Combinations comprising CD20 / CD47 blocking bifunctional fusion proteins and uses thereof

Abstract

The invention relates to a combination containing CD20 / CD47 blocking bifunctional fusion protein and application of the combination. The invention provides a scheme for treating cancers (especially non-Hodgkin's lymphoma) by combining the CD20 / CD47 double-blocking bifunctional fusion protein and lenalidomide or the salt thereof, realizes a better anti-tumor effect than a single drug (CD20 / CD47 double-blocking bifunctional fusion protein or lenalidomide), and has a remarkable synergistic effect.

Description

Technical Field

[0001] This application relates to the field of biopharmaceuticals, specifically to methods and applications of combining a CD20 / CD47 dual-blocking bifunctional fusion protein with an anticancer agent for the treatment of diseases, particularly methods and applications of combining a CD20 / CD47 dual-blocking bifunctional fusion protein with lenalidomide or its salts for the treatment of cancer (especially non-Hodgkin's lymphoma). Background Technology

[0002] Non-Hodgkin lymphoma (NHL) is predominantly B-cell lymphoma, accounting for 70%-85% of all cases. There are four main B-cell types: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MC), and mucosa-associated lymphoid tissue lymphoma (MAL); with diffuse large B-cell lymphoma being the most common. Clinically, lymphadenopathy is the most frequent local clinical manifestation. In recent years, the incidence of lymphoma in my country has been gradually increasing, currently reaching nearly 7 per 100,000 people per year.

[0003] Currently, R-CHOP (a combination therapy consisting of five drugs, where R stands for Rituximab (rituximab injection), C for cyclophosphamide, H for doxorubicin, O for vincristine, and P for prednisone) is a first-line treatment for patients with diffuse large B-cell lymphoma and other non-Hodgkin's lymphomas. Compared to the traditional CHOP regimen, the addition of rituximab increases the 5-year median overall survival from 45% to 58%; this suggests that immunochemotherapy can further improve treatment outcomes on top of existing chemotherapy regimens.

[0004] The mechanisms of action of rituximab include CDC (complement-dependent cytotoxicity), ADCC (antibody-dependent cell-mediated cytotoxicity), and ADCP (antibody-dependent cell-mediated phagocytosis), among which CDC plays a major role.

[0005] The reasons for resistance or relapse of non-Hodgkin lymphoma after R-CHOP treatment may involve the four CHOP chemotherapy drugs or rituximab.

[0006] Meanwhile, research has revealed that CD47 is a novel immune checkpoint that can bind to the SIRPα protein on macrophages, thereby inhibiting macrophage phagocytosis. Blocking the binding of CD47 to SIRPα can promote macrophage phagocytosis. Several anti-CD47 monoclonal antibodies have been developed, and further research has shown that the combination of anti-CD47 monoclonal antibodies and rituximab can further enhance macrophage phagocytosis. However, existing treatment regimens (whether combining anti-CD47 monoclonal antibodies with anti-CD20 monoclonal antibodies, or combining antibodies with chemical drugs) still cannot meet the treatment needs of relevant patients. Summary of the Invention

[0007] In previous studies, the inventors of this application developed a new generation of bifunctional fusion protein that blocks both CD20 and CD47, which significantly outperforms the SIRPα-Fc fusion protein in blocking the interaction between CD47 on tumor cells and SIRPα expressed on macrophages. Further research by the inventors showed that this type of fusion protein exhibits significantly stronger macrophage phagocytosis-promoting effects than rituximab, SIRPα fusion protein, and ovvarumumab; it also produces significant therapeutic effects in a rituximab-resistant diffuse large B-cell lymphoma (DLBCL) xenograft model.

[0008] Building upon this foundation, the inventors of this application, through extensive research and exploration, have discovered and provided combination drugs for the prevention or treatment of cancer, including a bifunctional fusion protein that blocks both CD20 and CD47 and an anticancer agent, particularly an anticancer agent such as lenalidomide, as well as the use of this combination drug in the treatment of cancer and in the preparation of cancer treatment drugs. In particular, a highly significant synergistic effect was discovered when the bifunctional fusion protein that blocks both CD20 and CD47 is combined with lenalidomide to treat lymphoma, especially diffuse large B-cell lymphoma.

[0009] Specifically, this application provides a pharmaceutical composition for the prevention or treatment of cancer, comprising a bifunctional fusion protein that blocks both CD20 and CD47 and an anticancer agent.

[0010] As used herein, the terms "pharmaceutical composition," "combination drug," "drug combination," and "drugs used in combination" are used interchangeably to refer to a combination of at least one drug and optional pharmaceutically acceptable carriers or excipients combined together to achieve a particular purpose. In some embodiments, the pharmaceutical composition comprises combinations that are separate in time and / or space, provided that they can work together to achieve the purpose of this application. For example, the components contained in the pharmaceutical composition (e.g., fusion proteins or anticancer agents according to this application) may be applied to the subject as a whole or separately. When the components contained in the pharmaceutical composition are applied to the subject separately, the components may be applied to the subject simultaneously or sequentially. For example, the pharmaceutically acceptable carrier may be water, a buffered aqueous solution, an isotonic saline solution such as PBS (phosphate-buffered saline), glucose, mannitol, dextran glucose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, or polyalkylene glycols such as polypropylene glycol, triglycerides, etc. The type of pharmaceutically acceptable carrier used depends particularly on whether the composition according to this application is formulated for inhalation, intranasal, oral, intravenous, subcutaneous, or intramuscular administration. The composition according to this application may contain wetting agents, emulsifiers, or buffering substances as additives.

[0011] The cancers / tumors treated in this application include hematologic malignancies, such as lymphomas, including Hodgkin's lymphoma or non-Hodgkin's lymphoma. In some embodiments, the cancer is non-Hodgkin's lymphoma. In some embodiments, the cancer is diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, or mucosa-associated lymphoid tissue lymphoma. In some embodiments, the cancer is diffuse large B-cell lymphoma. In some embodiments, the cancer is CD20-positive non-Hodgkin's lymphoma. In some embodiments, the cancer is CD20-positive diffuse large B-cell lymphoma.

[0012] The CD20 / CD47 dual-blocking bifunctional fusion protein described in this application comprises two functional moieties that block CD20 and CD47, respectively. In some instances of this application, the two functional moieties are also referred to as two "arms," ​​which can form a heterodimer in a homodimeric structure similar to that of natural antibodies. Those skilled in the art will understand that the bifunctional fusion protein of this application is not limited to a two-armed form, but can also be four-armed or other forms, similar to various conformations of bispecific antibodies in the art.

[0013] As used in this application, the term "antibody" broadly encompasses any form of molecule capable of specifically binding to an antigen, and may include the complete antibody and its antigen-binding fragment ("antigen-binding moiety") or single-chain form. The terms "antigen-binding moiety" or "antigen-binding fragment" may in particular refer to antibody fragments such as Fv, scFv, Fab, F(ab')2, Fab', VHH, etc.

[0014] In some implementations, the functional portion / arm that blocks CD20 is in the form of an antibody against CD20 or its antigen-binding portion.

[0015] In some embodiments, the functional portion / arm that blocks CD47 is an antibody against CD47 or its antigen-binding portion and / or a natural ligand of CD47 or a ligand-active portion derived from said natural ligand. In some embodiments, the natural ligand of CD47 is the SIRPα protein. The SIRPα protein is a transmembrane protein, and therefore its extracellular domain primarily performs its ligand-active function.

[0016] In some embodiments, the CD20 / CD47 dual-blocking bifunctional fusion protein and anticancer agent, the fusion protein comprising a first arm targeting CD20 and a second arm blocking the interaction between CD47 and SIRPα, the first arm comprising heavy chain CDR1-CDR3 and light chain CDR1-CDR3, the sequences of the heavy chain CDR1-CDR3 being shown in SEQ ID NO:1-3, the light chain CDR1-CDR3 being shown in SEQ ID NO:4-6, the CDRs being defined by Kabat encoding, and the second arm comprising the amino acid sequence shown in SEQ ID NO:15.

[0017] In some embodiments, the first arm comprises VH and VL, wherein the VH comprises an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 7, and the VL sequence comprises an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 8.

[0018] In some embodiments, the first arm of the CD20 targeting mechanism comprises the Fab or Fab' region and the Fc region of an immunoglobulin, and the second arm comprises SEQ ID NO:15 and the Fc region, wherein the Fc region comprises a natural Fc region sequence or a non-natural Fc region sequence; preferably, the Fc region is a human Fc region; more preferably, the Fc region is the Fc region of an IgG1, IgG2, IgG3 or IgG4 antibody. Preferably, the first arm of the CD20 target comprises 1) SEQ ID NO:9 and SEQ ID NO:10, or 2) SEQ ID NO:11 and SEQ ID NO:12; the second arm comprises SEQ ID NO:13 or SEQ ID NO:14.

[0019] The anticancer agent described in this application is a chemotherapeutic agent; preferably, the chemotherapeutic agent is lenalidomide or a pharmaceutically acceptable salt, solvate, or prodrug thereof; more preferably, the chemotherapeutic agent is lenalidomide. In some embodiments, the anticancer agent is a chemotherapeutic agent.

[0020] This application also provides the use of the CD20 / CD47 dual-blocking bifunctional fusion protein and anticancer agent described above in the preparation of pharmaceutical compositions for treating cancer (preferably CD20-positive tumors).

[0021] This application also provides a combination of a bifunctional fusion protein comprising the CD20 / CD47 dual blockade described above and an anticancer agent, said combination for the treatment of cancer (preferably CD20-positive tumors).

[0022] This application also provides a method for treating cancer (preferably a CD20-positive tumor) in an individual, the method comprising administering to the individual in need a therapeutically effective amount of the aforementioned CD20 / CD47 dual-blocking bifunctional fusion protein and an anticancer agent.

[0023] In some embodiments of the above-described uses, combinations, or methods, the CD20 / CD47 dual-blocking bifunctional fusion protein and the anticancer agent are formulated together or exist physically independently (e.g., as different units of a kit or pharmaceutical package). The CD20 / CD47 dual-blocking bifunctional fusion protein can be formulated for administration before, simultaneously with, or after the anticancer agent.

[0024] In some embodiments, the dosage of the bifunctional fusion protein is 1 mg / kg to 40 mg / kg, for example, 1 mg / kg, 3 mg / kg, 6 mg / kg, 12 mg / kg, 20 mg / kg, 30 mg / kg, or 40 mg / kg. In some embodiments, the bifunctional fusion protein is administered at least once every two weeks, for example, once every two weeks, once every three weeks, once every four weeks, once every five weeks, or once every six weeks, preferably once every two weeks, once every three weeks, or once every four weeks; or the administration frequency is more than once a week, for example, twice a week; preferably, it is administered once every two weeks, once every three weeks, or once every four weeks.

[0025] In some embodiments, the anticancer agent (e.g., lenalidomide) is administered at a dose of 15-25 mg / day, for example, 15 mg / day, 20 mg / day, 23 mg / day, or 25 mg / day. In some embodiments, the anticancer agent (e.g., lenalidomide) is administered once daily for 21 consecutive days; preferably, it is administered once daily on days D2-D22 of each cycle.

[0026] In some embodiments, the administration of the CD20 / CD47 dual-blocking bifunctional fusion protein and the anticancer agent is independently by inhalation, intranasal administration, oral administration, intravenous injection, subcutaneous or intramuscular injection.

[0027] Examples of the CD20 / CD47 dual-blocking bifunctional fusion protein described in this application can be found in Chinese Patent No. 201810430371.1 (CN 108864290B), the entire contents of which are incorporated herein by reference.

[0028] An example of the CD20 / CD47 dual-blocking bifunctional fusion protein described in this application comprises a left arm targeting CD20, consisting of the amino acid sequences shown in SEQ ID NO:11 and SEQ ID NO:12, and a right arm blocking the interaction of CD47 with SIRPα, consisting of the amino acid sequences shown in SEQ ID NO:14. This CD20 / CD47 dual-blocking bifunctional fusion protein is referred to as JMT601 in this application.

[0029] JMT601 can be prepared by referring to the preparation methods disclosed in Examples 1 and 2 of CN 108864290B, or by other methods in the prior art. Attached image description: Figure 1 Tumor volume change curves in different treatment groups in the WSU-DLCL2 human lymphoma subcutaneous model.

[0030] Note: Data are expressed as mean ± standard error.

[0031] Figure 2 Changes in body weight of mice in different treatment groups over time in the WSU-DLCL2 human lymphoma subcutaneous model.

[0032] Note: Data are expressed as mean ± standard error.

[0033] Figure 3 A schematic diagram of an embodiment of the CD20 / CD47 dual-blocking bifunctional fusion protein of this application, a representative example being JMT601, wherein the left arm is a single-arm form of a natural antibody targeting CD20, with the Fc region containing a hole mutation; the right arm is the extracellular D1 domain of the SIRPα protein, with the Fc region containing a knob mutation.

[0034] Figure 4 Statistical analysis of tumor weight data for each group (the columns from left to right represent JMT601, lenalidomide, JMT601 + lenalidomide, and rituximab, respectively). One-way ANOVA was used for multiple comparisons, and *p<0.05 indicated significant differences.

[0035] Figure 5 Statistical analysis of tumor volume data for each group (the columns from left to right represent JMT601, lenalidomide, JMT601 + lenalidomide, and rituximab, respectively). One-way ANOVA was used for multiple comparisons, and *p<0.05 indicated significant differences. Detailed implementation method: The embodiments listed below are provided to better illustrate the content of this application, but are not intended to limit the scope of this application to the illustrated embodiments. Non-essential improvements and adjustments made to the implementation schemes by those skilled in the art based on the above-described invention are still within the protection scope of this application.

[0036] Example 1: Pharmacodynamic study of combination therapy in a mouse model of WSU-DLCL2 human lymphoma subcutaneously transplanted with NOD SCID 1. Experimental Materials and Objectives The main experimental materials used in the experiment are shown in the table below: Table 1: Main experimental materials and reagents used .

[0037] This study evaluated the efficacy of combination therapy with test drugs in NOD SCID mice using the WSU-DLCL2 human lymphoma subcutaneous transplantation model. The pharmacodynamic assay design is as follows: Table 2: Dosing Regimen Note: 1. BIW = twice a week, QD = once a day, N = number of animals; 2. Dosage volume is 10 μl / g.

[0038] 2. Experimental Methods 2.1 Tumor inoculation and grouping WSU-DLCL2 cells (human diffuse large B-cell lymphoma cells) (cell number CL-00648, manufacturer: Crownbio) were cultured in RPMI 1640 medium containing 10% fetal bovine serum. WSU-DLCL2 cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration, and then used for subcutaneous tumor inoculation in mice.

[0039] 1×10 7 WSU-DLCL2 cells were resuspended in a 1:1 mixture of PBS and matrix gel (0.1 ml / mouse) and subcutaneously seeded into experimental mice. The seeding date was May 20, 2020. The average tumor volume was 150.53 mm². 3 At that time, the tumors were randomly grouped according to their size (see Table 2 for details).

[0040] 2.2 Preparation methods of test drug and control drug Table 3: Drug Preparation Methods and Storage Conditions .

[0041] 2.3 Observation All experimental protocols used in this study were reviewed and approved by the Crown Bio IACUC committee. Animal procedures were performed in accordance with AAALAC requirements. Post-tumor inoculation monitoring included assessing tumor growth and the impact of treatment on normal animal behavior, specifically monitoring activity levels, feeding and water intake, weight gain or loss, and any abnormalities observed in the eyes, coat, or other areas. All clinical symptoms observed during the experiment were recorded in the raw data.

[0042] Data was collected using StudyDirector™ (version 3.1.399.19, vendor Studylog System, Inc., S. San Francisco, CA, USA) software, including measurements of the tumor's major and minor diameters and animal weight.

[0043] The experiment ended 21 days after the mice were given the drugs. The tumor-bearing mice were photographed, and the tumors were weighed and photographed again.

[0044] 2.4 Data Analysis Compare the different treatment groups, performing one-way ANOVA while confirming homogeneity of variance. If the variances are homogeneous, continue using Dunnnett's test for pairwise comparisons; if the variances are not homogeneous, switch to one-way ANOVA using the non-parametric Kruskal-Wallis test, while using Dunn's test for pairwise comparisons.

[0045] All statistical analyses and graphing were performed using GraphPad Prism 8.4.2, and a p-value less than 0.05 was considered statistically significant.

[0046] 3. Experimental Results The mean tumor volume of mice in the solvent control IgG1 10 mg / kg group was 1629.39 mm 21 days after administration. 3 The tumor volume doubling time was 6 days. The therapeutic effect was evaluated at 21 days post-administration, comparing the relative tumor volume between the test drug group and the solvent control group. At 21 days post-administration, the mean tumor volume in the rituximab 10 mg / kg group was 1121.67 mm. 3The tumor inhibition rate was 33.39%, and the reduction in average tumor volume was significantly different compared to the solvent control group (p<0.0001), with a tumor volume doubling time of 8 days. The average tumor volume in the JMT601 3.3 mg / kg group was 825.07 mm. 3 The tumor inhibition rate was 50.00%, and the reduction in average tumor volume was significantly different compared to the solvent control group (p < 0.0001), with a tumor volume doubling time of 9 days. The average tumor volume in the lenalidomide 50 mg / kg group was 1509.39 mm. 3 The tumor inhibition rate was 9.69%, and the reduction in average tumor volume was not significantly different compared to the solvent control group. The tumor volume doubling time was 7 days. The average tumor volume in mice treated with lenalidomide 50 mg / kg combined with JMT601 3.3 mg / kg was 573.73 mm. 3 The tumor inhibition rate was 66.79%, and the average tumor volume reduction was significantly different from that of the solvent control group (p<0.0001). The tumor volume doubling time was 11 days.

[0047] The results of the tumor weight analysis were basically consistent with the results of the relative tumor volume analysis.

[0048] The tumor growth inhibition in mice of each treatment group and control group is shown in Table 4 and 5. Figure 1 .

[0049] Furthermore, tumor weight data were used to conduct a more detailed statistical analysis of tumor volume and weight in the combination therapy group and the single-drug group (see results). Figure 4 ), Figure 4 The four bars along the horizontal axis from left to right represent the JMT601 monotherapy group, the lenalidomide group, the JMT601 + lenalidomide group, and the rituximab monotherapy group, respectively. It can be seen that JMT601 monotherapy is significantly superior to rituximab monotherapy (p < 0.01); the JMT601 + lenalidomide groups are significantly superior to each of their respective monotherapy, with the JMT601 + lenalidomide group compared to JMT601 monotherapy (p < 0.01) and the JMT601 + lenalidomide group compared to lenalidomide monotherapy (p < 0.0001). JMT601 combined with lenalidomide has a synergistic effect.

[0050] Statistical analysis was performed using tumor volume data (see results). Figure 5 ), Figure 5The four bars along the horizontal axis from left to right represent the JMT601 monotherapy group, the lenalidomide group, the JMT601 + lenalidomide group, and the rituximab monotherapy group, respectively. It can be seen that JMT601 monotherapy is significantly more effective than rituximab monotherapy (p < 0.01); the JMT601 + lenalidomide group is more effective than either drug alone, and the JMT601 + lenalidomide group is significantly more effective than lenalidomide monotherapy (p < 0.0001). That is, JMT601 combined with lenalidomide has a synergistic effect.

[0051] The above analysis shows that, compared with the single application of JMT601 and lenalidomide, the combined application of JMT601 and lenalidomide can significantly improve the tumor inhibition rate and has a significant synergistic effect.

[0052] The following formula for evaluating drug synergy was used to analyze the combination of JMT601 and lenalidomide: Based on the above drug synergy evaluation formula and the tumor suppression data in Table 4, the combination index (CI) was calculated to be 1.22, indicating that the combination of JMT601 and lenalidomide has a synergistic therapeutic effect.

[0053] Example 2: Results and discussion of the safety study of the test drug in a WSU-DLCL2 human lymphoma subcutaneous transplantation model. In this experiment, the experimental materials and methods were the same as in Example 1. On days 15, 18, and 19 after administration, in the group receiving JMT601 3.3 mg / kg combined with lenalidomide 50 mg / kg, one mouse experienced a weight loss greater than 20%, reaching the humane endpoint and was euthanized. The weight loss in all mice was found to be due to decreased appetite; no significant abnormalities were found during necropsy, suggesting that the weight loss was likely due to individual differences among the mice. All other mice in each group tolerated all tested drugs well.

[0054] The changes in body weight after administration in the treatment group and the control group are shown in Table 5 and 6. Figure 2 .

[0055] Table 5: Drug Efficacy Data .

[0056] The above results indicate that, for WSU-DLCL2 cell subcutaneous xenografts in tumor-bearing mice, the combination therapy with JMT601 significantly increased the tumor-suppressive effect compared to lenalidomide alone. Tumor-bearing mice tolerated rituximab (10 mg / kg), JMT601 (3.3 mg / kg and 10 mg / kg), and lenalidomide (50 mg / kg). The combination therapy regimen showed no significant difference in tolerability compared to monotherapy, demonstrating the good safety profile of this combination therapy.

[0057] Example 3: A multicenter, phase II clinical study evaluating the efficacy and safety of JMT601 in combination with different chemotherapy regimens for the treatment of CD20-positive diffuse large B-cell lymphoma. 1. Drug Information Test drug 1: JMT601, provided by Shanghai Jinmante Biotechnology Co., Ltd. Specification: 100 mg (5 mL) / bottle Dosage form: Injection Storage conditions: Store at 2-8℃. Test drug 2: Lenalidomide capsules, provided by CSPC Ouyi Pharmaceutical Co., Ltd. Specification: 25mg / capsule Dosage form: Capsules Storage conditions: Sealed, store at room temperature (10-30℃).

[0058] 2. Dosage and administration method Participants were given JMT601 on the first day of each cycle.

[0059] The dosage of JMT601 is 12 mg / kg, 20 mg / kg, and 30 mg / kg; administered by intravenous infusion once every two weeks / three weeks / four weeks, with the first day of each cycle.

[0060] Lenalidomide was administered at a dose of 15-25 mg / day orally in this study, once daily from day 2 to day 22 of each cycle, for a total of 21 days. Each cycle consisted of four weeks.

[0061] 3. Study population Patients with CD20-positive diffuse large B-cell lymphoma.

[0062] 4. Inclusion criteria Participants must meet all of the following inclusion criteria to be eligible for this study: 1) CD20+ DLBCL confirmed by histopathology and / or cytology, including: diffuse large B-cell lymphoma, nonspecific type (DLBCL, NOS); high-grade B-cell lymphoma with MYC and BCL2 and / or BCL6 rearrangements; high-grade B-cell lymphoma, nonspecific type; 2) Relapsed or refractory CD20+ DLBCL after first-line standard therapy; Definition of relapse / refractory: Relapse is defined as disease progression after achieving CR or PR with adequate systemic therapy; refractory is defined as no response to adequate systemic therapy (disease progression during systemic therapy / disease progression within 6 months of the end of adequate therapy, or failure to achieve PR or CR after adequate cycles of systemic therapy). Adequate (cycles) of systemic therapy is defined as: DLBCL patients have received at least one rituximab-based combination therapy for at least two cycles. 3) Patients who have previously received autologous hematopoietic stem cell transplantation (ASCT) and have not achieved remission or have relapsed after ASCT; 4) There must be at least one measurable lesion that meets the Lugano 2014 criteria (measurable lesion: long axis of lymph node lesion >1.5 cm, or long diameter of extranodal lesion >1.0 cm; if the only measurable lesion has previously received radiotherapy, there must be evidence of radiographic progression after radiotherapy). 5) ECOG score for physical fitness ≤ 2 points; 6) Expected survival period is at least 3 months.

[0063] 5. Exclusion Criteria 1) The presence of lymphoma involving the central nervous system or leptomeninges (for participants suspected of having central nervous system lymphoma, lumbar puncture and MRI should be performed to rule out CNS lymphoma, depending on the investigator's decision). 2) Participants who have previously undergone allogeneic ASCT and other organ transplants; 3) Individuals who have previously received treatment targeting CD47 or signal regulatory protein α; 4) Individuals with a history of hemolytic anemia, Evans syndrome, arteritis, or progressive multifocal leukoencephalopathy; 5) History of other malignant tumors within 5 years or concurrently having other active malignant tumors (cured localized tumors, such as basal cell carcinoma of the skin, squamous cell carcinoma of the skin, superficial bladder cancer, prostate carcinoma in situ, cervical carcinoma in situ, breast carcinoma in situ, etc., are eligible for enrollment). 6) If the patient has received macromolecular antibody antitumor therapy within 28 days prior to the first dose of medication, or chemotherapy, small molecule targeted antitumor therapy, or any trial intervention within 14 days prior to the first dose of medication, unless it is an observational (non-interventional) clinical trial or is in the follow-up period of an interventional trial; 7) Adverse reactions to previous antitumor treatments have not recovered to CTCAE 5.0 grade ≤ 1 (excluding toxicities such as platelet count, hemoglobin, and hair loss that the investigators judged to pose no safety risk); 8) Participants who have undergone major surgery or invasive intervention within 28 days prior to the first dose, or who are scheduled to undergo systemic or local tumor resection during the study; 9) The presence of uncontrolled diseases, including but not limited to uncontrolled diabetes (glycated hemoglobin ≥8.0%), uncontrolled hypertension (systolic blood pressure ≥160 mmHg or diastolic blood pressure ≥100 mmHg), active infections (including bacterial, fungal, viral, etc.), active gastrointestinal ulcers, thromboembolic diseases requiring regular anticoagulation, and any other poorly controlled conditions that the investigator judges to increase the risk to the participant; 10) The participant's heart meets any of the following conditions: LVEF≤50%; NYHA Class II or higher congestive heart failure or active heart disease; Serious arrhythmias requiring treatment; Patients who have had a myocardial infarction or bypass / stent surgery within 6 months prior to administration; During the screening period, an electrocardiogram with a QTcF > 450 ms or a history of long QT syndrome was observed. Other heart diseases deemed unsuitable for inclusion by the researchers 11) Participants with a history of interstitial lung disease of any severity and / or severe lung function impairment; 12) History of immunodeficiency, including a positive HIV antibody test; 13) Patients with active HBV or hepatitis C (positive hepatitis C antibody and HCV-RNA quantitative test result > lower limit of detection range) confirmed by positive hepatitis B surface antigen (HBsAg) and anti-HBV test results. 14) Participants with a known history of allergic reactions to protein preparations or any investigational drug formulation; 15) Administer a live or attenuated live vaccine within 4 weeks prior to the first study dose, or an inactivated vaccine within 2 weeks prior to the first study dose; 16) Has spontaneous tumor lysis syndrome; 17) Breastfeeding women; 18) Other situations that may increase the risk of the investigational drug, interfere with the interpretation of research results, or affect trial compliance, which the investigators may deem unsuitable for participation in this trial.

[0064] 6. Effectiveness Assessment The oncology imaging efficacy assessment in this study will be conducted according to the investigator's criteria based on Lugano 2014. Enhanced CT and PET / CT scans of the chest, abdomen, pelvis, and other suspected metastatic sites will be performed during the screening period. After the start of treatment, the efficacy assessment methods and schedule are as follows: oncology imaging assessments will be performed every 8 weeks (±7 days), and every 12 weeks thereafter, until disease progression or death. Except for the screening period, at the end of combined therapy, when the participant achieves optimal response based on enhanced CT or the investigator's assessment, or when enhanced CT suggests residual lesions, suspected progression, or is inconclusive, only enhanced CT will be used. Except for the screening period, at week 12 (±7 days) after treatment, at the end of treatment, when the participant achieves optimal response based on enhanced CT or the investigator's assessment, or when enhanced CT suggests residual lesions, suspected progression, or is inconclusive, only enhanced CT will be used.

[0065] Efficacy endpoints include: objective response rate (ORR), time to relapse (TTR), progression-free survival (PFS), duration of response (DOR), and overall survival (OS).

[0066] 7. Security Assessment This study cohort will continuously assess participant safety during the screening period, treatment period (including end-of-treatment visits), and safety visits. Safety assessments will include vital signs, physical examination, laboratory tests, echocardiography, electrocardiography, and performance status scores.

[0067] 8. Test Results The results showed that JMT601 combined with lenalidomide had a synergistic effect in patients with CD20-positive diffuse large B-cell lymphoma, with definite efficacy and good clinical safety.

[0068] The relevant sequences of the fusion protein involved in this application are as follows: .

[0069] Although specific embodiments of this application have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and such changes are all within the scope of protection of this application. The full scope of this application is given by the appended claims and any equivalents.

Claims

1. A pharmaceutical composition for treating cancer, comprising a bifunctional fusion protein that blocks both CD20 and CD47 and an anticancer agent, said fusion protein comprising a first arm that targets CD20 and a second arm that blocks the interaction between CD47 and SIRPα, said first arm comprising heavy chain CDR1-CDR3 and light chain CDR1-CDR3, the sequences of said heavy chain CDR1-CDR3 being shown in SEQ ID NO:1-3, respectively, and the sequences of said light chain CDR1-CDR3 being shown in SEQ ID NO:4-6, respectively, said CDR being defined by Kabat encoding, and said second arm comprising the amino acid sequence shown in SEQ ID NO:

15.

2. Application of CD20 / CD47 dual-blocking bifunctional fusion protein and anticancer agent in the preparation of drugs for treating cancer.

3. A combination of a CD20 / CD47 dual-blocking bifunctional fusion protein and an anticancer agent, said combination for the treatment of cancer.

4. The pharmaceutical composition of claim 1, the application of claim 2, or the combination of claim 3, wherein the anticancer agent is a chemotherapeutic agent; preferably, the chemotherapeutic agent is lenalidomide or a pharmaceutically acceptable salt, solvate, or prodrug thereof; more preferably, the chemotherapeutic agent is lenalidomide.

5. The application or combination of any one of claims 2-4, wherein the fusion protein comprises a first arm targeting CD20 and a second arm blocking the interaction between CD47 and SIRPα, the first arm comprising heavy chain CDR1-CDR3 and light chain CDR1-CDR3, the sequences of the heavy chain CDR1-CDR3 being as shown in SEQ ID NO:1-3, the sequences of the light chain CDR1-CDR3 being as shown in SEQ ID NO:4-6, the CDRs being defined by Kabat encoding, and the second arm comprising the amino acid sequence shown in SEQ ID NO:

15.

6. The pharmaceutical composition or application or combination according to any one of claims 1-5, wherein the first arm comprises VH and VL, the VH comprising an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 7, and the VL sequence comprising an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 8; optionally, the first arm targeting CD20 comprises a Fab or Fab' region and an Fc region of an immunoglobulin, the second arm comprising SEQ ID NO: 15 and an Fc region, the Fc region comprising a natural Fc region sequence or a non-natural Fc region sequence; preferably, the Fc region is a human Fc region; more preferably, the Fc region is the Fc region of an IgG1, IgG2, IgG3, or IgG4 antibody; Preferably, the first arm of the CD20 target comprises 1) SEQ ID NO:9 and SEQ ID NO:10, or 2) SEQ ID NO:11 and SEQ ID NO:12; the second arm comprises SEQ ID NO:13 or SEQ ID NO:

14.

7. The pharmaceutical composition, application, or combination according to any one of claims 1-6, wherein the cancer includes hematologic malignancies, such as lymphoma, comprising: Hodgkin's lymphoma and non-Hodgkin's lymphoma; More preferably, non-Hodgkin's lymphoma, including: diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, and mucosa-associated lymphoid tissue lymphoma; most preferably, diffuse large B-cell lymphoma; or The cancers mentioned are CD20-positive cancers, such as CD20-positive non-Hodgkin lymphomas (e.g., diffuse large B-cell lymphoma).

8. The application or combination of any one of claims 2-7, wherein the CD20 / CD47 dual-blocking bifunctional fusion protein and the anticancer agent are formulated together or exist physically independently; Preferably, the CD20 / CD47 dual-blocking bifunctional fusion protein is administered before, simultaneously with, or after the anticancer agent.

9. The application or combination of any one of claims 2-8, wherein the dosage of the bifunctional fusion protein is 1 mg / kg to 40 mg / kg, for example 1 mg / kg, 3 mg / kg, 6 mg / kg, 12 mg / kg, 20 mg / kg, 30 mg / kg or 40 mg / kg; and / or The dosage of the anticancer agent is 15-25 mg / day, for example, 15 mg / day, 20 mg / day, 23 mg / day or 25 mg / day.

10. The application or combination of any one of claims 2-9, wherein the CD20 / CD47 dual-blocking bifunctional fusion protein is administered at least once every two weeks, for example, once every two weeks, once every three weeks, once every four weeks, once every five weeks, or once every six weeks, preferably once every two weeks, once every three weeks, or once every four weeks; or administered more than once a week, for example, twice a week; preferably, administered at a frequency of once every two weeks, once every three weeks, or once every four weeks; and / or The anticancer agent is administered once daily for 21 consecutive days; preferably once daily on days 2-22 of each cycle; and / or The administration methods for the CD20 / CD47 dual-blocking bifunctional fusion protein and the anticancer agent are independently inhalation, intranasal, oral, intravenous, subcutaneous or intramuscular injection.

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