii) comprising or consisting of the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 9, or comprising or consisting of an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 9, or comprising or consisting of an amino acid sequence that has 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: 7 or SEQ ID NO: 9, preferably, these amino acid modifications do not occur in the CDR regions, more preferably, these amino acid modifications occur in the FR regions, such as FR1, FR2, FR3 or FR4;

[0014] The first light chain variable region:

[0015] (i) comprising LCDR1, LCDR2, and LCDR3 derived from a light chain variable region consisting of the sequence shown in SEQ ID NO: 8 or SEQ ID NO: 10;

[0016] (ii) comprises LCDR1, LCDR2 and LCDR3, which respectively comprise or consist of the sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, or respectively comprise or consist of an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; or

[0017] (iii) comprising or consisting of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 10, or comprising or consisting of an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 10, or comprising or consisting of an amino acid sequence that has 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 or SEQ ID NO: 10, preferably, these amino acid modifications do not occur in the CDR regions, more preferably, these amino acid modifications occur in the FR regions, such as FR1, FR2, FR3 or FR4;

[0018] Wherein in SEQ ID NO: 8, X=S or G.

[0019] In one embodiment, the first antigen-binding region in the use involved in the present disclosure further comprises a first heavy chain constant region and / or a first light chain constant region, the first heavy chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO: 19 or 20, and the first light chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO: 21 or 22.

[0020] In one embodiment, the present disclosure relates to a use in which the first antigen binding region activates 4-1BB signaling and is derived from an anti-4-1BB monoclonal antibody. In some embodiments, the first antigen binding region is an scFv of an anti-4-1BB antibody.

[0021] In one embodiment, the second antigen-binding region that specifically binds to PD-L1 in the use involved in the present disclosure comprises a second heavy chain variable region and a second light chain variable region, wherein the second heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 consisting of the sequences shown in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively, and the second light chain variable region comprises LCDR1, LCDR2 and LCDR3 consisting of the sequences shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

[0022] In one embodiment, the second antigen-binding region in the use involved in the present disclosure further comprises a second heavy chain constant region and / or a second light chain constant region, the second heavy chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO: 19 or 20, and the second light chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO: 21 or 22.

[0023] In one embodiment, the bispecific antibody for use in the present disclosure comprises: (1) Chain 1: the heavy chain of the second antigen-binding region connected at the C-terminus with or without a linker to the antigen-binding portion of the first antigen-binding region; and

[0024] (2) Chain 2: the light chain of the second antigen-binding region.

[0025] In one embodiment, the present disclosure relates to uses in which the linker comprises an amino acid sequence (Gly4Ser)n or (GlySer4)n, wherein n is a positive integer of 1-7.

[0026] In one embodiment, the bispecific antibody for use in the present disclosure consists of two chains 1 and two chains 2.

[0027] In one embodiment, the antigen binding portion in the use related to the present disclosure is selected from: (i) a Fab fragment; (ii) a F(ab')2 fragment; (iii) a Fd fragment; (iv) an Fv fragment, (v) a dAb fragment; (vi) a nanobody; and (vii) a single-chain Fv (scFv); the scFv comprises the first heavy chain variable region and the first light chain variable region (e.g., connected via a linker).

[0028] In one embodiment, the first antigen-binding region and / or the second antigen-binding region in the use provided herein are derived from mouse antibodies, human antibodies, chimeric antibodies or humanized antibodies.

[0029] In one embodiment, the present disclosure relates to uses in which the first antigen-binding region and / or the second antigen-binding region is of IgG1, IgG2 or IgG4 isotype.

[0030] In one embodiment, the present disclosure relates to uses in which chain 1 of the bispecific antibody comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 25; and chain 2 of the bispecific antibody comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 26.

[0031] In one embodiment, the malignant tumor in the use involved in the present disclosure is bladder cancer, breast cancer, endometrioid cancer, cervical cancer, advanced solid tumors, renal cell carcinoma, neuroendocrine tumors, gastric cancer, transitional cell carcinoma, urothelial carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, colorectal cancer, non-small cell lung cancer, hepatocellular carcinoma. In a preferred embodiment, the neuroendocrine tumor in the use involved in the present disclosure is a neuroendocrine cancer or a neuroendocrine tumor. In a more preferred embodiment, the neuroendocrine tumor in the use involved in the present disclosure is an extrapulmonary neuroendocrine tumor. In a more preferred embodiment, the neuroendocrine tumor in the use involved in the present disclosure is an advanced neuroendocrine tumor.

[0032] In one embodiment, the neuroendocrine cancer in the use of the present disclosure is an advanced neuroendocrine cancer. In a preferred embodiment, the neuroendocrine cancer in the use of the present disclosure is an extrapulmonary neuroendocrine cancer, such as an advanced extrapulmonary neuroendocrine cancer, or a small cell lung cancer, such as an extensive-stage small cell lung cancer. In a more preferred embodiment, the neuroendocrine cancer in the use of the present disclosure is an unresectable locally advanced or metastatic neuroendocrine cancer.

[0033] In one embodiment, the use of the present disclosure involves administering the drug to a subject suffering from the malignant tumor, wherein the subject is a subject who has not received systemic treatment or a subject who has progressed after at least one line of chemotherapy. In an exemplary embodiment, the subject includes but is not limited to a subject who has progressed after receiving two or more lines of chemotherapy, for example, a subject who has progressed after receiving two lines of chemotherapy, a subject who has progressed after receiving three lines of chemotherapy, or a subject who has progressed after receiving four lines of chemotherapy.

[0034] In a specific embodiment, the subject can be a subject with small cell lung cancer who has not received systemic treatment before, a subject with advanced extrapulmonary neuroendocrine carcinoma who has progressed after receiving at least one line of chemotherapy, including but not limited to a subject with advanced extrapulmonary neuroendocrine carcinoma who has progressed after receiving two or more lines of chemotherapy, for example, a subject with advanced extrapulmonary neuroendocrine carcinoma who has progressed after receiving two lines of chemotherapy, a subject with advanced extrapulmonary neuroendocrine carcinoma who has progressed after receiving three lines of chemotherapy, and a subject with advanced extrapulmonary neuroendocrine carcinoma who has progressed after receiving four lines of chemotherapy.

[0035] In a more specific embodiment, the chemotherapy treatment received by the subject can be platinum-containing chemotherapy, for example, it can be first-line platinum-containing chemotherapy, second-line platinum-containing chemotherapy, or third-line platinum-containing chemotherapy.

[0036] In one embodiment, the uses involved in the present disclosure include administering 0.2 mg / kg to 200 mg / kg of the bispecific antibody to the subject per dosing cycle or each time. In a preferred embodiment, the uses involved in the present disclosure include administering 0.8 mg / kg to 25 mg / kg of the bispecific antibody to the subject per dosing cycle or each time. In a more preferred embodiment, the uses involved in the present disclosure include administering 3.2 mg / kg to 15 mg / kg of the bispecific antibody to the subject per dosing cycle or each time. In a further preferred embodiment, the uses involved in the present disclosure include administering 6 mg / kg to 15 mg / kg of the bispecific antibody to the subject per dosing cycle or each time. In the most preferred embodiment, the uses involved in the present disclosure include administering 10 mg / kg to 15 mg / kg of the bispecific antibody to the subject per dosing cycle or each time.

[0037] In one embodiment, the use involved in the present disclosure comprises administering 50mg to 5000mg of the bispecific antibody to a subject per dosing cycle or per dose. In a preferred embodiment, the use involved in the present disclosure comprises administering 60mg to 4000mg of the bispecific antibody to a subject per dosing cycle or per dose. In a more preferred embodiment, the use involved in the present disclosure comprises administering 80mg to 3750mg of the bispecific antibody to a subject per dosing cycle or per dose. In a most preferred embodiment, the use involved in the present disclosure comprises administering 100mg to 3500mg of the bispecific antibody to a subject per dosing cycle or per dose. In some specific embodiments, the use involved in the present disclosure comprises administering 200mg, 400mg, 600mg, 800mg, 1000mg, 1250mg, 1500mg, 1750mg, 2000mg, 2250mg, 2500mg, 2750mg, 3000mg, 3250mg, 3500mg of the bispecific antibody to a subject per dosing cycle or per dose.

[0038] In one embodiment, the bispecific antibody used in the present disclosure 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.

[0039] In one embodiment, the bispecific antibody for use in the present disclosure is formulated for intravenous infusion or subcutaneous injection.

[0040] In one embodiment, the bispecific antibody in the use of the present disclosure is further administered in combination with one or more chemotherapeutic agents. In a preferred embodiment, the chemotherapeutic agent in the use of the present disclosure is a platinum and / or a podophyllotoxin derivative. In a more preferred embodiment, the platinum in the use of the present disclosure is carboplatin or cisplatin. In a most preferred embodiment, the podophyllotoxin derivative in the use of the present disclosure is etoposide, etoposide glucuronide, or etoposide phosphate.

[0041] In one embodiment, the present disclosure relates to uses in which 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.

[0042] In one embodiment, the present disclosure relates to uses wherein the chemotherapeutic agent is formulated for administration as an intravenous infusion or oral formulation.

[0043] In one embodiment, the present disclosure relates to uses comprising administering the chemotherapeutic agent per dosing cycle or each time after administering the bispecific antibody to the subject.

[0044] In one embodiment, the use of the present disclosure comprises administering the podophyllotoxin derivative for 1-5 days per dosing cycle or each time after administration of the bispecific antibody, for example, administering the podophyllotoxin derivative for 3 days or 1 day. In a preferred embodiment, the use of the present disclosure comprises administering 100 mg / m2 per day. 2 or less of said podophyllotoxin derivative.

[0045] In one embodiment, the use of the present disclosure comprises administering the platinum compound for 1-5 days per dosing cycle or each time after the administration of the bispecific antibody, for example, administering the platinum compound for 3 days or 1 day. In a preferred embodiment, the use of the present disclosure comprises administering 75 mg / m2 per day. 2 or less of said platinum species, or AUC=5 mg / mL / min or less of said platinum species.

[0046] In a second aspect, the present disclosure relates to a medicament for treating a malignant tumor, said medicament comprising the bispecific antibody as defined in the first aspect.

[0047] In a third aspect, the present disclosure relates to a drug combination comprising a bispecific antibody as defined in the first aspect and one or more chemotherapeutic agents. In preferred embodiments, the chemotherapeutic agent is a platinum and / or a podophyllotoxin derivative. In a more preferred embodiment, the platinum is carboplatin or cisplatin. In a most preferred embodiment, the podophyllotoxin derivative is etoposide, etoposide glucuronide, or etoposide phosphate. In a preferred embodiment, the drug combination is used to treat a malignant tumor.

[0048] In one embodiment, the present disclosure relates to the drug or drug combination for treating a malignant tumor, wherein the malignant tumor is bladder cancer, breast cancer, endometrioid cancer, cervical cancer, advanced solid tumors, renal cell carcinoma, neuroendocrine tumors, gastric cancer, transitional cell carcinoma, urothelial carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, colorectal cancer, non-small cell lung cancer, or hepatocellular carcinoma. In a preferred embodiment, the neuroendocrine tumor is a neuroendocrine carcinoma or a neuroendocrine tumor. In a more preferred embodiment, the neuroendocrine tumor is an extrapulmonary neuroendocrine tumor. In a more preferred embodiment, the neuroendocrine tumor is an advanced neuroendocrine tumor.

[0049] In one embodiment, the neuroendocrine cancer is an advanced neuroendocrine cancer. In a preferred embodiment, the neuroendocrine cancer is an extrapulmonary neuroendocrine cancer, such as an advanced extrapulmonary neuroendocrine cancer, or a small cell lung cancer, such as an extensive-stage small cell lung cancer. In a more preferred embodiment, the neuroendocrine cancer is an unresectable locally advanced or metastatic neuroendocrine cancer.

[0050] In one embodiment, the medicament is administered to a subject having the malignant tumor, wherein the subject is a subject who has not received systemic treatment or a subject who has progressed after at least one line of platinum-containing chemotherapy.

[0051] In an exemplary embodiment, the subjects include but are not limited to subjects who have progressed after receiving two or more lines of chemotherapy, such as subjects who have progressed after receiving two lines of chemotherapy, subjects who have progressed after receiving three lines of chemotherapy, and subjects who have progressed after receiving four lines of chemotherapy.

[0052] In a specific embodiment, the subject can be a subject with small cell lung cancer who has not received systemic treatment before, a subject with advanced extrapulmonary neuroendocrine carcinoma who has progressed after receiving at least one line of chemotherapy, including but not limited to a subject with advanced extrapulmonary neuroendocrine carcinoma who has progressed after receiving two or more lines of chemotherapy, for example, a subject with advanced extrapulmonary neuroendocrine carcinoma who has progressed after receiving two lines of chemotherapy, a subject with advanced extrapulmonary neuroendocrine carcinoma who has progressed after receiving three lines of chemotherapy, and a subject with advanced extrapulmonary neuroendocrine carcinoma who has progressed after receiving four lines of chemotherapy.

[0053] In a more specific embodiment, the chemotherapy treatment received by the subject can be platinum-containing chemotherapy, for example, it can be first-line platinum-containing chemotherapy, second-line platinum-containing chemotherapy, or third-line platinum-containing chemotherapy.

[0054] In a fourth aspect, the present disclosure relates to a method for treating a malignant tumor, comprising administering to a subject a bispecific antibody comprising a first antigen-binding region that specifically binds to 4-1BB and a second antigen-binding region that specifically binds to PD-L1.

[0055] In one embodiment, the present disclosure relates to a method wherein the first antigen binding region comprises a first heavy chain variable region and / or a first light chain variable region, wherein:

[0056] The first heavy chain variable region:

[0057] (i) comprising HCDR1, HCDR2, and HCDR3 derived from the heavy chain variable region consisting of the sequence shown in SEQ ID NO: 7 or SEQ ID NO: 9;

[0058] (ii) comprises HCDR1, HCDR2 and HCDR3, which comprise or consist of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, or comprise or consist of an amino acid sequence 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, respectively; or

[0059] (iii) comprising or consisting of the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 9, or comprising or consisting of an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 9, or comprising or consisting of an amino acid sequence that has 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: 7 or SEQ ID NO: 9, preferably, these amino acid modifications do not occur in the CDR regions, more preferably, these amino acid modifications occur in the FR regions, such as FR1, FR2, FR3 or FR4;

[0060] The first light chain variable region:

[0061] (i) comprising LCDR1, LCDR2, and LCDR3 derived from a light chain variable region consisting of the sequence shown in SEQ ID NO: 8 or SEQ ID NO: 10;

[0062] (ii) comprises LCDR1, LCDR2 and LCDR3, which respectively comprise or consist of the sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, or respectively comprise or consist of an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; or

[0063] (iii) comprising or consisting of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 10, or comprising or consisting of an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 10, or comprising or consisting of an amino acid sequence that has 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 or SEQ ID NO: 10, preferably, these amino acid modifications do not occur in the CDR regions, more preferably, these amino acid modifications occur in the FR regions, such as FR1, FR2, FR3 or FR4;

[0064] Wherein in SEQ ID NO: 8, X=S or G.

[0065] In one embodiment, the present disclosure relates to a method in which the first antigen-binding region further comprises a first heavy chain constant region and / or a first light chain constant region, wherein the first heavy chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO: 19 or 20, and the first light chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO: 21 or 22.

[0066] In one embodiment, the present disclosure relates to methods wherein the first antigen binding region activates 4-1BB signaling and is a monoclonal antibody.

[0067] In one embodiment, the present disclosure relates to a method wherein the second antigen-binding region that specifically binds to PD-L1 comprises a second heavy chain variable region and a second light chain variable region, wherein the second heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 consisting of the sequences shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively, and the second light chain variable region comprises LCDR1, LCDR2, and LCDR3 consisting of the sequences shown in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively.

[0068] In one embodiment, the present disclosure relates to a method in which the second antigen-binding region further comprises a second heavy chain constant region and / or a second light chain constant region, wherein the second heavy chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO: 19 or 20, and the second light chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO: 21 or 22.

[0069] In one embodiment, the present disclosure relates to methods wherein the bispecific antibody comprises: (1) Chain 1: the heavy chain of the second antigen-binding region connected at the C-terminus with or without a linker to the antigen-binding portion of the first antigen-binding region; and

[0070] (2) Chain 2: the light chain of the second antigen-binding region.

[0071] In one embodiment, the present disclosure relates to methods wherein the linker comprises the amino acid sequence (Gly4Ser)n or (GlySer4)n, wherein n is a positive integer from 1-7.

[0072] In one embodiment, the present disclosure relates to methods wherein the bispecific antibody consists of two chain 1 and two chain 2.

[0073] In one embodiment, the present disclosure relates to a method wherein the antigen binding portion is selected from: (i) a Fab fragment; (ii) a F(ab')2 fragment; (iii) a Fd fragment; (iv) an Fv fragment, (v) a dAb fragment; (vi) a nanobody; and (vii) a single-chain Fv (scFv); the scFv comprising the first heavy chain variable region and the first light chain variable region.

[0074] In one embodiment, the present disclosure relates to methods wherein the first antigen-binding region and / or the second antigen-binding region is a mouse antibody, a human antibody, a chimeric antibody, or a humanized antibody.

[0075] In one embodiment, the present disclosure relates to methods wherein the first antigen binding region and / or the second antigen binding region is of the IgG1, IgG2, or IgG4 isotype.

[0076] In one embodiment, the present disclosure relates to a method wherein said chain 1 of said bispecific antibody comprises, or consists of, an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 25; and said chain 2 of said bispecific antibody comprises, or consists of, an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 26.

[0077] In one embodiment, the malignant tumor in the method of the present disclosure is bladder cancer, breast cancer, endometrioid cancer, cervical cancer, advanced solid tumors, renal cell carcinoma, neuroendocrine tumors, gastric cancer, transitional cell carcinoma, urothelial carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, colorectal cancer, non-small cell lung cancer, hepatocellular carcinoma. In a preferred embodiment, the neuroendocrine tumor in the method of the present disclosure is a neuroendocrine carcinoma or a neuroendocrine tumor. In a more preferred embodiment, the neuroendocrine tumor in the method of the present disclosure is an extrapulmonary neuroendocrine tumor. In a more preferred embodiment, the neuroendocrine tumor in the method of the present disclosure is an advanced neuroendocrine tumor.

[0078] In one embodiment, the neuroendocrine cancer described in the methods of the present disclosure is an advanced neuroendocrine cancer. In another embodiment, the neuroendocrine cancer described in the methods of the present disclosure is an extrapulmonary neuroendocrine cancer or a small cell lung cancer. In a specific embodiment, the neuroendocrine cancer described in the methods of the present disclosure is an unresectable locally advanced or metastatic neuroendocrine cancer.

[0079] In one embodiment, the present disclosure relates to a method comprising administering the bispecific antibody to a subject having the malignant tumor, wherein the subject is a subject who has not received systemic treatment or a subject who has progressed after at least one line of platinum-containing chemotherapy.

[0080] In an exemplary embodiment, the subjects include but are not limited to subjects who have progressed after receiving two or more lines of chemotherapy, such as subjects who have progressed after receiving two lines of chemotherapy, subjects who have progressed after receiving three lines of chemotherapy, and subjects who have progressed after receiving four lines of chemotherapy.

[0081] In a specific embodiment, the subject can be a subject with small cell lung cancer who has not received systemic treatment before, a subject with advanced extrapulmonary neuroendocrine carcinoma who has progressed after receiving at least one line of chemotherapy, including but not limited to a subject with advanced extrapulmonary neuroendocrine carcinoma who has progressed after receiving two or more lines of chemotherapy, for example, a subject with advanced extrapulmonary neuroendocrine carcinoma who has progressed after receiving two lines of chemotherapy, a subject with advanced extrapulmonary neuroendocrine carcinoma who has progressed after receiving three lines of chemotherapy, and a subject with advanced extrapulmonary neuroendocrine carcinoma who has progressed after receiving four lines of chemotherapy.

[0082] In a more specific embodiment, the chemotherapy treatment received by the subject can be platinum-containing chemotherapy, for example, it can be first-line platinum-containing chemotherapy, second-line platinum-containing chemotherapy, or third-line platinum-containing chemotherapy.

[0083] In one embodiment, the method to which the present disclosure relates comprises administering 0.2 mg / kg to 200 mg / kg of the bispecific antibody to the subject per dosing cycle or at a time. In a preferred embodiment, the method to which the present disclosure relates comprises administering 0.8 mg / kg to 25 mg / kg of the bispecific antibody to the subject per dosing cycle or at a time. In a more preferred embodiment, the method to which the present disclosure relates comprises administering 3.2 mg / kg to 20 mg / kg of the bispecific antibody to the subject per dosing cycle or at a time. In an even more preferred embodiment, the method to which the present disclosure relates comprises administering 6.0 mg / kg to 15 mg / kg of the bispecific antibody to the subject per dosing cycle or at a time. In a most preferred embodiment, the method to which the present disclosure relates comprises administering 10 mg / kg to 15 mg / kg of the bispecific antibody to the subject per dosing cycle or at a time.

[0084] In one embodiment, the method of the present disclosure comprises administering 50 mg to 5000 mg of the bispecific antibody to the subject per dosing cycle or at a time. In a preferred embodiment, the method of the present disclosure comprises administering 60 mg to 4000 mg of the bispecific antibody to the subject per dosing cycle or at a time. In a more preferred embodiment, the method of the present disclosure comprises administering 80 mg to 3750 mg of the bispecific antibody to the subject per dosing cycle or at a time. In a most preferred embodiment, the method of the present disclosure comprises administering 100 mg to 3500 mg of the bispecific antibody to the subject per dosing cycle or at a time. In some specific embodiments, the method of the present disclosure comprises administering 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, 3500 mg of the bispecific antibody to the subject per dosing cycle or at a time.

[0085] In one embodiment, the bispecific antibody in the methods of the present disclosure 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.

[0086] In one embodiment, the present disclosure relates to methods wherein the bispecific antibody is formulated for intravenous infusion or subcutaneous injection.

[0087] In one embodiment, the methods of the present disclosure further comprise administering one or more chemotherapeutic agents to the subject. In preferred embodiments, the chemotherapeutic agents in the methods of the present disclosure are platinums and / or podophyllotoxin derivatives. In more preferred embodiments, the platinums in the methods of the present disclosure are carboplatin or cisplatin. In most preferred embodiments, the podophyllotoxin derivatives in the methods of the present disclosure are etoposide, etoposide glucuronide, or etoposide phosphate.

[0088] In one embodiment, the present disclosure relates to methods wherein 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.

[0089] In one embodiment, the present disclosure relates to methods wherein the chemotherapeutic agent is formulated for administration as an intravenous infusion or an oral formulation.

[0090] In one embodiment, the present disclosure relates to methods comprising administering the chemotherapeutic agent with each dosing cycle or each time the bispecific antibody is administered to the subject.

[0091] In one embodiment, the method of the present disclosure comprises administering the podophyllotoxin derivative for 1-5 days per dosing cycle or each time after administration of the bispecific antibody, for example, administering the podophyllotoxin derivative for 3 days or 1 day. In a preferred embodiment, the method of the present disclosure comprises administering 100 mg / m 2 or less of said podophyllotoxin derivative.

[0092] In one embodiment, the method of the present disclosure comprises administering the platinum compound for 1-5 days per dosing cycle or each time after the administration of the bispecific antibody, for example, administering the platinum compound for 3 days or 1 day. In a preferred embodiment, the method of the present disclosure comprises administering 75 mg / m2 per day. 2 or less of said platinum species, or AUC=5 mg / mL / min or less of said platinum species. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] FIG1 shows the structure of the P4B-3 bispecific antibody.

[0094] FIG2 shows the curves of the serum concentration of P4B-3 antibody changing with time in different administration cycles, wherein FIG2A is the curve of the serum concentration changing with time in the first cycle; FIG2B is the curve of the serum concentration changing with time in the fifth cycle.

[0095] FIG3 shows the level of peripheral cytokine IFN-γ release after a single administration of P4B-3 antibody.

[0096] Figure 4 shows the correlation analysis between P4B-3 antibody, PD-L1 expression and therapeutic efficacy. DETAILED DESCRIPTION

[0097] In order that the present disclosure may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0098] The term "4-1BB" refers to tumor necrosis factor receptor superfamily member 9. The term "4-1BB" includes variants, isoforms, homologs, orthologs and paralogs. For example, antibodies specific for human 4-1BB protein may cross-react with 4-1BB proteins from species other than humans (such as monkeys) in some cases. In other embodiments, antibodies specific for human 4-1BB protein may be completely specific to human 4-1BB protein and do not show cross-reactivity to other species or other types, or may cross-react with 4-1BB from certain other species but not all other species.

[0099] The term "human 4-1BB" refers to a 4-1BB protein having an amino acid sequence from a human, such as the amino acid sequence of human 4-1BB with Genbank accession number NP_001552.2. In one embodiment, human 4-1BB comprises the amino acid sequence set forth in SEQ ID NO: 48. The terms "monkey or rhesus 4-1BB" and "mouse 4-1BB" refer to monkey and mouse 4-1BB sequences, respectively, such as monkey and mouse 4-1BB sequences having amino acid sequences with Genbank accession numbers NP_001253057.1 and NP_033430.1, respectively.

[0100] The term "antibody" as referred to herein includes intact antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. In addition, the term "antibody" as referred to herein also encompasses multispecific antibodies, such as bispecific antibodies or trispecific antibodies. 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 comprises a heavy chain variable region (abbreviated herein as V H ) and the heavy chain constant region (abbreviated herein as C H The heavy chain constant region comprises three domains, namely CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as V L ) and the light chain constant region (abbreviated herein as C L The light chain constant region comprises one domain, namely CL. The VH region and V L The V domains can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), alternating with regions that are more conserved, termed framework regions (FRs). H and VL It is composed of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domain that interacts with the antigen. The constant region of an antibody can mediate the binding of the immunoglobulin 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.

[0101] As used herein, the term "antigen-binding portion" of an antibody (or simply "antibody portion") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., 4-1BB 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 encompassed within the term "antigen-binding portion" of an antibody include (i) Fab fragments, i.e., fragments consisting of V L 、V H 、C L and C H1 (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of a VH domain and a CH1 domain; (iv) an Fv fragment consisting of a single-armed VL domain and a VH domain; (v) a dAb fragment consisting of a VH domain (Ward et al. (1989) Nature 341:544-546); (vi) isolated complementarity determining regions (CDRs); and (vii) nanobodies, heavy chain variable regions containing a single variable domain and two constant domains. In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be connected by a synthetic linker using recombinant methods, which enables them to be made into a single protein chain, wherein the VL region and the VH region pair to form a monovalent molecule (referred to as 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 an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and fragments are screened for utility in the same manner as intact antibodies.

[0102] The term "bispecific" refers to an antibody that is capable of specifically binding to at least two different antigenic determinants. Typically, a bispecific antibody comprises two antigen-binding domains, wherein each antigen-binding domain is specific for different antigenic determinants. In certain embodiments, a bispecific antibody is capable of simultaneously binding to two antigenic determinants, particularly two antigenic determinants expressed on two different cells. As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope," and refers to the site (for example, a continuous segment of amino acids or a conformational configuration consisting of different regions of non-continuous amino acids) of the formation antigen-binding domain-antigen complex to which the antigen-binding portion is bound on a polypeptide macromolecule. Useful antigenic determinants may be present in, for example, free matter and / or extracellular matrix (ECM) on the surface of tumor cells, virus-infected cells, other disease cell surfaces, immune cell surfaces, serum.

[0103] As used herein, the term "antigen binding region" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, the antigen binding region is capable of guiding the subject to which it is connected (e.g., the second antigen binding region) to a target site, for example, to a specific type of tumor cell carrying an antigenic determinant. In another embodiment, the antigen binding region (e.g., the first antigen binding region) is capable of activating signal transduction through its target antigen, such as a T cell receptor antigen. The antigen binding region includes an antibody as defined herein or its antigen-binding fragment. A specific antigen binding region includes an antigen-binding fragment of an antibody, which includes an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, the antigen binding region may include an antibody constant region as defined herein and known in the art.

[0104] "Specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific 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) technology (e.g., analysis on a BIAcore instrument).

[0105] Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., a monoclonal antibody and an antigen, an antigen binding region and an antigen, or a receptor and its ligand). Affinity can be determined by recognized methods known in the art, including the methods described herein.

[0106] As used herein, "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an antibody that specifically binds to the separation of 4-1BB protein is substantially free of antibodies that specifically bind to antigens other than 4-1BB protein). However, the antibody that specifically binds to the separation of human 4-1BB protein can have cross-reactivity to other antigens, such as 4-1BB proteins from other species. In addition, the isolated antibody can be substantially free of other cellular substances and / or chemicals.

[0107] The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0108] The term "human antibody" as used herein is intended to include antibodies with variable regions, in which the framework regions and CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from another mammalian germline have been transplanted onto human framework sequences.

[0109] The term “agonistic 4-1BB antibody” or “agonistic anti-4-1BB antibody” refers to an anti-4-1BB antibody that binds to 4-1BB and activates or induces 4-1BB signaling to promote activation and / or proliferation of immune cells (such as T cells).

[0110] The term "isotype" refers to the antibody class (eg, IgM or IgG1) encoded by the heavy chain constant region genes.

[0111] The phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0112] As used herein, an antibody that "specifically binds to 4-1BB" means an antibody that binds to 4-1BB protein (including 4-1BB protein from humans and possibly from one or more non-human species) but does not substantially bind to non-4-1BB proteins. Preferably, the antibody is expressed at 1.0×10 -6 M or less, preferably 5.0×10 -7 M or less, more preferably 1.0×10 -7 Binds to human 4-1BB protein with a KD of M or lower.

[0113] As used herein, the term "does not substantially bind" to a protein or cell means that the protein or cell is not specifically bound or does not bind with high affinity, i.e., with an affinity of 1.0×10 -6 M or higher, preferably 1.0×10 -5 M or higher, preferably 1.0×10 -4 M or higher, more preferably 1.0×10 -3 M or higher, even more preferably 1.0×10 -2 Binds to proteins or cells with a KD of M or higher.

[0114] For IgG antibodies or antigen-binding regions thereof, the term "specifically binds to a second antigen (PD-L1) with high affinity" means that the antibody or antigen-binding region has a 1.0 × 10 -8 M or less, preferably 5.0×10 -9 M or less, more preferably 1.0×10 -9 M or lower KD.

[0115] As used herein, the term "K association" or "Ka" is intended to refer to the association rate of a particular antibody-antigen interaction, while the term "K dissociation" or "Kd" is intended to refer to the dissociation rate of a particular antibody-antigen interaction. As used herein, 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 a Biacore™ system.

[0116] The term "EC50," also known as half maximal effective concentration, refers to the concentration of an antibody that elicits a response halfway between baseline and maximum after a specified exposure time.

[0117] The term "IC50," also known as half-maximal inhibitory concentration, refers to the concentration of an antibody that inhibits a specific biological or biochemical function by 50% relative to the absence of the antibody.

[0118] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals are preferred, such as non-human primates, sheep, dogs, cats, cows, and horses.

[0119] The term "therapeutically effective amount" refers to an amount of an antibody of the present disclosure sufficient to prevent or ameliorate symptoms associated with a disease or condition (such as cancer) and / or lessen the severity of the disease or condition. A therapeutically effective amount is understood to be relevant to the condition being treated, wherein the actual effective amount is readily discernible to those skilled in the art.

[0120] 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 to immune checkpoint molecules), anti-infective agents, immunomodulators, and small molecule drugs.

[0121] The term "chemotherapeutic agent" includes compounds used to treat cancer.

[0122] The term "anti-infective agent" includes any molecule that specifically inhibits or eliminates the growth of microorganisms such as viruses, bacteria, fungi, or protozoa, eg, parasites, at the concentrations and intervals of administration, but is not lethal to the host.

[0123] As used herein, the term anti-infective agent includes antibiotics, antibacterial agents, antiviral agents, antifungal agents, and antiprotozoal agents. In a specific aspect, the anti-infective agent is non-toxic to the host at the concentrations and intervals of administration.

[0124] Immunomodulators include immune checkpoint inhibitors and co-stimulatory molecule activators.

[0125] The term "small molecule drug" refers to a low molecular weight organic compound that can modulate biological processes. "Small molecule" is defined as a molecule having a molecular weight generally less than 2 kD, preferably less than 1 kD, and more preferably about 0.5 kD or less. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimetics.

[0126] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.

[0127] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorders," "proliferative disorders," and "tumor" as referred to herein are not mutually exclusive.

[0128] The term "neuroendocrine neoplasm" (NEN) refers to a class of tumors that arise from peptidergic neurons and neuroendocrine cells, exhibit neuroendocrine differentiation, and express neuroendocrine markers. NENs can arise throughout the body and are highly heterogeneous, with the lung and gastroenteropancreatic NENs being the most common. Based on the degree of differentiation, NENs are classified into well-differentiated, slow-growing neuroendocrine tumors (NETs), poorly differentiated, highly malignant neuroendocrine carcinomas (NECs), and mixed neuroendocrine-non-neuroendocrine tumors (MiNENs). NECs include small cell (SCNEC) and large cell (LCNEC) types. NECs are further divided into pulmonary NECs (including small cell lung cancer (SCLC) and large cell lung NECs (p-LCNEC)) and extrapulmonary NECs (EP-NECs). Most NECs are diagnosed at an advanced stage or with distant metastases, resulting in a poor prognosis. An analysis of data from 162,983 patients with neuroendocrine cancer in the US SEER database showed that the 5-year survival rates of patients with NEC from the lung, gastrointestinal tract, and other sites were 5.6%, 13.1%, and 26.0%, respectively.

[0129] The term "non-small cell lung cancer" (NSCLC) has its ordinary meaning in the art and is the most common type of lung cancer, including three major types: squamous cell carcinoma, large cell carcinoma, and adenocarcinoma.

[0130] The term "hepatocellular carcinoma" (HCC) has its general meaning in the art and is a malignant tumor of liver cells and one of the common primary liver cancers.

[0131] The term "infection" refers to a disease caused by a pathogen, including, for example, viral infection, bacterial infection, fungal infection, or infection by protozoa such as parasites.

[0132] The term "tumor immune escape" refers to a tumor's ability to evade immune recognition and clearance. Therefore, as a therapeutic concept, when this escape is reduced, tumor immunity is "cured" and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

[0133] The term "drug combination" or "drug combination product" refers to a non-fixed combination product or a fixed combination product. The term "non-fixed combination" means that the active ingredients (e.g., (i) an antibody of the invention and a platinum and / or podophyllotoxin derivative) are administered to a patient simultaneously, without specific time restrictions, or at the same or different time intervals, in a separate entity, wherein such administration provides a preventive or therapeutically effective level of the two active agents in the patient's body, and an exemplary non-fixed combination product is a kit. In some embodiments, the antibody of the invention and the platinum and / or podophyllotoxin derivative used in the drug combination are administered at a level that does not exceed that when they are 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. The dosage and / or time interval of the two active agents are preferably selected so that the combined use of the parts can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in the form of a separate formulation, which formulation can be the same or different, and each component can also be a pharmaceutical composition.

[0134] The term "pharmaceutical composition" refers to a mixture of one or more active ingredients and pharmaceutically acceptable excipients.

[0135] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiotherapy or surgery) to treat diseases described herein. This administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed ratio of active ingredients. Alternatively, this administration includes co-administration of each active ingredient in a variety of or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids can be reconstituted or diluted to the desired dose before administration. In addition, this administration also includes using each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the therapeutic regimen will provide the beneficial effects of the drug combination in treating disorders or conditions described herein.

[0136] "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 from fresh, frozen and / or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood component; body fluids, such as tears, vitreous humor, cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time during the subject's pregnancy or development. In some embodiments, the tissue sample is tumor tissue. The tissue sample may contain compounds that are not naturally mixed with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0137] The term "cycle" refers to a specific time period expressed in days or weeks that is repeated on a conventional 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) per cycle. The components of the drug combination of the present invention can be applied on the same day or on different days of the cycle, that is, (i) and (ii) of the drug combination of the present invention are applied separately, simultaneously or sequentially within the cycle.

[0138] The term "administer" refers to any one of the various methods and delivery systems known to those skilled in the art that the active ingredient in the medicine of the present invention or drug combination is physically introduced to an individual. The route of administration of each active ingredient in the medicine of the present invention or drug combination includes oral, intravenous (e.g., infusion (also known as drip) or injection), intramuscular, subcutaneous, intraperitoneal, spinal, local or other parenteral routes of administration. Phrase "parenteral administration" as used herein refers to the mode of administration beyond gastrointestinal and topical administration, usually by intravenous, and includes, but is not limited to, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardial, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and electroporation in vivo. Accordingly, each active ingredient in the medicine of the present invention or drug combination can be formulated into capsules, tablets, injections (including infusions or injections), syrups, sprays, lozenges, liposomes or suppositories etc.

[0139] The term "continuous administration" refers to daily administration. In the case of continuous administration, the drug can be administered once or more per day, for example, once a day, twice a day, three times a day, preferably once a day.

[0140] The term "dose" refers to the amount of a drug required to elicit a therapeutic effect. Unless otherwise indicated, the dose is relative to the amount of the drug in free form. If the drug is in the form of a pharmaceutically acceptable salt, the amount of drug is proportionally increased compared to the amount of the drug in free form. For example, the dose will be stated on the product packaging or product information sheet.

[0141] The term "pharmaceutically acceptable salt" includes, but is not limited to, acid addition salts or base addition salts, for example, acid addition salts formed with inorganic acids such as hydrochloride, hydrobromide, carbonate, bicarbonate, phosphate, sulfate, sulfite, nitrate, etc., and acid addition salts formed with organic acids such as formate, acetate, malate, maleate, fumarate, tartrate, succinate, citrate, lactate, methanesulfonate, p-toluenesulfonate, 2-hydroxyethanesulfonate, benzoate, salicylate, stearate, and salts with organic acids of the formula HOOC-(CH2) n"Pharmaceutically acceptable salts" also include 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.

[0142] The term "pharmaceutically acceptable" 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 response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0143] The term "adverse event" (AE) is any unfavorable and generally unexpected or unwanted sign (including abnormal laboratory findings), symptom, or disease 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 the treatment or expansion of immune system cells (e.g., T cells) in response to the treatment. A medical treatment may have one or more associated AEs, and each AE may have the same or different levels of severity.

[0144] The term "overall survival" or "OS" is the time from the first dose of the study drug to the patient's death from any cause.

[0145] The term "progression-free survival" or "PFS" is the time from the first dose of the study drug to the time of disease progression or death from any cause.

[0146] All numerical ranges herein should be understood to disclose every value and subset of values ​​within the range, regardless of whether they are specifically disclosed otherwise. For example, when any numerical range is mentioned, it should be considered to mention every value within the numerical range, such as every integer within the numerical range. The present invention relates to all values ​​falling within these ranges, all smaller ranges, and the upper or lower limits of the numerical ranges.

[0147] The term "therapeutically effective amount" is an amount sufficient to provide a therapeutic benefit in the treatment or management of cancer or to delay or minimize one or more symptoms associated with cancer. A therapeutically effective amount of a compound refers to an amount of a therapeutic agent, alone or in combination with other therapeutic agents, that provides a therapeutic benefit in the treatment or management of cancer. The term "therapeutically effective amount" can encompass an amount that improves the overall therapy of cancer, alleviates or avoids the symptoms or causes of cancer, or enhances the therapeutic efficacy of another therapeutic agent. An example of an "effective amount" is an amount sufficient to help treat, prevent, or alleviate one or more symptoms of a disease, which may also be referred to as a "therapeutically effective amount." "Alleviation" of a symptom means a reduction 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 a "therapeutically effective amount") will depend on the purpose of the treatment and can be determined by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (Volumes 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, Gennaro ed., Lippincott, Williams & Wilkins).

[0148] The term "AUC" (area under the curve) refers to the total amount of drug absorbed or exposed to a subject, and can also refer to the partial AUC for a specific time interval. AUC can be derived based on a curve of drug concentration over time in a subject.

[0149] The term "first-line treatment" refers to the first treatment given for a condition or disease. It is usually part of a group of standard treatments, such as chemotherapy, radiation therapy, and / or immunotherapy followed by surgery. It is also called initial treatment, etc. Generally speaking, surgical resection of a malignant tumor does not count as a line of treatment. First-line treatment alone is generally recognized as the best treatment for the condition or disease. If it does not cure the condition or disease (for example, the treatment fails, stops working) or causes serious side effects, other treatments may be added or used.

[0150] Accordingly, the term "second-line treatment" refers to the treatment given when first-line treatment is unsuccessful, the term "third-line treatment" refers to the treatment or treatment regimen given when first-line treatment and subsequent second-line treatment are unsuccessful, and the term "multi-line treatment" refers to second-line treatment and above.

[0151] Various aspects of the invention are described in more detail in the following subsections.

[0152] II. Antibodies

[0153] The applicant has developed a bispecific antibody that specifically binds to 4-1BB and PD-L1 based on its 4-1BB antibody platform "X-Body". The 4-1BB antibody platform "X-Body" and the antibodies suitable for the present invention developed based on this platform are disclosed in PCT / CN2020 / 119388.

[0154] Bispecific antibodies

[0155] The exemplary bispecific antibodies of the present disclosure include a first antigen binding region that specifically binds 4-1BB and a second antigen binding region that specifically binds PD-L1. Bispecific antibodies can have several structural forms (see, for example, Aran F.Labrijn et al., Bispecific antibodies:a mechanistic review of the pipeline, Nature Reviews Drug Discovery, Vol. 18, pp. 585-608 (2019)). For example, bispecific antibodies can be bispecific antibodies of IgG samples, i.e., full-length bispecific antibodies, or bispecific antibodies of non-IgG samples, which are not full-length antibody constructs.

[0156] The bispecific antibodies of the present disclosure may comprise one or two or more first antigen-binding regions and / or one or two or more second antigen-binding regions.

[0157] In one embodiment, the first antigen-binding region is from or derived from an anti-4-1BB antibody or antigen-binding fragment, for example, from or derived from an anti-4-1BB antibody or antigen-binding fragment in PCT / CN2020 / 119388.

[0158] In one aspect, the first antigen binding region comprises a first heavy chain variable region and / or a first light chain variable region.

[0159] In another embodiment, the first antigen binding region can bind to human 4-1BB. In a specific embodiment, the first antigen binding region comprises the V of the above-mentioned anti-4-1BB antibody or antigen binding fragment. H and / or V L In one embodiment, the first heavy chain variable region in the first antigen binding region comprises the V H Or by the V H The first light chain variable region in the composition and / or the first antigen binding region comprises the VL Or by the V L composition. In another embodiment, the first antigen binding region further comprises a constant region. In another embodiment, the constant region in the first antigen binding region comprises a first heavy chain constant region and / or a first light chain constant region. In another embodiment, the first antigen binding region further comprises the constant region of the above-mentioned anti-4-1BB antibody or antibody binding fragment, such as the heavy chain constant region and / or light chain constant region of the above-mentioned anti-4-1BB antibody or antibody binding fragment. In one embodiment, the first antigen binding region comprises the scFv of the above-mentioned anti-4-1BB antibody.

[0160] In one aspect, the first heavy chain variable region in the first antigen binding region comprises three heavy chain variable region HCDRs: HCDR1, HCDR2 and / or HCDR3. In one embodiment, the three heavy chain variable region HCDRs: HCDR1, HCDR2 and HCDR3 are or comprise the three heavy chain variable region HCDRs: HCDR1, HCDR2 and HCDR3 of the above-mentioned anti-4-1BB antibody or antigen-binding fragment.

[0161] On the other hand, the first light chain variable region in the first antigen binding region comprises three light chain variable regions LCDR: LCDR1, LCDR2 and / or LCDR3. In one embodiment, the three light chain variable regions LCDR: LCDR1, LCDR2 and LCDR3 are or comprise the three light chain variable regions LCDR: LCDR1, LCDR2 and LCDR3 of the above-mentioned anti-4-1BB antibody or antigen binding fragment.

[0162] In another aspect, the first heavy chain variable region in the first antigen binding region comprises the three heavy chain variable regions HCDR1, HCDR2 and / or HCDR3 of the above-mentioned anti-4-1BB antibody or antigen-binding fragment; and the first light chain variable region in the first antigen binding region comprises LCDR1, LCDR2 and / or LCDR3 of the three light chain variable regions of the above-mentioned anti-4-1BB antibody or antigen-binding fragment.

[0163] In one embodiment, the first heavy chain variable region in the first antigen binding region

[0164] (i) comprises, or consists of, an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:9; or

[0165] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 9; or

[0166] (iii) comprises or consists 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: 7 or SEQ ID NO: 9, 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.

[0167] In a specific embodiment, the HCDR1, HCDR2 and HCDR3 of the first heavy chain variable region in the first antigen binding region are:

[0168] (i) HCDR1, HCDR2, and HCDR3 derived from the heavy chain variable region consisting of the sequence shown in SEQ ID NO: 7 or SEQ ID NO: 9;

[0169] (ii) The HCDR1, HCDR2 and HCDR3 of (i) further comprise at least one and no more than five amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) in total compared to the three HCDRs of (i).

[0170] In a specific embodiment, the first heavy chain variable region HCDR1 in the first antigen binding region comprises or consists of the amino acid sequence of SEQ ID NO: 1, or the HCDR1 comprises or consists of an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 1.

[0171] In a specific embodiment, the first heavy chain variable region HCDR2 in the first antigen binding region comprises or consists of the amino acid sequence of SEQ ID NO: 2, or the HCDR2 comprises or consists of an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 2.

[0172] In a specific embodiment, the first heavy chain variable region HCDR3 in the first antigen binding region comprises or consists of the amino acid sequence of SEQ ID NO: 3, or the HCDR3 comprises or consists of an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 3.

[0173] In one embodiment, the HCDR1, HCDR2 and HCDR3 of the first heavy chain variable region in the first antigen binding region are:

[0174] (i) HCDR1, HCDR2 and HCDR3 consisting of the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, or

[0175] (ii) The HCDR1, HCDR2 and HCDR3 of (i) further comprise at least one and no more than five amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) in total compared to the three HCDRs of (i).

[0176] In another embodiment, the first light chain variable region in the first antigen binding region

[0177] (i) comprises, or consists of, an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 10; or

[0178] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 10; or

[0179] (iii) 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: 8 or SEQ ID NO: 10, 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;

[0180] Wherein in SEQ ID NO: 8, X=S or G.

[0181] In one embodiment, LCDR1, LCDR2 and LCDR3 of the first light chain variable region in the first antigen binding region are:

[0182] (i) LCDR1, LCDR2 and LCDR3 derived from the light chain variable region consisting of the sequence shown in SEQ ID NO: 8 or SEQ ID NO: 10, or

[0183] (ii) LCDR1, LCDR2 and LCDR3 of (i), which further comprise 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);

[0184] Wherein in SEQ ID NO: 8, X=S or G.

[0185] In a specific embodiment, the first light chain variable region LCDR1 in the first antigen binding region comprises or consists of the amino acid sequence of SEQ ID NO: 4, or the LCDR1 comprises or consists of an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 4.

[0186] In a specific embodiment, the first light chain variable region LCDR2 in the first antigen binding region comprises or consists of the amino acid sequence of SEQ ID NO: 5, or the LCDR2 comprises or consists of an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 5.

[0187] In a specific embodiment, the first light chain variable region LCDR3 in the first antigen binding region comprises or consists of the amino acid sequence of SEQ ID NO: 6, or the LCDR3 comprises or consists of an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 6.

[0188] In one embodiment, LCDR1, LCDR2 and LCDR3 of the first light chain variable region in the first antigen binding region are:

[0189] (i) LCDR1, LCDR2 and LCDR3 consisting of the sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively, or

[0190] (ii) LCDR1, LCDR2 and LCDR3 of (i), further comprising at least one and no more than five amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) in total compared to the three LCDRs of (i).

[0191] In another embodiment, the first antigen binding region comprises: a first heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 consisting of the sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and a first light chain variable region comprising HCDR1, HCDR2 and HCDR3 consisting of the sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0192] In another embodiment, the first antigen binding region comprises:

[0193] (i) a first heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 7, and a first light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 8 (wherein X = S or G); or

[0194] (ii) a first heavy chain variable region comprising or consisting of the amino acid sequence shown in SEQ ID NO: 9, and a first light chain variable region comprising or consisting of the amino acid sequence shown in SEQ ID NO: 10.

[0195] In yet another aspect, the first antigen binding region optionally further comprises a constant region, and the constant region in the first antigen binding region comprises a first heavy chain constant region and / or a first light chain constant region.

[0196] In one embodiment, the first heavy chain constant region is or is derived from a human IgG constant region, such as an IgG1, IgG2, IgG3 or IgG4 constant region, preferably an IgG1 constant region, an IgG2 constant region or an IgG4 constant region.

[0197] In another embodiment, the first heavy chain constant region

[0198] (i) comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 19 or SEQ ID NO: 20; or

[0199] (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO: 19 or SEQ ID NO: 20; or

[0200] (iii) comprises or consists 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 shown in SEQ ID NO: 19 or SEQ ID NO: 20.

[0201] In one embodiment, the first light chain constant region is or is derived from a kappa or lambda light chain constant region, eg, a human kappa or lambda light chain constant region, eg, a human lambda light chain constant region.

[0202] In another embodiment, the first light chain constant region

[0203] (i) comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:21 or SEQ ID NO:22; or

[0204] (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO: 21 or SEQ ID NO: 22; or

[0205] (iii) comprises or consists 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 shown in SEQ ID NO: 21 or SEQ ID NO: 22.

[0206] In one embodiment, the constant region of the antibody can be mutated to improve the preparation and purification of the antibody. For example, the IgG4 constant region can have a mutation of S228P (EU numbering). The IgG1 constant region can have a mutation of L234A, L235A, D265A, P329A (EU numbering).

[0207] In one embodiment, the first antigen binding region is an scFv fragment. In another embodiment, the scFv fragment comprises HCDR1, HCDR2 and / or HCDR3 of the heavy chain variable region of the anti-4-1BB antibody or antibody binding fragment disclosed herein; and / or LCDR1, LCDR2 and / or LCDR3 of the light chain variable region. For example, the scFv fragment comprises a first light chain variable region and a first heavy chain variable region connected by a joint (or alternatively not via a joint).

[0208] In one embodiment, the linker is a flexible linker, such as a linker having a single glycine and / or serine residue or a combination thereof. In one embodiment, the linker comprises the amino acid sequence (Gly4Ser)n or (GlySer4)n, wherein 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, 6. In one embodiment, n is 1, 2, 3 or 4. In a specific embodiment, the linker comprises or consists of the amino acid sequence shown in SEQ ID NO:47 or SEQ ID NO:24 or SEQ ID NO:23 ((GGGGS)n, n is 1, 2, 3, 4, 5 or 6).

[0209] In another embodiment, the second antigen-binding region is from or derived from an anti-PD-L1 antibody or an antigen-binding fragment thereof, such as the anti-PD-L1 antibody or an antigen-binding fragment thereof disclosed in CN109021107A.

[0210] In another aspect, the second antigen binding region comprises a second heavy chain variable region and / or a second light chain variable region.

[0211] In another embodiment, the second antigen binding region can bind to human PD-L1. In a specific embodiment, the second antigen binding region comprises a V-binding region for anti-PD-L1 or antigen binding fragment as described above. H and / or V L In one embodiment, the second heavy chain variable region in the second antigen binding region comprises the V H or consisting thereof and / or the second light chain variable region in the second antigen binding region comprises the V L Or consisting thereof. In another embodiment, the second antigen-binding region further comprises a constant region. In another embodiment, the constant region in the second antigen-binding region comprises a second heavy chain constant region and / or a second light chain constant region. In another embodiment, the second antigen-binding region further comprises the constant region of the above-mentioned anti-PD-L1 antibody or antibody binding fragment, for example, the heavy chain constant region or light chain constant region of the above-mentioned anti-PD-L1 antibody or antibody binding fragment.

[0212] In another aspect, the second heavy chain variable region in the second antigen-binding region comprises three heavy chain variable region HCDRs: HCDR1, HCDR2, and / or HCDR3. In one embodiment, the three heavy chain variable region HCDRs: HCDR1, HCDR2, and HCDR3 are or comprise the three heavy chain variable region HCDRs: HCDR1, HCDR2, and / or HCDR3 of the above-mentioned anti-PD-L1 antibody or antigen-binding fragment.

[0213] In another aspect, the second light chain variable region in the second antigen-binding region comprises three light chain variable regions LCDR: LCDR1, LCDR2 and / or LCDR3. In one embodiment, the three light chain variable regions LCDR: LCDR1, LCDR2 and LCDR3 are or comprise the three light chain variable regions LCDR: LCDR1, LCDR2 and LCDR3 of the above-mentioned anti-PD-L1 antibody or antigen-binding fragment, respectively.

[0214] In another aspect, the second heavy chain variable region in the second antigen-binding region comprises the three heavy chain variable regions HCDR1, HCDR2 and / or HCDR3 of the above-mentioned anti-PD-L1 antibody or antigen-binding fragment; and the second light chain variable region in the second antigen-binding region comprises the three light chain variable regions LCDR1, LCDR2 and / or LCDR3 of the above-mentioned anti-PD-L1 antibody or antigen-binding fragment.

[0215] In one embodiment, the second heavy chain variable region in the second antigen binding region

[0216] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 17; or

[0217] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 17; or

[0218] (iii) comprises or consists 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: 17, 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.

[0219] In a specific embodiment, the second heavy chain variable region HCDR1, HCDR2 and HCDR3 in the second antigen binding region are:

[0220] (i) HCDR1, HCDR2 and HCDR3 derived from a heavy chain variable region comprising or consisting of the sequence shown in SEQ ID NO: 17, or

[0221] (ii) The HCDR1, HCDR2 and HCDR3 of (i) further comprise at least one and no more than five amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) in total compared to the three HCDRs of (i).

[0222] In a specific embodiment, the second heavy chain variable region HCDR1 in the second antigen binding region comprises or consists of the amino acid sequence of SEQ ID NO: 11, or the HCDR1 comprises or consists of an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 11.

[0223] In a specific embodiment, the second heavy chain variable region HCDR2 in the second antigen binding region comprises or consists of the amino acid sequence of SEQ ID NO: 12, or the HCDR2 comprises or consists of an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 12.

[0224] In a specific embodiment, the second heavy chain variable region HCDR3 in the second antigen binding region comprises or consists of the amino acid sequence of SEQ ID NO: 13, or the HCDR3 comprises or consists of an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 13.

[0225] In one embodiment, the second heavy chain variable region HCDR1, HCDR2 and HCDR3 in the second antigen binding region are:

[0226] (i) HCDR1, HCDR2 and HCDR3 consisting of the sequences shown in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively, or

[0227] (ii) The HCDR1, HCDR2 and HCDR3 of (i) further comprise at least one and no more than five amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) in total compared to the three HCDRs of (i).

[0228] In one embodiment, the second light chain variable region in the second antigen binding region

[0229] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 18; or

[0230] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 18; or

[0231] (iii) comprises or consists 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: 18, preferably, these amino acid modifications do not occur in the CDR region, more preferably, these amino acid changes occur in the FR region, such as FR1, FR2, FR3 or FR4.

[0232] In a specific embodiment, LCDR1, LCDR2 and LCDR3 of the second light chain variable region in the second antigen binding region are:

[0233] (i) LCDR1, LCDR2 and LCDR3 derived from a light chain variable region, wherein the light chain variable region comprises or consists of the sequence shown in SEQ ID NO: 18, or

[0234] (ii) LCDR1, LCDR2 and LCDR3 of (i), further comprising at least one and no more than five amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) in total compared to the three LCDRs of (i).

[0235] In a specific embodiment, the second light chain variable region LCDR1 in the second antigen binding region comprises or consists of the amino acid sequence of SEQ ID NO: 14, or the LCDR1 comprises or consists of an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 14.

[0236] In a specific embodiment, the second light chain variable region LCDR2 in the second antigen binding region comprises or consists of the amino acid sequence of SEQ ID NO: 15, or the LCDR2 comprises or consists of an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 15.

[0237] In a specific embodiment, the second light chain variable region LCDR3 in the second antigen binding region comprises or consists of the amino acid sequence of SEQ ID NO: 16, and the LCDR3 comprises or consists of an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 16.

[0238] In one embodiment, the three light chain variable regions LCDR1, LCDR2 and LCDR3 of the second light chain variable region in the second antigen binding region are:

[0239] (i) LCDR1, LCDR2 and LCDR3 consisting of the sequences shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively, or

[0240] (ii) LCDR1, LCDR2 and LCDR3 of (i), further comprising at least one and no more than five amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) in total compared to the three LCDRs of (i).

[0241] In another embodiment, the second antigen binding region comprises: a second heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 consisting of the sequences shown in SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13, and a second light chain variable region comprising HCDR1, HCDR2 and HCDR3 consisting of the sequences shown in SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16.

[0242] In another embodiment, the second antigen binding region comprises: a second heavy chain variable region comprising or consisting of the amino acid sequence shown in SEQ ID NO: 17, and a second light chain variable region comprising or consisting of the amino acid sequence shown in SEQ ID NO: 18.

[0243] In yet another aspect, the second antigen binding region optionally further comprises a constant region, and the constant region in the second antigen binding region comprises a second heavy chain constant region and / or a second light chain constant region.

[0244] In one embodiment, the second heavy chain constant region is or is derived from a human IgG constant region, such as an IgG1, IgG2, IgG3 or IgG4 constant region, preferably an IgG1 constant region, an IgG2 constant region or an IgG4 constant region.

[0245] In another embodiment, the second heavy chain constant region

[0246] (i) comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 19 or SEQ ID NO: 20; or

[0247] (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO: 19 or SEQ ID NO: 20; or

[0248] (iii) comprises or consists 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 shown in SEQ ID NO: 19 or SEQ ID NO: 20.

[0249] In one embodiment, the second light chain constant region is or is derived from a kappa or lambda light chain constant region, eg, a human kappa or lambda light chain constant region, preferably a human kappa light chain constant region.

[0250] In another embodiment, the second light chain constant region

[0251] (i) comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:21 or SEQ ID NO:22; or

[0252] (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO: 21 or SEQ ID NO: 22; or

[0253] (iii) comprises or consists 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 shown in SEQ ID NO: 21 or SEQ ID NO: 22.

[0254] V of known bispecific antibodies H and / or V L The V sequences of the monoclonal antibodies from which they are derived and used to construct bispecific antibodies may differ in one or several amino acids. H and / or V L For example, the derived V H and V L Can have one or more mutations to make the scFv it constitutes more stable. In one embodiment, V H and / or V L The antibodies or antigen-binding portions thereof may be more stable by forming disulfide bonds with each other. For example, mutations may be made to form a disulfide bond between the first heavy chain variable region and the first light chain variable region. For example, the mutation in the first heavy chain variable region is G44C (Eu numbering) and / or the mutation in the first light chain variable region is T104C (Eu numbering).

[0255] In a specific embodiment, the bispecific antibody comprises the following chains:

[0256] (1) Chain 1: the heavy chain of the second antigen-binding region connected at the C-terminus with or without a linker to the antigen-binding portion of the first antigen-binding region; and

[0257] (2) Chain 2: the light chain of the second antigen-binding region.

[0258] In a specific embodiment, the bispecific antibody comprises two chains 1 and two chains 2 connected by disulfide bonds.

[0259] In another specific embodiment, the structure of the bispecific antibody is shown in FIG1 .

[0260] In one embodiment, the antigen binding portion in the first antigen binding region is selected from: (i) a Fab fragment; (ii) a F(ab')2 fragment; (iii) a Fd fragment; (iv) an Fv fragment, (v) a dAb fragment; (vi) a nanobody; and (vii) a single-chain Fv (scFv); the scFv comprises the first heavy chain variable region and the first light chain variable region.

[0261] In one embodiment, the present disclosure relates to methods wherein the first antigen-binding region and / or the second antigen-binding region is a mouse antibody, a human antibody, a chimeric antibody, or a humanized antibody.

[0262] In one embodiment, the heavy chain of the second antigen-binding region comprises the second heavy chain variable region. In another embodiment, the heavy chain of the second antigen-binding region comprises the HCDR1, HCDR2, and / or HCDR3 of the second heavy chain variable region. In another embodiment, the light chain of the second antigen-binding region comprises the second light chain variable region. In another embodiment, the light chain of the second antigen-binding region comprises the LCDR1, LCDR2, and / or LCDR3 of the second heavy chain variable region.

[0263] In a specific embodiment, the heavy chain of the second antigen binding region further comprises the second heavy chain constant region.

[0264] In a specific embodiment, the light chain of the second antigen binding region further comprises the second light chain constant region.

[0265] In one embodiment, the linker in the bispecific antibody of the present invention is a flexible linker, such as a linker having a separate glycine and / or serine residue or a combination thereof. In one embodiment, the linker comprises the amino acid sequence (Gly4Ser)n or (GlySer4)n, wherein n is a positive integer equal to or greater than 1, for example, n is a positive integer of 1-7, for example, n is 2, 3, 4, 5, 6. In one embodiment, n is 1, 2, 3 or 4, preferably 1 or 3. In a specific embodiment, the linker of the bispecific antibody comprises or consists of the amino acid sequence shown in SEQ ID NO:47, SEQ ID NO:24 or SEQ ID NO:23. In a preferred embodiment, the linker of the bispecific antibody comprises or consists of the amino acid sequence shown in SEQ ID NO:23, wherein n=1 or 3.

[0266] In one embodiment, chain 1 of the bispecific antibody

[0267] (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 25; or

[0268] (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO: 25; or

[0269] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20, more preferably no more than 10 or 5) amino acid modifications (preferably amino acid substitutions, more preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO: 25.

[0270] In one embodiment, chain 2 of the bispecific antibody

[0271] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 26; or

[0272] (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO: 26; or

[0273] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20, more preferably no more than 10 or 5) amino acid modifications (preferably amino acid substitutions, more preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO: 26.

[0274] conservative modifications

[0275] In another embodiment, the bispecific antibodies of the present disclosure comprise a first antigen-binding region and a second antigen-binding region that differ from those described above in that one or more conservative modifications are present. It is understood in the art that certain conservative sequence modifications can be made without abolishing antigen binding. See, e.g., 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.

[0276] Thus, in one embodiment, the bispecific antibodies of the present disclosure comprise a first heavy chain variable region sequence and / or a first light chain variable region sequence that differs from those described above in that it has one or more conservative modifications, preferably, the modifications do not occur in the CDRs, preferably, the modifications occur in the FRs.

[0277] In some embodiments, modifications can 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 that can be used to construct a bispecific antibody.

[0278] In some embodiments, the modifications are substitutions, additions, and / or deletions.

[0279] In some embodiments, the substitution is a conservative substitution. A conservative substitution refers to the replacement of one amino acid by another amino acid of the same class, for example, an acidic amino acid is replaced by another acidic amino acid, a basic amino acid is replaced by another basic amino acid, or a neutral amino acid is replaced by another neutral amino acid. Exemplary substitutions are shown in the table below:

[0280] In various embodiments, the antibody can be, for example, a mouse antibody, a human antibody, a humanized antibody, or a chimeric antibody.

[0281] As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or change the binding properties of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibody of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are amino acid substitutions in which the amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), 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 non-polar 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). Thus, one or more amino acid residues within the CDR regions of the antibodies of the invention can be replaced by other amino acid residues from the same side chain family and the retained function (i.e., the above-mentioned function) of the altered antibodies can be tested using the functional assays described herein.

[0282] Engineered and modified antibodies

[0283] The V domains having the first and second antigen-binding regions of the bispecific antibodies of the present disclosure can be used. H Sequence / V L The antibodies of the present invention can be engineered by modifying one or more of the variable regions (i.e., V H and / or V L ), for example, one or more CDR regions and / or one or more framework regions. Additionally or alternatively, an 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.

[0284] In certain embodiments, CDR transplantation can be used to engineer the variable region of an antibody. Antibodies interact with the target antigen primarily through the amino acid residues located in the heavy chain complementary determining region and the light chain complementary determining region (CDR). For this reason, the amino acid sequence within the CDR between individual antibodies is more diverse than the sequence outside the CDR. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a specific naturally occurring antibody by constructing expression vectors that include CDR sequences from a naturally occurring antibody of that specificity grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann et al. (1998) Nature 332:323-327; Jones et al. (1986) Nature 321:522-525; Queen et al. (1989) Proc. Natl. Acad. See. USA 86:10029-10033; U.S. Pat. Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0285] Therefore, another embodiment of the present disclosure relates to an isolated monoclonal antibody or antigen-binding portion thereof, comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 sequences having the sequences of the present disclosure as described above and / or a light chain variable region having LCDR1, LCDR2 and LCDR3 sequences having the sequences of the present disclosure as described above. Although these antibodies contain the V sequences of the monoclonal antibodies of the present disclosure, H and V L CDR sequences, but they may contain different framework sequences.

[0286] Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk / vbase), as well as Kabat et al. (1991) (cited above); Tomlinson et al. (1992), J. Mol. Biol. 227: 776-798; and Cox et al. (1994), Eur. J. Immunol. 24: 827-836, the contents of each of which are expressly incorporated herein by reference. As another example, the germline DNA sequences of human heavy and light chain variable region genes can be found in the GenBank database. For example, the following heavy chain germline sequences found in the HCo7 HuMAb mouse are available under the accompanying GenBank Accession Nos.: 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 the HCo12 HuMAb mouse are available under the accompanying GenBank Accession Nos.: 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).

[0287] The antibody protein sequence is compared to compiled protein sequence databases using one of the sequence similarity search methods known to those skilled in the art as Gapped BLAST (Altschul et al. (1997) supra).

[0288] Preferred framework sequences for use in the antibodies of the present invention are those that are structurally similar to the framework sequences used in the antibodies of the present invention. HThe HCDR1, HCDR2, and HCDR3 sequences of the present invention can be grafted onto framework regions having sequences identical to those present in the germline immunoglobulin gene from which the framework sequences are derived, or the CDR sequences can be grafted onto framework regions that contain one or more mutations compared to the germline sequences. For example, it has been found that in certain circumstances it is beneficial to mutate residues within the framework regions to maintain or enhance the antigen-binding ability of the antibody (see, e.g., U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0289] Another type of variable region modification is to make V H and / or V L The amino acid residue mutations in the CDR1, CDR2 and / or CDR3 regions of the antibody of interest can be used 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 impact on antibody binding or other functional properties of interest can be evaluated in the in vitro or in vivo assays provided as described herein and in the Examples. Preferably, conservative modifications (e.g., known in the art) are introduced. The mutation can be an amino acid substitution, addition or deletion, but preferably a substitution. In addition, typically no more than 1, 2, 3, 4 or 5 residues are changed in the CDR regions.

[0290] Thus, in another embodiment, the present invention provides an isolated anti-PD-L1 monoclonal antibody, or antigen-binding portion thereof, comprising a heavy chain variable region comprising: (a) a V comprising a sequence of the present invention or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions compared to a sequence of the present invention; H (b) a V region comprising a sequence of the present invention or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions compared to the sequence of the present invention; H (c) comprising a V sequence of the present invention or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions compared to the sequence of the present invention; H (d) a V region comprising a sequence of the present invention or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions compared to the sequence of the present invention; L LCDR1 region; (e) V comprising a sequence of the present invention or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions compared to the sequence of the present invention; LLCDR2 region; and (f) V comprising a sequence of the present invention or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions compared to the sequence of the present invention. L LCDR3 area.

[0291] The engineered antibodies of the present invention include those wherein V H and / or V L In some embodiments, the invention relates to antibodies that modify framework residues within the antibody, for example, to improve the properties of the antibody. Typically, such framework modifications are performed to reduce the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, antibodies that have undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody was derived. Such residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody was derived.

[0292] Another type of framework modification involves mutating one or more residues within the framework region or even within 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.

[0293] In addition to or as an alternative to modifications in the framework or CDR regions, the antibodies of the invention can be engineered to include modifications in 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 cellular cytotoxicity. In addition, the antibodies of the invention can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody), or modified to alter its glycosylation, thereby again altering one or more functional properties of the antibody.

[0294] In one embodiment, the hinge region of CH1 is modified such that the number of cysteine ​​residues in the hinge region is altered, e.g., increased or decreased. This approach is further described in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of CH1 is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

[0295] In another embodiment, the Fc hinge region of the antibody is mutated to shorten the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc hinge fragment so that the antibody has impaired Staphylococcal protein A (SpA) binding relative to native Fc hinge domain SpA binding. This approach is described in more detail in U.S. Patent No. 6,165,745.

[0296] In another embodiment, the glycosylation of the antibody is modified. For example, an antibody without glycosylation can be prepared (i.e., the antibody lacks glycosylation). Glycosylation can be changed to, for example, increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be achieved by, for example, changing one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that cause the elimination of one or more variable region framework glycosylation sites, thereby eliminating the glycosylation at the site. Such aglycosylation can increase the affinity of the antibody for the antigen. See, for example, U.S. Patent Nos. 5,714,350 and 6,350,861.

[0297] Additionally or alternatively, antibodies with altered glycosylation patterns can be prepared, such as low-fucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. It has been demonstrated that such altered glycosylation profiles increase the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation machinery. Cells with altered glycosylation machinery 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, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (α(1,6)-fucosyltransferase), and therefore, antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8- / - cell lines were generated by targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement 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 (encoding a fucosyltransferase) such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating enzymes involved in α-1,6 linkages. EP 1,176,195 also describes cell lines with low or no activity for the enzyme that adds fucose to N-acetylglucosamine bound to the Fc region of antibodies, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). PCT Publication WO 03 / 035835 describes a variant CHO cell line, Lec13 cells, which have a reduced ability to attach fucose to Asn(297)-linked carbohydrates, thereby also resulting in 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. Methods for producing antibodies in plant systems are disclosed in U.S. patent application filed August 11, 2006, corresponding to Alston & Bird LLP attorney docket number 040989 / 314911.PCT publication WO 99 / 54342 describes a cell line engineered to express glycosyltransferases that modify glycoproteins, such as β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), whereby antibodies expressed in the engineered cell line exhibit increased bisecting GlcNac structures, which results in increased ADCC activity of the antibody (see also Umana et al. (1999) Nat. Biotech. 17: 176-180). Alternatively, fucosidases can be used to cleave off fucose residues from antibodies; for example, fucosidase α-L-fucosidase removes fucosyl residues from antibodies (Tarentino et al. (1975) Biochem. 14: 5516-23).

[0298] Another modification of the antibody herein that the present disclosure contemplates is pegylation. Antibodies can be pegylated to, for example, extend the biological (e.g., serum) half-life of the antibody. In order to pegylate the antibody, typically under conditions wherein one or more PEG groups are attached to the antibody or antibody fragment, the antibody or its fragment is reacted with polyethylene glycol (PEG), such as the reactive ester or aldehyde derivatives of PEG. Preferably, pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any one of the forms of PEG that have been used to derivatize other proteins, such as single (C1-C10) alkoxypolyethylene glycol or aryloxypolyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an antibody without glycosylation. Methods for pegylating proteins are known in the art and can be applied to antibodies of the present invention. See, for example, EPO 154 316 and EP 0 401 384.

[0299] II. Antibody Physical Properties

[0300] The antibodies of the present invention can be characterized by their various physical properties in order to detect and / or distinguish between their different classes.

[0301] For example, the antibody may contain one or more glycosylation sites in the light chain variable region or the heavy chain variable region. Such glycosylation sites may cause the immunogenicity of the antibody to increase or the pK of the antibody to change 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 a motif containing an NXS / T sequence. In some cases, it is preferred to have an anti-PD-L1 antibody that does not contain variable region glycosylation. 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.

[0302] In a preferred embodiment, the antibody does not contain an asparagine isomerization site. Deamidation of asparagine may occur on NG or DG sequences and result in the generation of an isoaspartic acid residue, which introduces a knot into the polypeptide chain and reduces its stability (isoaspartic acid effect).

[0303] 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 generally falls within the pH range of 7-9.5 and the pI value of IgG4 antibodies generally falls within the pH range of 6-8. It is speculated that antibodies with pI values ​​outside the normal range may have some unfolding and instability under in vivo conditions. Therefore, it is preferred to have anti-4-1BB antibodies containing pI values ​​falling within the normal range. This can be achieved by selecting antibodies with pI values ​​within the normal range or by mutating charged surface residues.

[0304] III. Pharmaceutical Compositions

[0305] In another aspect, the present disclosure provides pharmaceutical compositions comprising a bispecific antibody of the present disclosure formulated together with a pharmaceutically acceptable excipient and optionally one or more chemotherapeutic agents.

[0306] The pharmaceutical composition can include any number of excipients. Operable excipients include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersion or suspension aids, solubilizers, coloring agents, flavorings, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof. The selection and use of suitable excipients are described in Gennaro, Remington: The Science and Practice of Pharmacy, 20th edition (Lippincott Williams & Wilkins 2003), which is incorporated herein by reference.

[0307] 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 can be coated in a material to protect it from the effects of acid and other natural conditions that may inactivate it. As used herein, the phrase "parenteral administration" means the mode of administration except enteral and topical administration, usually by injection, and includes but is not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, the antibody of the present invention can be via non-parenteral route, such as topical, epidermal or mucosal administration route, for example, intranasal, oral, vaginal, rectal, sublingual or topical. Preferably, the antibody is formulated into intravenous infusion or subcutaneous injection formulations.

[0308] The pharmaceutical compositions can be in the form of sterile aqueous solutions or dispersions. They can also be formulated as microemulsions, liposomes, or other ordered structures suitable for high drug concentration.

[0309] 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 particular mode of administration, and will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01% to about 99% of the active ingredient, preferably from about 0.1% to about 70%, and most preferably from about 1% to about 30% of the active ingredient, combined with a pharmaceutically acceptable carrier.

[0310] The pharmaceutical composition can be a controlled release formulation, including implants, transdermal patches and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0311] The therapeutic agent can be administered via medical devices, such as (1) needle-free subcutaneous injection devices (e.g., U.S. Pat. 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. Pat. No. 4,487,603); (3) transdermal devices (U.S. Pat. No. 4,486,194); (4) infusion sets (U.S. Pat. Nos. 4,447,233 and 4,447,224); and (5) osmotic devices (U.S. Pat. Nos. 4,439,196 and 4,475,196); the disclosures of which are incorporated herein by reference.

[0312] In some embodiments, the pharmaceutical composition comprises (i) a bispecific antibody of the invention; (ii) a podophyllotoxin derivative, such as etoposide, etoposide glucuronide, or etoposide phosphate; and (iii) a platinum, such as carboplatin or cisplatin.

[0313] IV. Combination Therapies and Drug Combinations

[0314] In another aspect, the disclosure provides combination therapies in which the bispecific molecules of the disclosure are co-administered with one or more other therapeutic agents (i.e., drug combinations) and / or treatment modalities, such as additional antibodies or chemotherapeutic agents, that are effective in ameliorating cancer, infection, or autoimmune disease in a subject.

[0315] In some embodiments, the disclosure provides combination therapies for treating a cancer disease in a subject, comprising administering to the subject an antibody of the invention and one or more other therapies (e.g., standard disease treatments, e.g., standard cancer treatments), e.g., therapeutic modalities and / or other therapeutic agents.

[0316] In some embodiments, treatment modalities include surgery, radiation, cryosurgery, and / or thermal therapy.

[0317] In some embodiments, the additional therapeutic agent is, for example, an additional antibody, such as an anti-PD-1 antibody and / or an anti-CTLA-4 antibody.

[0318] In some embodiments, the therapeutic agent is selected from a chemotherapeutic agent, an additional antibody, a cytotoxic agent, a vaccine, an anti-infective agent, a small molecule drug, or an immunomodulatory agent.

[0319] In one aspect, the invention provides pharmaceutical combinations comprising a bispecific molecule of the disclosure and one or more additional therapeutic agents.

[0320] For example, the antibodies of the present invention can be combined with standard cancer treatments. For example, antibodies can be effectively combined with chemotherapy regimens. In these cases, the dose of the chemotherapeutic agent administered can be reduced (Mokyr, M. et al., (1998) Cancer Research 58: 5301-5304). In certain embodiments, the methods and antibodies described herein 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 drugs), cytotoxic agents, immune-based therapies (e.g., cytokines), surgery and / or radiation.

[0321] In certain embodiments, the antibodies of the invention are used in combination with standard cancer treatment chemotherapeutic agents, including, but not limited to, platinums and / or podophyllotoxin derivatives; more preferably, the platinums are carboplatin or cisplatin; and most preferably, the podophyllotoxin derivatives are etoposide, etoposide glucuronide, or etoposide phosphate. In some embodiments, the drug combination comprises a bispecific antibody of the invention; a podophyllotoxin derivative, such as etoposide, etoposide glucuronide, or etoposide phosphate; and a platinum, such as carboplatin or cisplatin.

[0322] In some embodiments, an antibody of the invention is combined with carboplatin and etoposide, or with cisplatin and etoposide.

[0323] In some embodiments, the bispecific molecule and one or more other therapeutic agents in the pharmaceutical combination of the present disclosure are administered to a patient simultaneously, without specific time limits, or sequentially at the same or different time intervals, as separate entities, wherein such administration provides prophylactically or therapeutically effective levels of the two active agents in the patient. Exemplarily, the bispecific molecule of the present disclosure and one or more other therapeutic agents are a kit.

[0324] In some specific embodiments, the bispecific molecules and one or more other therapeutic agents in the pharmaceutical combination of the present invention are administered to the patient simultaneously as a single entity. The dosage and / or time interval of the bispecific molecules of the present invention and one or more other therapeutic agents are preferably selected so that the combined use of the components can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in the form of a separate formulation, which can be the same or different. The bispecific antibody of the present invention and / or other therapeutic agent in the pharmaceutical combination of the present invention can be a pharmaceutical dosage unit, such as a single pharmaceutical dosage unit.

[0325] In other embodiments, the bispecific molecule and one or more other therapeutic agents in the pharmaceutical combinations of the present disclosure are present in the same pharmaceutical composition.

[0326] In one aspect, the present invention also relates to a kit comprising the pharmaceutical combination of the present invention, preferably in the form of a pharmaceutical dosage unit, whereby the dosage unit can be provided according to a dosing regimen or interval of drug administration.

[0327] In one embodiment, the kit of parts of the present invention comprises in the same package:

[0328] - a first container containing the antibody or pharmaceutical composition of the present invention;

[0329] - A second container containing one or more other therapeutic agents, such as chemotherapeutic agents, such as standard cancer treatment chemotherapeutic agents. In some embodiments, the bispecific antibody of the present invention and the other therapeutic agent, such as the chemotherapeutic agent, in the pharmaceutical combination of the present invention can 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, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments, creams and injections, etc., and the dosage forms can be different or the same. In some embodiments, the bispecific antibody of the present invention is preferably formulated as an intravenous infusion or subcutaneous injection formulation. In some embodiments, the chemotherapeutic agent of the present invention is formulated for intravenous infusion or oral administration. In some embodiments, in the pharmaceutical combination of the present invention, the bispecific antibody is administered intravenously and the chemotherapeutic agent is also administered intravenously.

[0330] V. Use / Treatment Method

[0331] The bispecific antibodies of the present disclosure have numerous in vitro and in vivo uses, and these in vitro and in vivo uses relate to, for example, the treatment of cancer, infection, and autoimmune diseases. The bispecific antibodies of the present disclosure can be administered to human subjects, for example, in vivo, to treat these diseases. In some embodiments, the methods of treatment of the present disclosure include administering the bispecific antibodies of the present disclosure to the subject. In other embodiments, the methods of treatment of the present disclosure include administering the bispecific antibodies of the present disclosure to the subject and one or more other therapies (e.g., standard disease treatments, such as standard cancer treatments), such as treatment modalities and / or other therapeutic agents.

[0332] In one aspect, the invention provides methods of modulating an immune response in a subject.

[0333] In some embodiments, the present invention provides methods for activating T cells or inducing T cell-mediated anti-tumor activity. In one embodiment, the T cells are CD8 + In another aspect, the present invention provides a method for reducing Treg cells, such as CD4 + T cell approach.

[0334] In some embodiments, activation of T cells comprises stimulating cytokine secretion by T cells, such as IL-12 secretion by T cells.

[0335] In some embodiments, the present invention provides methods of activating the 4-1BB signaling pathway.

[0336] In some embodiments, the above methods include administering a bispecific antibody of the present disclosure or a pharmaceutical combination / combination therapy comprising the same. Specifically, the above methods include administering a bispecific antibody of the present disclosure, a pharmaceutical composition or formulation comprising the bispecific antibody of the present disclosure, a combination product, or a nucleic acid encoding the bispecific antibody of the present disclosure.

[0337] In another aspect, the present invention provides a method for treating a tumor in a subject, comprising administering a bispecific antibody that binds to 4-1BB and PD-L1 of the present disclosure, or a pharmaceutical composition or pharmaceutical combination comprising the same.

[0338] In some embodiments, the tumor is a malignant tumor, such as a cancer.

[0339] In some embodiments, the cancer is an immune evasion tumor.

[0340] In some embodiments, cancer includes solid cancers and non-solid cancers and metastatic lesions. In one embodiment, examples of solid cancers include malignant tumors. The cancer can be early, mid-stage, or late-stage or metastatic cancer. In some embodiments, the cancer is a cancer that requires T cell activation, such as a cancer with T cell dysfunction.

[0341] In some embodiments, the cancer is one that has increased expression levels of PD-L1 protein or increased levels of a nucleic acid encoding PD-L1, e.g., as compared to the levels in a normal subject or normal cells.

[0342] In some embodiments, the malignant tumor is selected from bladder cancer, breast cancer, endometrioid cancer, cervical cancer, advanced solid tumors, renal cell carcinoma, neuroendocrine tumors, transitional cell carcinoma, urothelial carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cancer in the gastrointestinal tract (such as colorectal cancer, rectal cancer or colon cancer), non-small cell lung cancer, hepatocellular carcinoma; preferably, the neuroendocrine tumor is a neuroendocrine carcinoma or a neuroendocrine tumor, more preferably, the neuroendocrine tumor is an extrapulmonary neuroendocrine tumor; more preferably, the neuroendocrine tumor is an advanced neuroendocrine tumor.

[0343] In some embodiments, the neuroendocrine cancer is an advanced neuroendocrine cancer. In other embodiments, the neuroendocrine cancer is an extrapulmonary neuroendocrine cancer, such as an advanced extrapulmonary neuroendocrine cancer, or a small cell lung cancer, such as an extensive-stage small cell lung cancer. In an exemplary embodiment, the neuroendocrine cancer is an unresectable locally advanced or metastatic neuroendocrine cancer.

[0344] In some embodiments, treatment of cancer will benefit from:

[0345] (i) inhibition of PD-L1 nucleic acid or protein levels;

[0346] (ii) blocking the binding of PD-L1 to its receptors such as PD-1,

[0347] (iii) activation of the 4-1BB signaling pathway;

[0348] (iv) activation of T cells, such as increasing the proliferation of CD8+ T cells, or decreasing Treg cells, such as CD4+ T cells;

[0349] (v) any one or more combinations of the above.

[0350] In some embodiments, the cancer is a cancer that expresses a biomarker in a sample, such as a tumor tissue or cell, or blood, compared to a control, or has an altered expression of the biomarker, such as an increased expression of the biomarker. In some embodiments, the control is a corresponding tissue or blood or cell or tissue of a healthy subject, or healthy tissue or healthy cells surrounding the tumor tissue.

[0351] In some embodiments, the biomarker is selected from PD-L1, d-MMR, MSI-H, TMB and / or 4-1BB.

[0352] In some embodiments, the biomarkers can be detected by immunohistochemistry or NGS.

[0353] In some embodiments, the cancer is a tumor with high biomarker levels or expression and / or tumor infiltrating lymphocytes (TIL)+. In certain embodiments, the cancer is a tumor with high PD-L1 levels or expression and TIL+.

[0354] In some embodiments, the tumor of TIL+ is positive for CD8 and / or IFNγ. In some embodiments, the cancer or the subject suffering from the cancer has a high percentage of cells that are positive for one or more of the biomarker, CD8 and / or IFNγ. In certain embodiments, the cancer or the subject suffering from the cancer has or is identified as having a high percentage of cells that are positive for both the biomarker and CD8.

[0355] In some embodiments, the methods described herein further describe identifying the cancer or a subject having the cancer based on having a high percentage of cells that are positive for one or more of a biomarker, CD8, and / or IFNγ. In certain embodiments, the methods described herein further describe identifying the cancer or a subject having the cancer based on having a high percentage of cells that are positive for both a biomarker and CD8. In some embodiments, the cancer or a subject having the cancer has or is identified as having one or more of a biomarker and CD8.

[0356] In some embodiments, the treatment methods using the bispecific antibodies of the present invention can be used for first-line treatment, second-line treatment, or multiple-line treatment of malignancies or cancers.

[0357] In some specific embodiments, the subject with the malignant tumor or cancer has not received systemic treatment, for example, has not received other cancer therapies such as immunotherapy or standard cancer treatment methods. In other specific embodiments, the subject with the malignant tumor or cancer is a subject who has progressed after at least one first-line chemotherapy, for example, the subject includes but is not limited to a subject who has progressed after receiving two or more lines of chemotherapy, for example, a subject who has progressed after receiving two lines of chemotherapy, a subject who has progressed after receiving three lines of chemotherapy, or a subject who has progressed after receiving four lines of chemotherapy.

[0358] In some exemplary embodiments, the treatment methods using the bispecific antibodies of the present invention can be used for the first-line treatment, second-line treatment, or multi-line treatment of neuroendocrine cancer (e.g., extrapulmonary neuroendocrine cancer or small cell lung cancer); specifically, for the first-line treatment, second-line treatment, or multi-line treatment of advanced neuroendocrine cancer; more specifically, for the first-line treatment, second-line treatment, or multi-line treatment of advanced extrapulmonary neuroendocrine cancer or extensive-stage small cell lung cancer.

[0359] In a specific embodiment, the subject can be a subject with small cell lung cancer who has not received systemic treatment before, a subject with advanced extrapulmonary neuroendocrine carcinoma who has progressed after receiving at least one line of chemotherapy before, a subject with advanced extrapulmonary neuroendocrine carcinoma who has progressed after receiving at least one line of chemotherapy before, including but not limited to a subject with advanced extrapulmonary neuroendocrine carcinoma who has progressed after receiving two or more lines of chemotherapy before, for example, a subject with advanced extrapulmonary neuroendocrine carcinoma who has progressed after receiving two lines of chemotherapy before, a subject with advanced extrapulmonary neuroendocrine carcinoma who has progressed after receiving three lines of chemotherapy before, and a subject with advanced extrapulmonary neuroendocrine carcinoma who has progressed after receiving four lines of chemotherapy before.

[0360] In a more specific embodiment, the chemotherapy treatment received by the subject can be platinum-containing chemotherapy, for example, it can be first-line platinum-containing chemotherapy, second-line platinum-containing chemotherapy, or third-line platinum-containing chemotherapy.

[0361] V. Dosage Regimen

[0362] In the therapeutic methods of the present invention, the bispecific antibodies of the present disclosure are administered to a human subject at a therapeutically effective dose.

[0363] A "therapeutically effective dose" of the bispecific antibody of the invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevention of impairment or disability due to the disease. For example, for treating a tumor-bearing subject, a "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%, relative to an untreated subject. A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise ameliorate symptoms in a subject, which is typically a human or can be another mammal.

[0364] The dosage regimen is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased, as indicated by the urgency of the therapeutic situation. It is particularly advantageous to formulate the parenteral composition in a dosage unit form for ease of administration and maintaining uniform dosage. As used herein, a dosage unit form refers to a physically discrete unit suitable for use as a unit dose for a 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 pharmaceutical carrier. Alternatively, the antibody can be administered as a sustained release formulation, in which case less frequent administration is required.

[0365] The dosing regimen for the bispecific antibody of the present invention described below is applicable to both treatment methods in which only the bispecific antibody of the present invention is administered and to the drug combination / combination therapy of the present invention.

[0366] In the dosing regimen of the present invention, the bispecific antibodies of the present invention can be administered to an individual in need thereof in one or more doses.

[0367] In some embodiments, approximately 50 mg to 5000 mg of the bispecific antibody, preferably 60 mg to 4000 mg of the bispecific antibody, more preferably 80 mg to 3750 mg of the bispecific antibody, and most preferably 100 mg to 3500 mg of the bispecific 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, or 3500 mg of the bispecific antibody, is administered to the subject in each dosing cycle or each time.

[0368] In some embodiments, approximately 0.2 mg / kg to 200 mg / kg of the bispecific antibody is administered to the subject per dosing cycle or each time, preferably 0.8 mg / kg to 25 mg / kg of the bispecific antibody, more preferably 3.2 mg / kg to 20 mg / kg of the bispecific antibody, still more preferably 6 mg / kg to 15 mg / kg of the bispecific antibody, and most preferably 10 mg / kg to 15 mg / kg of the bispecific antibody. In some embodiments, 0.8 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3.2 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 8 mg / kg, 10 mg / kg, 12 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, or 25 mg / kg of the bispecific antibody is administered to the subject during each dosing cycle or at each time.

[0369] In some embodiments, administration of the bispecific 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.

[0370] In some embodiments, when multiple doses are administered, the next dose is administered about 2 days to 84 days after the previous dose, for example, each administration cycle of the bispecific antibody of the present invention is about 2 days to 84 days, for example, the administration cycle 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 or twice a week, three times a week. In some embodiments, the bispecific antibody of the present invention is administered to the subject for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 administration cycles, preferably, the bispecific antibody is administered for no more than two years.

[0371] The other therapeutic agents in the pharmaceutical combination of the present invention, such as chemotherapeutic agents, are administered about 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 1-5 days, such as 1 day or 3 days.

[0372] In some embodiments, the chemotherapeutic agent in the drug combination / combination therapy of the present invention is a podophyllotoxin derivative, which is administered at a dose of less than or equal to about 100 mg / m2 per administration cycle or per administration. 2or less, such as less than or equal to about 90, 80, 75, 70, 65, 60, 55, or 50 mg / m 2 , or between the stated ranges.

[0373] In some embodiments, the chemotherapeutic agent in the drug combination / combination therapy of the present invention is a platinum-based agent, and the dose in each administration cycle or each administration is less than or equal to about 75 mg / m 2 or less, such as less than or equal to about 70, 65, 60, 55, 50, 45, 42.5, 40, 37.5, or 35 mg / m 2 , or between the stated ranges; or AUC ≤ about 5 mg / mL / min or less, or less than or equal to about 4, 3, 2, or 1 mg / mL / min, or between the stated ranges. Wherein, AUC ≤ about 5 mg / mL / min means that the dosage can be calculated according to the following formula:

[0374] Dose (mg) = AUC (mg / ml / min) × [creatinine clearance (ml / min) + 25], where AUC ≤ approximately 5 mg / mL / min. The dosages expressed herein as AUC (mg / mL / min) are calculated according to this publication.

[0375] The bispecific antibodies of the present invention or the bispecific antibodies in the drug combination can be administered before, simultaneously with, or after the administration of other therapeutic agents. When the bispecific antibodies of the present invention are administered "before" the administration of other therapeutic agents, the interval between the administration of the bispecific antibodies of the present invention and the other therapeutic agents can 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 4 hours, about 2 hours, about 1 hour, or about 30 minutes, about 15 minutes or about 10 minutes or no interval time. When administered after the start of administration of other therapeutic agents, the interval between the administration of the bispecific antibodies of the present invention and the other therapeutic agents can be no interval, about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours or more than 72 hours. Administration "simultaneously" with the start of administration of the other therapeutic agent means that the bispecific antibody of the present invention is administered to the individual within less than 10 minutes of the start of administration of the other therapeutic agent (before, after, or at the same time). Preferably, the bispecific antibody of the present invention or the bispecific antibody of the drug combination is administered before the administration of the other therapeutic agent. For example, the interval between the administration of the bispecific antibody of the present invention and the other therapeutic agent can be no interval, about 1 hour, about 3 hours, about 6 hours, about 12 hours, about 15 hours, or about 24 hours.

[0376] In some embodiments, the drug combination / combination therapy is administered in cycles, for example, each administration cycle is at least 14-35 days, for example, 2, 3, 4 or 5 weeks, preferably 3 weeks.

[0377] 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 of the bispecific antibody and other therapeutic agents in the drug combination / combination therapy can be different, for example, the number of administration cycles of the bispecific antibody is no more than two years, and the number of administration cycles of the other therapeutic agent (such as a chemotherapeutic agent) is 2 to 6 cycles, preferably 4 to 6 cycles.

[0378] Preferably, the bispecific antibody of the present invention is administered once or twice per cycle, or the bispecific antibody of the present invention is administered per cycle; and / or

[0379] The other therapeutic agent is administered on at least days 1 to 5 of each cycle, for example on day 1 or on days 1 to 3, and then rested until the end of the cycle.

[0380] The pharmaceutical combination / combination therapy of the present invention may be administered in every 5 weeks, every 4 weeks, every 3 weeks, every 2 weeks, or weekly dosing.

[0381] In some embodiments, the drug combination / combination therapy comprises

[0382] (i) the bispecific antibodies of the present invention;

[0383] (ii) podophyllotoxin derivatives, such as etoposide, etoposide glucuronide or etoposide phosphate; and

[0384] (iii) Platinums, such as carboplatin or cisplatin.

[0385] In some embodiments of the described drug combination / combination therapy,

[0386] The single administration dose of (i) is about 0.2 mg / kg to 200 mg / kg, preferably 0.8 mg / kg to 25 mg / kg, more preferably 3.2 mg / kg to 20 mg / kg, still more preferably 6 mg / kg to 15 mg / kg, most preferably 10 mg / kg to 15 mg / kg, for example 0.8 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3.2 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 8 mg / kg, 10 mg / kg, 12 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg. / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg or 25 mg / kg; or about 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, 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, 3500 mg;

[0387] (ii) a single administration dose of less than or equal to about 100 mg / m 2 , such as less than or equal to about 90, 80, 75, 70, 65, 60, 55, or 50 mg / m 2 , or between the stated ranges; and / or

[0388] (iii) a single administration dose of less than or equal to about 75 mg / m 2 , such as less than or equal to about 70, 65, 60, 55, 50, 45, 42.5, 40, 37.5, or 35 mg / m 2 , or between the stated ranges; or AUC ≤ about 5 mg / mL / min or less, or less than or equal to about 4, 3, 2, or 1 mg / mL / min, or between the stated ranges.

[0389] The other therapeutic agents in the pharmaceutical combination / combination therapy of the present invention, such as chemotherapeutic agents, are administered about 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 1-5 days, such as 1 day or 3 days.

[0390] In some embodiments of the drug combination, the drug combination is administered in a cycle of three weeks, wherein each cycle comprises one (i) administration, one (ii) administration for 1-5 days, preferably one (ii) administration for 3 days, and one (iii) administration, preferably, the first administration of (ii) and the administration of (iii) are performed after (i) administration (e.g., immediately or about 15, 30, 60 minutes, 1 hour, 2 hours or 3 hours later), wherein the first administration of (ii) and (iii) can be administered simultaneously or sequentially (e.g., about 0, 15, 30, 60 minutes, 1 hour, 2 hours or 3 hours apart). In some embodiments, the single dose of (i) is 5-20 mg / kg, for example, about 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mg / kg; the single dose of (ii) is 100 mg / m 2 (body surface area), preferably (ii) is etoposide and its single dose is 100 mg / m 2 , preferably (ii) is administered once a day for 3 days; (iii) a single dose is 100 mg / m 2 , or AUC = 5 mg / mL / min, preferably a single dose of (iii) is 75 mg / m 2 Cisplatin or carboplatin AUC = 5 mg / mL / min.

[0391] In a preferred embodiment, the drug combination is administered for 4 to 6 cycles.

[0392] In one embodiment, after all dosing cycles of the drug combination are completed, the bispecific antibody of the invention is continuously administered, for example, once every three weeks, every four weeks, every five weeks, or every six weeks, with a single dose of 0.2-25 mg / kg each time, for example, about 0.8 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3.2 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 8 mg / kg, 10 mg / kg, 12 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg or 25 mg / kg.

[0393] In a specific embodiment, after all cycles of drug combination administration are completed, the bispecific antibody of the invention is continuously administered, for example, every three weeks, every four weeks, every five weeks, every six weeks, for example, 1, 3, 5, 7, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 dosing cycles. Preferably, the total duration of administration of the drug combination and the bispecific antibody alone does not exceed two years.

[0394] In a preferred embodiment, the bispecific antibody of the present invention (e.g., P4B-3) is used in combination with etoposide and cisplatin, with a dosing cycle of 3 weeks. On the first day of each dosing cycle, the bispecific antibody is intravenously infused at a dose of 6 mg / kg, 10 mg / kg, or 15 mg / kg; etoposide is intravenously infused at a dose of 100 mg / m 2 or lower; intravenous cisplatin at a dose of 75 mg / m 2 or lower; the order of administration is to first infuse the bispecific antibody, then administer etoposide after the completion of the bispecific antibody infusion, and then administer cisplatin after the completion of etoposide administration; etoposide is administered intravenously alone on the second and third days, with a daily dose of 100 mg / m 2 or lower; a maximum of 6 cycles.

[0395] In another preferred embodiment, the bispecific antibody of the present invention (e.g., P4B-3) is used in combination with etoposide and carboplatin, with a dosing cycle of every 3 weeks. On the first day of each dosing cycle, the bispecific antibody is intravenously infused at a dose of 6 mg / kg, 10 mg / kg, or 15 mg / kg; etoposide is intravenously infused at a dose of 100 mg / m 2 or lower; intravenous infusion of carboplatin at a dose of AUC = 5 mg / mL / min or lower; the order of administration is first infusion of the bispecific antibody, followed by etoposide administration after the completion of the bispecific antibody infusion, and then carboplatin administration after the completion of the etoposide administration; etoposide is administered intravenously alone on days 2 and 3 at a daily dose of 100 mg / m 2 or lower; a maximum of 6 cycles.

[0396] In some embodiments, the monotherapy of the bispecific antibody of the present invention or the combination therapy of administration with other therapeutic agents results in an increase, preferably a synergistic increase in the progression-free survival (PFS) or overall survival (OS) of the patient. In some embodiments, the bispecific antibody of the present invention or drug combination administered for at least one cycle results in an increase in the PFS of the patient 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 some embodiments, the bispecific antibody of the present invention or drug combination administered for at least one cycle results in an increase in the OS of the patient 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.

[0397] The bispecific antibodies or drug combinations of the present invention result in increased, preferably synergistic, inhibition of tumor growth. In some embodiments, the bispecific antibody drug combination of the present invention results in inhibition of tumor growth by 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.

[0398] In some embodiments, the bispecific antibody drug combination of the present invention 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 can increase progression-free survival or overall survival.

[0399] In some embodiments, administration of a bispecific antibody or drug combination of the invention to an individual with cancer results in complete disappearance of the tumor (a "complete response"). In some embodiments, administration of a bispecific antibody or drug combination of the invention to an individual with cancer results in a reduction in tumor cells or tumor size of at least 30% or more (a "partial response"). Tumor reduction 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), cytology, histology, or molecular genetic analysis.

[0400] In some embodiments, the bispecific antibodies or drug combinations of the present invention can reduce adverse events caused by the administration of known therapies, such as hematological toxic reactions, non-hematological toxic reactions, or other toxic reactions, such as pneumonia, diarrhea, enterocolitis, renal insufficiency, rash, hepatitis, endocrine diseases and peripheral or central neuritis, abnormal liver function, etc.

[0401] Example

[0402] Example 1:

[0403] The bispecific antibody P4B-3 was constructed and expressed as described in CN 114555638 A, FIG1 .

[0404] The bispecific P4B-3 was constructed as follows: two scFvs of the 4-1BB antibody were linked to the C-termini of the two heavy chains of the intact PD-L1 antibody to construct a heavy chain consisting of the following from the N-terminus to the C-terminus:

[0405] VH from a PD-L1 antibody (VH of anti-PD-L1) - heavy chain constant region - linker - VH from a 4-1BB antibody (VH of anti-4-1BB VH) - linker - VL from a 4-1BB antibody (VL of anti-4-1BB); and

[0406] A bispecific antibody derived from the light chain of a PD-L1 antibody (the light chain of anti-PD-L1, i.e., the VL of the anti-PD-L1 light chain constant region).

[0407] The nucleotides encoding the bispecific antibody and the control were generated by gene synthesis (Kinsher) and cloned into the expression vector pcDNA3.1 (Invitrogen). The nucleic acid encoding the light chain variable region was inserted into the expression vector pcDNA3.1 (Invitrogen) containing the nucleic acid encoding the light chain constant region to construct a vector expressing the antibody light chain, and the nucleic acid encoding the heavy chain variable region was inserted into the expression vector pcDNA3.1 (Invitrogen) containing the nucleic acid encoding the heavy chain constant region to construct a vector expressing the antibody heavy chain. The expression vectors were expressed using ExpiCHO according to the manufacturer's instructions. TM The obtained vector was co-transfected into CHO-S cells at a molar ratio of 1:1 using the ExpiCHO expression system (Thermo Fisher Scientific, catalog number: A29133). The transfected cells were cultured in the ExpiCHO TM After culturing in expression medium for 12 days, the culture supernatant was harvested and purified by protein A affinity chromatography (GE Healthcare).

[0408] The amino acid sequences and nucleic acid sequences of different regions can be found in Table 1.

[0409] Table 1. SEQ ID NOs of regions / domains of bispecific antibodies 1 4-1BB VH is derived from the VH of the monoclonal antibody 41BB-2 and forms a disulfide bond with VL via G44C (EU numbering), thereby improving the stability of the scFv. 2 4-1BB VL is derived from the VL of the monoclonal antibody 41BB-2 and forms a disulfide bond with VH via T104C (EU numbering), thereby improving the stability of the scFv.

[0410] Example 2: Preclinical toxicology studies

[0411] 2.1. GLP toxicity study of P4B-3 administered intravenously to rats for 29 days with repeated intermittent administration and a 28-day recovery period

[0412] Male and female Sprague-Dawley rats (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) received the study drug P4B-3 once weekly for five consecutive days at 30, 100, or 300 mg / kg. Results showed that P4B-3 was well tolerated by the SD rats after weekly intravenous infusion of 30, 100, or 300 mg / kg / dose for 29 consecutive days, followed by a 28-day recovery period. No clinical symptoms, injection site irritation, ophthalmological changes, clinical pathological changes (hematology, blood biochemistry, coagulation, urinalysis), changes in organ weights, gross ocular lesions, or histopathological changes related to the study drug were observed. The no-observed-adverse-effect level (NOAEL) for this study was 300 mg / kg.

[0413] 2.2. GLP toxicology study of P4B-3 administered intravenously to cynomolgus monkeys for repeated 29 days and a 28-day recovery period

[0414] Cynomolgus monkeys (Guangdong Chunsheng Biotechnology Development Co., Ltd.) were treated with the study drug P4B-3 at doses of 20, 60, or 200 mg / kg weekly for five consecutive days. Results showed no drug-related clinical symptoms or changes in body weight, food intake, ophthalmological examinations, body temperature, safety pharmacology parameters (quantitative and qualitative electrocardiographic, cardiovascular, respiratory, and neurological changes), coagulation, urinalysis, or organ weights. The highest non-serious toxic dose (HNSTD) in this study was 200 mg / kg.

[0415] Example 3: Clinical pharmacology study

[0416] A preliminary pharmacokinetic (PK) analysis of P4B-3 was conducted. The dosing frequency was once every 3 weeks (Q3W), with each 3-week dosing cycle being one.

[0417] As of September 4, 2023, based on five cycles of PK data collection, P4B-3 plasma concentrations increased with increasing doses after single and repeated dosing at doses of 0.2-25 mg / kg, with similar trends in the concentration-time curves across all doses. Plasma concentrations after repeated dosing remained similar to those after the initial dose (see Figures 2A and B).

[0418] After a single dose, Cmax and AUC increased with increasing dose within the 0.2-25 mg / kg dose range, with mean half-lives of approximately 1.886-6.732 days in the 0.2-25 mg / kg dose group. Dose-corrected Cmax and AUC0-inf distributions were similar across the 0.2-25 mg / kg dose group, suggesting a dose-proportional relationship between exposure and dose.

[0419] After multiple dosing, Cmax and AUC increased with increasing dose within the 0.8-25 mg / kg dose range. The Rac ranged from 1.02 to 1.30 after multiple dosing of C5D1, suggesting some drug accumulation in the high-dose group. Furthermore, pre-dose trough concentrations of C2D1, C3D1, and C5D1 were similar across all dose groups, suggesting steady-state.

[0420] During the dose-escalation phase of the P4B-3 clinical study, doses were 0.2 mg / kg, 0.8 mg / kg, 3.2 mg / kg, 6 mg / kg, 10 mg / kg, 15 mg / kg, and 25 mg / kg. At low doses from 0.2 mg / kg to 3.2 mg / kg, P4B-3 serum exposure exhibited TMDD, a nonlinear pharmacokinetic profile. At high doses from 6 mg / kg to 25 mg / kg, P4B-3 serum exposure exhibited linear dose-proportional pharmacokinetic characteristics, with Ctrough values ​​of 9.9, 16.3, 31.8, and 45.5 μg / mL, respectively. The EC90 range for the PDL1 / P4B-3 / 4-1BB trimer was achieved and maintained at 6 mg / kg to 15 mg / kg. In patients with NEC, P4B-3 achieved ORRs of 0, 20, and 41%, respectively, at doses of 6 mg / kg to 15 mg / kg. This trend increased with increasing dose, demonstrating a dose-response relationship, reaching an ORR of 41% at 15 mg / kg. P4B-3 was well tolerated clinically, with the MTD not reached up to 25 mg / kg. No dose-related increases in liver function, cardiac function, safety, or hematologic toxicity were observed.

[0421] Example 4: PD marker detection

[0422] 4.1 Serum cytokine release

[0423] As of August 14, 2023, valid test data were available for 58 Phase I subjects. The results showed that IFN-γ release was most pronounced 24 hours after C1D1 administration and before C2D1 administration. Of the 54 subjects with pre-dose baseline data, 49 (90.7%) showed an increase in IFN-γ concentrations 24 hours after C1D1 administration compared to pre-dose levels; 34 (63.0%) showed an increase in IFN-γ concentrations before C2D1 administration compared to pre-dose levels. Based on the mechanism of action of P4B-3, it is speculated that intravenous administration of P4B-3 activated T lymphocytes in the vast majority of subjects, promoting IFN-γ release. See Figure 3.

[0424] The test results of the other four cytokines (IL-2, IL-6, IL-10 and TNF-α) showed that the number of subjects who obtained valid test data was relatively small, and there was no significant change in the release of each factor compared with the concentration level before administration.

[0425] 4.2 PD-L1 immunohistochemical staining of tumor tissue

[0426] As of January 10, 2024, PD-L1 immunohistochemistry staining was performed on tumor tissue samples from 73 subjects. Results were interpreted using the CPS, with a preliminary cutoff of ≥1 defined as positive. Of these, 26 subjects had extrapulmonary neuroendocrine carcinoma (with measurable lesions). Only four subjects were PD-L1 positive, with two achieving a complete response (PR). Twenty-two subjects were PD-L1 negative, with 12 achieving a complete response (PR). Both PD-L1-negative and PD-L1-positive subjects with extrapulmonary neuroendocrine carcinoma benefited from P4B-3 monotherapy, with similar ORRs (54.5% vs 50%) in both groups. (See Figure 4.)

[0427] Example 5: Clinical Study on the Safety, Tolerability and Preliminary Efficacy of P4B-3 in Subjects with Advanced Malignant Tumors

[0428] Drug name and strength: P4B-3 100mg / bottle Prepared by PCT / CN2020 / 119388 Name: P4B-3 for injection or P4B-3;

[0429] Specifications: 100 mg / bottle, each bottle contains 100 mg of P4B-3 protein;

[0430] Preparation: lyophilized preparation;

[0431] Excipients: histidine, histidine hydrochloride, trehalose dihydrate, mannitol, polysorbate 80;

[0432] Security

[0433] Safety Overview: A total of 175 subjects were enrolled, including 20 in the Phase I dose-escalation phase. No DLTs were observed, and the MTD was not reached. Common TRAEs (incidence ≥ 10%) included: increased aspartate aminotransferase, anemia, increased alanine aminotransferase, decreased white blood cell count, decreased neutrophil count, and decreased platelet count. Most were grade 1-2 in severity, and the safety profile was manageable.

[0434] Effectiveness

[0435] As of February 19, 2024, 154 subjects were evaluable for efficacy, with 1 CR, 20 PR, and 49 SD, ORR: 13.6%, DCR: 45.4%.

[0436] Pharmacokinetic (PK) evaluation

[0437] PK parameters mainly include peak concentration (C max ), peak time (T max ), elimination half-life (T 1 / 2 ), clearance (CL), volume of distribution (Vd), area under the plasma concentration-time curve (AUC 0-t ), the area under the plasma concentration-time curve (AUC 0-inf ), area under the steady-state plasma concentration-time curve (AUCss), steady-state peak plasma concentration (Cmax,ss), steady-state trough plasma concentration (C min,ss ), steady-state blood drug peak time (T max,ss ), accumulation factor, etc.

[0438] Immunogenicity evaluation

[0439] Immunogenicity evaluation indicators are the incidence of anti-drug antibodies (ADA) and neutralizing antibodies (if applicable) in subjects. If applicable, the impact of ADAs on the efficacy, PK characteristics and safety of the investigational drug will be further evaluated.

[0440] Biomarker evaluation

[0441] The biomarker evaluation indicator is PD-L1, and the expression level of PD-L1 in tumor tissue will be evaluated.

[0442] Statistical analysis

[0443] (1)1. Sample size estimation

[0444] The sample size calculated from the exact test for the one-sample binomial distribution with a one-sided α = 0.025 was 98 subjects.

[0445] (2) Statistical analysis

[0446] 1. General Methods

[0447] All statistical analyses will be performed using SAS version 9.4 or above.

[0448] 2. Subject Distribution

[0449] The subject distribution includes a summary of the screening, grouping, dropout or exclusion of subjects in each group, as well as the division of data sets, and is presented using a subject distribution flow chart.

[0450] 3. Baseline Statistical Analysis

[0451] Descriptive statistics were used to analyze the baseline characteristics of each group, including demographics and medical history, treatment history, medication history, disease characteristics, etc.

[0452] 4. Security Analysis

[0453] Adverse events will be coded using the Medical Dictionary for Regulatory Activities (MedDRA) version 24.1 or above.

[0454] 5. Effectiveness Analysis

[0455] Descriptive statistics were used to summarize the number and proportion of subjects with the best response of complete remission (CR), partial remission (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.

[0456] The Kaplan-Meier method will be used to draw the progression-free survival (PFS) survival curve of patients after treatment with the trial drug, distinguishing different dose groups and different cancer types. The median, first quartile, and third quartile will be calculated. The Brookmeyer & Crowley method will be used to calculate the median of the time-to-event indicators and each quantile and their 95% CI, and the Greenwood formula will be used to calculate the variance.

[0457] Primary efficacy endpoint: ORR assessed by IRC

[0458] The primary efficacy endpoint of this study phase is the ORR assessed by IRC. The primary analysis of the ORR assessed by IRC will be based on the FAS analysis set, and the Clopper-Pearson method will be used to provide the ORR estimate and 95% confidence interval.

[0459] Secondary efficacy endpoints: Secondary efficacy endpoints of this study phase include ORR assessed by the investigator, DOR assessed by the investigator and IRC, DCR, PFS and OS.

[0460] ORR and DCR

[0461] The number of subjects achieving the best objective response was summarized in different categories: CR, PR, SD, and PD. The Clopper-Pearson method was used to calculate the ORR, DCR, and their 95% CIs.

[0462] PFS, OS, and DOR

[0463] PFS, OS, and DOR were analyzed using the Kaplan-Meier method, and medians and 95% confidence intervals were provided.

[0464] Expected timing of primary efficacy endpoint analysis: The primary efficacy analysis will be conducted when mature ORR data are observed, which is expected to be no later than 6 months after the first dose of the last subject.

[0465] 6. Pharmacokinetic Analysis

[0466] Individual PK data should be presented, and group mean PK data should be summarized descriptively. The plasma concentration-time curve for P4B-3 should be plotted. Descriptive statistics should be used to summarize PK parameters. For multiple-dose PK studies, PK parameters, drug accumulation, and time to steady state should be discussed.

[0467] 7. Immunogenicity Analysis

[0468] The incidence of anti-P4B-3 antibodies and neutralizing antibodies (if applicable) will be summarized for all subjects who received at least one dose of the study drug. If applicable, the impact of ADA on the PK, efficacy, and safety of P4B-3 will be assessed.

[0469] 10. Biomarker Analysis

[0470] The expression level of PD-L1 in tumor tissue samples was assessed.

[0471] Example 6: Clinical study of the safety, tolerability, pharmacokinetic characteristics and efficacy of P4B-3 combined with etoposide and platinum (cisplatin or carboplatin) in subjects with advanced neuroendocrine cancer

[0472] An open, multicenter phase Ib / II clinical study evaluating the P4B-3 antibody combined with etoposide and platinum (cisplatin or carboplatin) as first-line treatment for subjects with advanced neuroendocrine carcinoma.

[0473] Purpose

[0474] The primary objective of this study is to evaluate the safety, tolerability, and efficacy of P4B-3 antibody combined with etoposide and platinum (cisplatin or carboplatin) as first-line treatment for subjects with advanced neuroendocrine carcinoma.

[0475] Other objectives include evaluating the pharmacokinetic (PK) characteristics of P4B-3 combined with etoposide and platinum as first-line treatment for subjects with advanced neuroendocrine carcinoma; evaluating the immunogenicity of P4B-3 combined with etoposide and platinum as first-line treatment for subjects with advanced neuroendocrine carcinoma; evaluating the correlation between biomarkers in tumor tissue and efficacy, etc.

[0476] Study endpoints

[0477] Phase Ib clinical study

[0478] Primary endpoints included: dose-limiting toxicity (DLT), incidence and severity of adverse events (AEs) and serious adverse events (SAEs), and abnormal laboratory test indicators.

[0479] Secondary endpoints include: objective response rate (ORR), disease control rate (DCR), duration of response (DOR), progression-free survival (PFS) and overall survival (OS) (based on Response Evaluation Criteria in Solid Tumors version 1.1 [RECIST 1.1] and Immunotherapy Response Evaluation Criteria in Solid Tumors [iRECIST]); PK, indicators include Ctrough, etc.; immunogenicity evaluation, indicators are the incidence of anti-drug antibodies (ADA) and neutralizing antibodies (if applicable) in subjects; expression levels of programmed death receptor-ligand 1 (PD-L1), tumor mutation burden (TMB), mismatch repair deficiency (dMMR), microsatellite high instability (MSI-H) and 4-1BB in tumor tissues, as well as the correlation between CD8+ T cell levels and efficacy.

[0480] Phase II clinical study

[0481] The primary endpoint included investigator-assessed ORR according to RECIST 1.1.

[0482] Secondary endpoints include: the incidence and severity of adverse events (AEs) and serious adverse events (SAEs), as well as abnormal laboratory test indicators; DCR, DOR, PFS and OS (based on RECIST 1.1 and iRECIST) and ORR (based on iRECIST); immunogenicity evaluation, with indicators such as the incidence of anti-drug antibodies (ADA) and neutralizing antibodies (if applicable) in subjects; the expression levels of PD-L1, TMB, dMMR, MSI-H and 4-1BB in tumor tissues, and the correlation between CD8+ T cell levels and efficacy.

[0483] Study design and duration

[0484] This trial included a dose-escalation phase (Phase Ib) and an expansion phase (Phase II) of the combination.

[0485] Phase Ib (safety lead-in phase) plans to enroll subjects with advanced neuroendocrine carcinoma who have not received systemic treatment. According to the "Rolling six design" dose escalation method, the safety and tolerability of 6mg / kg, 10mg / kg or 15mg / kg P4B-3 in combination with etoposide and platinum (cisplatin or carboplatin) will be evaluated. Each dose group will include 3 to 6 subjects. In this study phase, for subjects with extrapulmonary NEC, P4B-3 combined with EP / EC was used, and the etoposide was administered at a dose of 100mg / m 2 , continuous administration on days 1 to 3 of each cycle, Q3W; cisplatin is administered at 75 mg / m 2 , administered on the first day of each cycle, Q3W; carboplatin is administered at AUC=5mg / mL / min, administered on the first day of each cycle, Q3W; on D1 of each cycle of combination therapy, the order of administration is to first infuse P4B-3, then administer etoposide after the infusion of P4B-3, and then administer cisplatin or carboplatin after the administration. On D2 and D3 of each cycle of combination therapy, the subjects will receive etoposide alone. Chemotherapy is used for 6 cycles. After the end of chemotherapy, the subjects can continue to receive P4B-3 treatment (Q3W). For subjects with SCLC, P4B-3 is combined with EC, and the administration of etoposide is 100mg / m 2 , administered continuously on days 1 to 3 of each cycle, Q3W; carboplatin is administered at AUC = 5 mg / mL / min on day 1 of each cycle, Q3W; on D1 of each cycle of the combination, the order of administration is to first infuse P4B-3, then administer carboplatin after the completion of the P4B-3 infusion, and then administer etoposide after the completion of the carboplatin. On D2 and D3 of each cycle of the combination, the subjects will receive etoposide alone. Chemotherapy is used for 4 cycles. After the completion of chemotherapy, the subjects can continue to receive P4B-3 treatment (Q3W) until intolerable toxicity occurs, disease progression or death, voluntary withdrawal, loss to follow-up, or continuous treatment for 2 years or termination of the study (whichever occurs first). If the subject cannot tolerate the first dose level of P4B-3, the SMC will evaluate the safety and tolerability of the combination therapy by reducing it to 3.2 mg / kg.

[0486] The Phase II (combination therapy expansion phase) is planned to enroll approximately 50 patients with advanced extrapulmonary neuroendocrine carcinoma and approximately 50 patients with small cell lung cancer who have not undergone systemic treatment. Patients with extrapulmonary NEC will receive P4B-3 combined with EP / EC, using the same dosing regimen as in Phase Ib. Patients with SCLC will receive P4B-3 combined with EC, using the same dosing regimen as in Phase Ib.

[0487] Main inclusion criteria

[0488] (1) Stage Ib: Unresectable locally advanced or metastatic neuroendocrine carcinoma (NEC) confirmed by histology and / or cytology, including extrapulmonary and intrapulmonary;

[0489] Stage II: Histologically and / or cytologically confirmed unresectable locally advanced or metastatic extrapulmonary NEC; histologically and / or cytologically confirmed extensive-stage small cell lung cancer (ES-SCLC) (according to the Veterans Administration Lung Group (VALG) disease staging system)

[0490] (2) Patients who have not received systemic treatment for advanced NEC in the past. Subjects who have received neoadjuvant or adjuvant treatment in the past and whose recurrence or metastasis occurred more than 6 months after the last dose can be enrolled (except for subjects who have received anti-PD(L)-1 antibody treatment in the past); patients who have not received systemic treatment for ES-SCLC in the past and have received chemoradiotherapy for limited-stage SCLC must be treated for the purpose of cure, and there must be at least 6 months of treatment-free interval from the diagnosis of extensive-stage SCLC to the last course of chemotherapy, radiotherapy or chemoradiotherapy.

[0491] (3) Eastern Cooperative Oncology Group (ECOG) performance status score of 0 to 1;

[0492] (4) expected survival time is at least 12 weeks;

[0493] (5) According to the RECIST 1.1 standard, the subjects were required to have at least one measurable target lesion;

[0494] Dosage adjustments

[0495] Dosage adjustments for P4B-3

[0496] No P4B-3 dose escalation or reduction was allowed in this trial.

[0497] If one drug in the combination therapy regimen needs to be discontinued, treatment with the other drugs in the combination can be continued. If the investigator deems it appropriate to continue P4B-3 when chemotherapy is delayed, P4B-3 should be administered according to the established schedule. A maximum of six cycles of EP / EC regimens refers to a maximum of six administrations of P4B-3 plus EP / EC. Reasons for dose adjustments, treatment delays, and supportive measures should be documented in the medical record and CRF.

[0498] Duration of drug treatment

[0499] The subjects will receive long-term treatment with the trial drug until intolerable toxicity occurs, disease progression, death, voluntary withdrawal of the subject, or continuous treatment for 2 years or termination of the study (including completion of the trial or early termination of the trial) (whichever occurs first).

[0500] Safety evaluation

[0501] During the trial, subjects will undergo safety checks at specified times, including laboratory tests (blood count, urine count, blood biochemistry, coagulation function, and thyroid function), vital signs, physical examinations, and 12-lead electrocardiograms (12-ECGs). The clinical manifestations, severity, onset, end time, duration, treatment, and outcomes of any adverse events will be recorded, and their relevance to P4B-3 will be determined. Adverse events will be graded using the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) version 5.0. The relevance of adverse events to P4B-3 will be assessed by the investigator according to the attribution assessment criteria specified in the protocol.

[0502] This trial will also evaluate drug exposure, including drug exposure duration, actual dosing intensity, relative dosing intensity, and actual total amount of drug administration.

[0503] Effectiveness evaluation

[0504] Anti-tumor efficacy was evaluated according to the revised Response Evaluation Criteria in Solid Tumors (RECIST version 1.1) and the Immunotherapy Response Evaluation Criteria in Solid Tumors (iRECIST). 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.

[0505] Objective response rate (ORR): includes partial response (PR) and complete response (CR), and is defined as the proportion of subjects with complete response and partial response. For subjects with solid tumors, anti-tumor efficacy was evaluated according to RECIST 1.1. For subjects with an initial PR or CR, efficacy was confirmed at least 4 weeks later.

[0506] Duration of response (DOR) was defined as the time from the first confirmed objective response to disease progression or death from any cause, whichever occurred first.

[0507] Disease control rate (DCR): defined as the proportion of subjects who achieved remission (PR+CR) and stable disease (SD) after treatment.

[0508] Progression-free survival (PFS) was defined as the time from the date of the subject's first dose to the first tumor assessment confirming disease progression or death from any cause.

[0509] Overall survival (OS) was defined as the time from the enrollment of the subjects to death from any cause.

[0510] Pharmacokinetic (PK) evaluation

[0511] PK parameters mainly include Ctrough, etc. PK blood samples will be sent for analysis and testing.

[0512] Immunogenicity evaluation

[0513] Immunogenicity assessment measures the incidence of anti-drug antibodies (ADA) and, if applicable, neutralizing antibodies in the subjects. If applicable, the impact of ADAs on the efficacy, PK characteristics, and safety of the investigational drug will be further evaluated. Blood samples for immunogenicity testing will be sent for analysis.

[0514] Biomarker evaluation

[0515] The biomarker evaluation indicators are the expression of PD-L1, d-MMR, MSI-H, TMB and 4-1BB in tumor tissues and CD8 + The expression of PD-L1, dMMR and B-1BB in tumor tissues and the expression of CD8 T cells were detected by immunohistochemistry. + T cell levels, MSI-H and TMB levels in tumor tissues detected by NGS. Tumor tissue samples and paired whole blood biomarker assays for TMB / MSI-H are required.

[0516] Statistical analysis

[0517] (1)1. Sample size estimation

[0518] No formal statistical testing was performed in Phase Ib of this study, and the sample size was not derived from statistical calculations. Phase Ib was a dose-escalation study with three dose groups using a "rolling six" escalation method, so the planned enrollment was 9 to 12 subjects.

[0519] The primary endpoint of the Phase II study is ORR. Phase II plans to include approximately 50 subjects to preliminarily observe the efficacy of P4B-3 combined with EP / EC.

[0520] Therefore, a total of approximately 62 subjects were included in Phase Ib and Phase II.

[0521] (2) Statistical analysis

[0522] 1. General Methods

[0523] All statistical analyses will be performed using SAS version 9.4 or above.

[0524] 2. Subject Distribution

[0525] The subject distribution includes a summary of the screening, grouping, dropout or exclusion of subjects in each group, as well as the division of data sets, and is presented using a subject distribution flow chart.

[0526] 3. Baseline Statistical Analysis

[0527] Descriptive statistics were used to analyze the baseline characteristics of each group, including demographics and medical history, treatment history, medication history, disease characteristics, etc.

[0528] 4. Drug Exposure Data Analysis

[0529] To reflect exposure to the study drug, the following data will be summarized:

[0530] The number of treatment cycles and days, the total amount of study drug received, and the number of subjects requiring dose reductions, treatment interruptions, and treatment discontinuation due to adverse events.

[0531] 5. Compliance and concomitant medication analysis

[0532] Descriptive statistical analysis of planned and actual medication dosages was performed using the following formula:

[0533] Compliance = actual dosage / planned dosage × 100%.

[0534] For concomitant medications that occurred during the study treatment period, the frequency and percentage were summarized. A table provides details of the subjects' concomitant medications.

[0535] 6. Security Analysis

[0536] Adverse events will be coded using the Medical Dictionary for Regulatory Activities (MedDRA) version 26.0 or above.

[0537] 7. Effectiveness Analysis

[0538] The effectiveness analysis of solid tumors was mainly based on RECIST 1.1, and iRECIST was used as an exploratory analysis.

[0539] The number of subjects who achieved the best objective response was summarized in different categories: complete response (CR), partial response (PR), stable disease (SD), progressive disease (PD), and not evaluable (NE). The Clopper-Pearson method was used to calculate the ORR, DCR, and their 95% CIs.

[0540] The Kaplan-Meier method was used to plot survival curves (KM curves) for PFS and OS, and the median, first quartile, and third quartile were calculated. Two-sided 95% confidence intervals for these statistics were calculated using the Greenwood formula. Similar statistical methods will be used to analyze and summarize other survival endpoints (DOR, etc.).

[0541] 8. Pharmacokinetic Analysis

[0542] Individual PK data should be presented, along with descriptive summaries of group mean PK data. A plasma concentration-time curve for P4B-3 should be plotted. Descriptive statistics should be used to summarize PK parameters and dose-PK proportionality. Repeated-dose PK studies should discuss PK parameters, drug accumulation, and time to steady state.

[0543] Population PK will be analyzed by building relevant models (if applicable).

[0544] 9. Immunogenicity Analysis

[0545] The incidence of anti-P4B-3 antibodies and neutralizing antibodies (if applicable) will be summarized for all subjects who received at least one dose of the study drug. If applicable, the impact of ADA on the PK, efficacy, and safety of P4B-3 will be assessed.

[0546] 10. Biomarker Analysis

[0547] The expression of PD-L1, dMMR, MSI-H, TMB, and 4-1BB, as well as CD8+ T cell levels in tumor tissue samples and TMB / MSI-H levels in paired whole blood samples were evaluated for correlation with efficacy. The expression of PD-L1, dMMR, and B-1BB, as well as the level of C8+ T cells, in tumor tissues were assessed by immunohistochemistry, and the levels of MSI-H and TMB in tumor tissues were assessed by NGS.

[0548] Example 7: Phase IIb clinical study of the efficacy and safety of P4B-3 in patients with advanced extrapulmonary neuroendocrine carcinoma who have progressed after receiving two or more lines of chemotherapy

[0549] Purpose

[0550] The primary objective of this study was to evaluate the efficacy of P4B-3 according to ORR assessed by RECIST 1.1;

[0551] Other objectives include evaluating the efficacy of P4B-3 using other efficacy indicators assessed by the investigator (based on RECIST 1.1 and iRECIST) or IRC (based on RECIST 1.1), evaluating the safety of P4B-3 in subjects with advanced extrapulmonary neuroendocrine carcinoma, evaluating the pharmacokinetic (PK) characteristics of P4B-3 in subjects with advanced extrapulmonary neuroendocrine carcinoma; evaluating the immunogenicity of P4B-3 in subjects with advanced extrapulmonary neuroendocrine carcinoma; and evaluating the correlation between biomarkers (PD-L1) in tumor tissue and efficacy.

[0552] Study endpoints

[0553] Primary endpoints included: ORR assessed by IRC.

[0554] Secondary endpoints include: 1) DCR, PFS, and DOR assessed by IRC; ORR, DCR, DOR, PFS assessed by investigators; 6-month OS rate and OS; 2) adverse events (laboratory tests, physical examinations, vital signs, and electrocardiograms, etc.); 3) PK parameters, including but not limited to Cmax, Tmax, T1 / 2, CL, Vd, AUC, etc.; 4) Immunogenicity: The immunogenicity evaluation indicators are the incidence of ADA and the incidence of neutralizing antibodies (if applicable) in the subjects; 5) PD-L1 expression in tumor tissue

[0555] Study design and duration

[0556] The efficacy and safety of P4B-3 continue to be evaluated in patients with advanced extrapulmonary neuroendocrine carcinoma who have progressed after two or more lines of chemotherapy.

[0557] Based on analysis of previously obtained safety, efficacy, PK, and PD data, the dose level of P4B-3 in this phase of the study is 15 mg / kg every three weeks. All subjects are required to provide tumor tissue for pathological type verification. Only subjects diagnosed with extrapulmonary neuroendocrine carcinoma by a central laboratory are eligible for enrollment in this study. Tumor tissue samples should be collected from all subjects whenever possible to evaluate PD-L1 expression levels. Blood samples will be collected from all subjects participating in the sparse PK study to evaluate the immunogenicity of P4B-3.

[0558] Dose group design

[0559] In the Phase IIb study, subjects will receive a dose level of 15 mg / kg Q3W.

[0560] Measure the subject's weight before each dose and calculate the dosage based on weight. If the subject's weight changes within ±10% (inclusive) relative to baseline, the dosage will still be calculated based on the baseline weight. If the weight change is >10%, the dosage will need to be calculated based on the new weight, which will serve as the baseline for subsequent weight measurements.

[0561] Main inclusion criteria

[0562] (1) Patients with locally advanced or metastatic extrapulmonary neuroendocrine carcinoma confirmed by histology or cytology and who have progressed after first-line platinum-based chemotherapy or second-line chemotherapy;

[0563] Note: Refers to disease progression during or after chemotherapy. For subjects who have previously received neoadjuvant chemotherapy, concurrent chemoradiotherapy, or adjuvant chemotherapy, if recurrence / metastasis occurs within 6 months of the last treatment, the original treatment regimen is defined as the subject's first-line treatment regimen.

[0564] (2) Eastern Cooperative Oncology Group (ECOG) performance status score of 0 to 1 (see Appendix 1);

[0565] (3) expected survival time is at least 12 weeks;

[0566] (4) The subject has at least one measurable tumor lesion.

[0567] Dosage adjustments

[0568] Dosage adjustments for P4B-3

[0569] In this trial, the dose increase or decrease of the trial drug is not allowed. If the subject experiences a grade ≥3 adverse event (AE) related to the trial drug, repeated grade 2 AEs related to the trial drug, grade ≥3 infusion adverse reaction, or serious life-threatening adverse event, the investigator will assess whether the trial drug needs to be suspended or permanently discontinued. Subjects who have not received the trial drug for three consecutive dosing cycles will be considered for withdrawal from the study.

[0570] Duration of drug treatment

[0571] The subjects will receive long-term treatment with the trial drug until intolerable toxicity occurs, disease progression, death, voluntary withdrawal of the subject, or continued treatment for more than 24 months or termination of the study (including completion of the trial or early termination of the trial) (whichever occurs first).

[0572] Safety evaluation

[0573] During the trial, subjects will undergo safety checks at specified times, including laboratory tests (blood count, urine count, blood biochemistry, coagulation function, and thyroid function), vital signs, physical examinations, and 12-lead electrocardiograms (12-ECGs). The clinical manifestations, severity, onset, end time, duration, treatment, and outcomes of any adverse events will be recorded, and their relevance to P4B-3 will be determined. Adverse events will be graded using the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) version 5.0. The relevance of adverse events to P4B-3 will be assessed by the investigator according to the attribution assessment criteria specified in the protocol.

[0574] Effectiveness evaluation

[0575] Anti-tumor efficacy was evaluated according to the revised Response Evaluation Criteria in Solid Tumors (RECIST version 1.1) and the Immunotherapy Response Evaluation Criteria in Solid Tumors (iRECIST). 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.

[0576] Objective response rate (ORR): includes partial response (PR) and complete response (CR), and is defined as the proportion of subjects with complete response and partial response. For subjects with solid tumors, anti-tumor efficacy was evaluated according to RECIST 1.1. For subjects with an initial PR or CR, efficacy was confirmed at least 4 weeks later.

[0577] Duration of response (DOR) was defined as the time from the first confirmed objective response to disease progression or death from any cause, whichever occurred first.

[0578] Disease control rate (DCR): defined as the proportion of subjects who achieved remission (PR+CR) and stable disease (SD) after treatment.

[0579] Progression-free survival (PFS) was defined as the time from the date of the subject's first dose to the first tumor assessment confirming disease progression or death from any cause.

[0580] Overall survival (OS) was defined as the time from the enrollment of the subjects to death from any cause.

[0581] As of February 19, 2024, a total of 47 patients with measurable lesions who had progressed after at least one line of platinum-based chemotherapy received P4B-3. Of these, 45 patients underwent at least one efficacy assessment, with 15 achieving a PR and 9 achieving SD, resulting in an ORR of 33.3% and a DCR of 53.3%. Among 25 patients with EP-NEC who had progressed after two or more lines of systemic therapy, 24 were evaluable for efficacy, with 7 achieving a PR and 5 achieving SD, resulting in an ORR and DCR of 29.2% and 50.0%, respectively. As of September 19, 2024, the median follow-up for these 45 patients who underwent at least one efficacy assessment was 12.3 months, and the median OS was 15 months (95% CI: 10.4, NE).

[0582] Study results demonstrate that P4B-3 monotherapy demonstrates promising efficacy in patients with EP-NEC who have progressed after at least one prior line of platinum-based chemotherapy. This is particularly true for patients in third-line or higher-line settings, where guidelines lack recommended treatment options. P4B-3 monotherapy is expected to address unmet clinical needs in this population.

Claims

1. Use of a bispecific antibody in the preparation of a medicament for treating a malignant tumor, the bispecific antibody comprising a first antigen binding region that specifically binds to 4-1BB and a second antigen binding region that specifically binds to PD-L1.

2. The use according to claim 1, wherein the first antigen binding region comprises a first heavy chain variable region and / or a first light chain variable region, wherein: The first heavy chain variable region: (i) comprising HCDR1, HCDR2 and HCDR3 derived from the heavy chain variable region consisting of the sequence shown in SEQ ID NO: 7 or SEQ ID NO: 9; (ii) comprises HCDR1, HCDR2 and HCDR3, which comprise or consist of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, or comprise or consist of an amino acid sequence 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, respectively; or (iii) comprising or consisting of the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:9, or comprising 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 to the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:9, or 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:7 or SEQ ID NO:9, preferably, these amino acid modifications do not occur in the CDR regions, more preferably, these amino acid modifications occur in the FR regions, such as FR1, FR2, FR3 or FR4 regions; The first light chain variable region: (i) comprising LCDR1, LCDR2 and LCDR3 derived from the light chain variable region consisting of the sequence shown in SEQ ID NO: 8 or SEQ ID NO: 10; (ii) comprises LCDR1, LCDR2 and LCDR3, which respectively comprise or consist of the sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, or respectively comprise or consist of an amino acid sequence having one, two or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6; or (iii) comprises or consists of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 10, or comprising or consisting of an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 10, or comprising or consisting of an amino acid sequence that has 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 or SEQ ID NO: 10, preferably, these amino acid modifications do not occur in the CDR regions, more preferably, these amino acid modifications occur in the FR regions, such as FR1, FR2, FR3 or FR4 regions; Wherein in SEQ ID NO:8, X=S or G.

3. The use according to claim 1 or 2, wherein the first antigen binding region further comprises a first heavy chain constant region and / or a first light chain constant region, the first heavy chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO: 19 or 20, and the first light chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO: 21 or 22.

4. The method of any one of claims 1 to 3, wherein the first antigen binding region activates 4-1BB signaling and is a monoclonal antibody.

5. The use of any one of claims 1 to 4, wherein the second antigen-binding region that specifically binds to PD-L1 comprises a second heavy chain variable region and a second light chain variable region, wherein the second heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 consisting of sequences shown in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively, and the second light chain variable region comprises LCDR1, LCDR2 and LCDR3 consisting of sequences shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

6. The use according to claim 5, wherein the second antigen-binding region further comprises a second heavy chain constant region and / or a second light chain constant region, the second heavy chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO: 19 or 20, and the second light chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO: 21 or 22.

7. The use according to any one of claims 1 to 6, wherein the bispecific antibody comprises: (1) Chain 1: the heavy chain of the second antigen-binding region connected at the C-terminus with or without a linker to the antigen-binding moiety in the first antigen-binding region; and (2) Chain 2: The light chain of the second antigen-binding region.

8. The use as claimed in claim 7, wherein the linker comprises an amino acid sequence (Gly4Ser)n or (GlySer4)n, wherein n is a positive integer of 1-7.

9. The use as claimed in claim 7 or 8, wherein the bispecific antibody consists of two chains 1 and two chains 2.

10. The use of any one of claims 7-9, wherein the antigen binding portion is selected from: (i) a Fab fragment; (ii) a F(ab')2 fragment; (iii) a Fd fragment; (iv) an Fv fragment, (v) a dAb fragment; (vi) a nanobody; and (vii) a single-chain Fv (scFv); the scFv comprising the first heavy chain variable region and the first light chain variable region.

11. The use according to any one of claims 7 to 9, wherein the first antigen-binding region and / or the second antigen-binding region is a mouse antibody, a human antibody, a chimeric antibody or a humanized antibody.

12. The use according to any one of claims 7 to 9, wherein the first antigen binding region and / or the second antigen binding region is of IgG1, IgG2 or IgG4 isotype.

13. The use of claim 7, wherein the chain 1 of the bispecific antibody comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 25; and the chain 2 of the bispecific antibody comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:

26.

14. The use according to any one of claims 1 to 13, wherein the malignant tumor is bladder cancer, breast cancer, endometrioid carcinoma, cervical cancer, advanced solid tumors, renal cell carcinoma, neuroendocrine tumors, gastric cancer, transitional cell carcinoma, urothelial carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, colorectal cancer, non-small cell lung cancer, hepatocellular carcinoma; preferably, the neuroendocrine tumor is a neuroendocrine carcinoma or a neuroendocrine tumor, more preferably, the neuroendocrine tumor is an extrapulmonary neuroendocrine tumor; more preferably, the neuroendocrine tumor is an advanced neuroendocrine tumor.

15. The use according to claim 14, wherein the neuroendocrine cancer is an advanced neuroendocrine cancer, preferably, the neuroendocrine cancer is an extrapulmonary neuroendocrine cancer or a small cell lung cancer; more preferably, the neuroendocrine cancer is an unresectable locally advanced or metastatic neuroendocrine cancer.

16. The use according to claim 14 or 15, wherein the drug is administered to a subject suffering from the malignant tumor, wherein the subject is a subject who has not received systemic treatment or a subject whose disease has progressed after receiving at least a first-line chemotherapy in the past, for example, the subject is a subject whose disease has progressed after receiving at least a first-line chemotherapy containing platinum; further, the subject includes a subject whose disease has progressed after receiving two or more lines of chemotherapy in the past.

17. The use according to any one of claims 1 to 16, comprising administering to the subject 0.2 mg / kg to 200 mg / kg of the bispecific antibody per dosing cycle or each time, preferably 0.8 mg / kg to 25 mg / kg of the bispecific antibody, more preferably 3.2 mg / kg to 20 mg / kg of the bispecific antibody, still more preferably 6 mg / kg to 15 mg / kg of the bispecific antibody, most preferably 10 mg / kg to 15 mg / kg of the bispecific antibody.

18. The use according to any one of claims 1 to 16, comprising administering to a subject 50 mg to 5000 mg of the bispecific antibody per dosing cycle or each time, preferably 60 mg to 4000 mg of the bispecific antibody, more preferably 80 mg to 3750 mg of the bispecific antibody, most preferably 100 mg to 3500 mg of the bispecific antibody, for example 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, 3500 mg of the bispecific antibody.

19. The use according to claim 17 or 18, wherein the administration cycle of the bispecific 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.

20. The use according to any one of claims 1 to 19, wherein the bispecific antibody is formulated for intravenous infusion or subcutaneous injection.

21. The use of any one of claims 1-20, wherein the bispecific antibody is also administered in combination with one or more chemotherapeutic agents; preferably the chemotherapeutic agent is a platinum and / or a podophyllotoxin derivative; more preferably the platinum is carboplatin or cisplatin; most preferably the podophyllotoxin derivative is etoposide, etoposide glucuronide or etoposide phosphate.

22. The use according to claim 21, wherein 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.

23. The use according to claim 21 or 22, wherein the chemotherapeutic agent is formulated for administration as an intravenous infusion or an oral formulation.

24. The use according to any one of claims 21 to 23, comprising administering the chemotherapeutic agent per dosing cycle or each time after administering the bispecific antibody to the subject.

25. The use according to claim 24, comprising administering the podophyllotoxin derivative for 1-5 days (e.g., 3 days) per dosing cycle or each time after the administration of the bispecific antibody, once a day, preferably 100 mg / m 2 or less of said podophyllotoxin derivative.

26. The use according to claim 24, comprising administering the platinum for 1-5 days (e.g., 3 days) per dosing cycle or each time after the administration of the bispecific antibody, preferably 75 mg / m per day. 2 or less of said platinum, or AUC=5 mg / mL / min or less of said platinum.

27. A medicament for treating a malignant tumor, said medicament comprising a bispecific antibody as defined in claims 1-13.

28. A drug combination comprising a bispecific antibody as defined in any one of claims 1-13, and one or more chemotherapeutic agents; preferably, the chemotherapeutic agent is a platinum and / or a podophyllotoxin derivative; more preferably, the platinum is carboplatin or cisplatin; most preferably, the podophyllotoxin derivative is etoposide, etoposide glucuronide or etoposide phosphate; preferably, the drug combination is used to treat malignant tumors.

29. The drug of claim 27 or the drug combination of claim 28, wherein the malignant tumor is bladder cancer, breast cancer, endometrioid carcinoma, cervical cancer, advanced solid tumors, renal cell carcinoma, neuroendocrine tumors, gastric cancer, transitional cell carcinoma, urothelial carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, colorectal cancer, colorectal cancer, non-small cell lung cancer, hepatocellular carcinoma; preferably, the neuroendocrine tumor is a neuroendocrine carcinoma or a neuroendocrine tumor, more preferably, the neuroendocrine tumor is an extrapulmonary neuroendocrine tumor; more preferably, the neuroendocrine tumor is an advanced neuroendocrine tumor.

30. The drug or drug combination according to claim 29, wherein the neuroendocrine cancer is an advanced neuroendocrine cancer, preferably, the neuroendocrine cancer is an extrapulmonary neuroendocrine cancer or a small cell lung cancer; more preferably, the neuroendocrine cancer is an unresectable locally advanced or metastatic neuroendocrine cancer.

31. The drug or drug combination of claim 29 or 30, wherein the drug is administered to a subject suffering from the malignant tumor, the subject being a subject who has not received systemic treatment or a subject whose disease has progressed after receiving at least a first-line chemotherapy treatment in the past, for example, the subject is a subject whose disease has progressed after receiving at least a first-line chemotherapy treatment containing platinum; further, the subject includes a subject whose disease has progressed after receiving two or more lines of chemotherapy in the past.

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