Combination medicine containing CD20 / CD47 blocking bifunctional fusion protein and application thereof

Abstract

The invention provides a method and application for treating cancer by combining a CD20 / CD47 double-blocking bifunctional fusion protein with a bifunctional alkylating agent, in particular a method and application for treating cancer (particularly non-Hodgkin's lymphoma) by combining the CD20 / CD47 double-blocking bifunctional fusion protein with bendamustine or a salt thereof.

Description

Combination drug containing CD20 / CD47 blocking bifunctional fusion protein and its use

[0001] Related applications

[0002] This application claims priority to Chinese patent application No. 202310194436.8, filed on March 1, 2023, entitled “Combination drug comprising CD20 / CD47 blocking bifunctional fusion protein and its use”, and the full text of the patent application is incorporated herein by reference for all purposes. Technical Field

[0003] The present application relates to the field of biopharmaceuticals, and specifically to methods and applications of combining a CD20 / CD47 dual-blocking bifunctional fusion protein with a bifunctional alkylating agent for the treatment of cancer, especially methods and applications of combining a CD20 / CD47 dual-blocking bifunctional fusion protein with bendamustine or its salts for the treatment of cancer (particularly non-Hodgkin's lymphoma). Background Art

[0004] Non-Hodgkin lymphoma (NHL) is predominantly B-cell, accounting for 70%-85% of all cases. There are four main B-cell subtypes: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MC), and mucosa-associated lymphoid tissue lymphoma (MAL). DLBCL is the most common clinical manifestation. Clinically, lymphadenopathy is the most common local manifestation. In recent years, the incidence of lymphoma in my country has gradually increased, currently reaching nearly 7 per 100,000 people per year.

[0005] Currently, R-CHOP (a combination therapy consisting of five drugs: R stands for rituximab injection, C for cyclophosphamide, H for doxorubicin, O for vincristine, and P for prednisone) is the first-line treatment for patients with non-Hodgkin's lymphoma, including diffuse large B-cell lymphoma. Compared to the traditional CHOP regimen, the addition of rituximab improves patients' five-year median overall survival from 45% to 58%, demonstrating that immunochemotherapy can further improve patient outcomes beyond existing chemotherapy regimens.

[0006] The mechanism of action of rituximab includes CDC (complement-dependent cytotoxicity), ADCC (antibody-dependent cell-mediated cytotoxicity) and ADCP (antibody-dependent cell-mediated phagocytosis), among which CDC plays a major role.

[0007] The reasons for non-Hodgkin's lymphoma resistance or recurrence after R-CHOP treatment may involve both the four chemotherapy drugs of CHOP and rituximab.

[0008] Bendamustine is a bifunctional alkylating agent that can overcome the cross-resistance of previous alkylating agents (such as cyclophosphamide). Bendamustine was developed in combination with rituximab and has been approved for the second-line treatment of non-Hodgkin's lymphoma (NHL), achieving good therapeutic effects.

[0009] At the same time, research has discovered that CD47 is a new immune checkpoint that can bind to the SIRPα protein on macrophages, thereby preventing macrophage phagocytosis. Blocking the binding of CD47 and SIRPα can promote macrophage phagocytosis. Currently, several anti-CD47 monoclonal antibodies have been developed, and research has further demonstrated that the combination of anti-CD47 monoclonal antibodies and rituximab can further enhance macrophage phagocytosis. However, existing treatment options still cannot meet the treatment needs of patients with this disease.

[0010] Summary of the Invention

[0011] In previous studies, the inventors of this application developed a new generation of CD20 / CD47 dual-blocking bifunctional fusion proteins that significantly outperformed SIRPα-Fc fusion proteins in blocking the interaction between CD47 on tumor cells and SIRPα expressed on macrophages. Further research by the inventors demonstrated that this fusion protein significantly enhances macrophage phagocytosis compared to rituximab, SIRPα fusion protein, and ofavumab, and also exhibited significant therapeutic effects in rituximab-resistant diffuse large B-cell lymphoma (DLBCL) xenograft models.

[0012] On this basis, the inventors of the present application, through extensive research and exploration, have discovered and provided a combination drug for preventing or treating cancer, comprising a CD20 / CD47 dual-blocking bifunctional fusion protein and a bifunctional alkylating agent, particularly the bifunctional alkylating agent bendamustine (including bendamustine hydrochloride), as well as uses of the combination drug in treating cancer and preparing a drug for treating cancer. In particular, it was discovered that the combination of the CD20 / CD47 dual-blocking bifunctional fusion protein and a bifunctional alkylating agent, particularly the bifunctional alkylating agent bendamustine (including bendamustine hydrochloride), exhibits a highly significant synergistic effect in treating lymphoma, particularly diffuse large B-cell lymphoma.

[0013] Specifically, the present application provides a pharmaceutical composition for preventing or treating cancer, comprising a CD20 / CD47 dual-blocking bifunctional fusion protein and a bifunctional alkylating agent.

[0014] As used herein, the terms "pharmaceutical composition", "combination drug", "drug combination", "combination drug" and the like are used interchangeably and refer to a combination of at least one drug and an optional pharmaceutically acceptable carrier or excipient that are combined together to achieve a specific purpose. In certain embodiments, the pharmaceutical composition includes a combination separated in time and / or space, as long as they can work together to achieve the purpose of the present application. For example, the ingredients contained in the pharmaceutical composition (e.g., the fusion protein, bifunctional alkylating agent according to the present application) can be administered to the subject as a whole, or separately. When the ingredients contained in the pharmaceutical composition are administered separately to the subject, the ingredients can be administered to the subject simultaneously or sequentially. For example, the pharmaceutically acceptable carrier can be water, a buffered aqueous solution, an isotonic saline solution such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol or a polyalkylene glycol such as polypropylene glycol, triglyceride, etc. The type of pharmaceutically acceptable carrier used depends inter alia on whether the composition according to the present application is formulated for inhaled, intranasal, oral, intravenous, subcutaneous or intramuscular administration.The composition according to the present application may contain wetting agents, emulsifiers or buffer substances as additives.

[0015] Cancers / tumors treated herein include, but are not limited to, lymphoma, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, esophageal cancer, endometrial cancer, ovarian cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, myeloma, leukemia, thyroid cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protrusio, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, or myelodysplastic syndrome. In some embodiments, the cancer is a CD20-positive tumor. In some embodiments, the cancer is a lymphoma. In some embodiments, the cancer is Hodgkin lymphoma or non-Hodgkin lymphoma. In some embodiments, the cancer is non-Hodgkin 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 lymphoma. In some embodiments, the cancer is CD20-positive diffuse large B-cell lymphoma.

[0016] The CD20 / CD47 dual-blocking bifunctional fusion protein described in this application comprises two functional parts that block CD20 and CD47, respectively. In some examples of this application, the two functional parts are also referred to as two "arms," ​​which can form a heterodimer in a homodimeric structure similar to a natural antibody. Those skilled in the art will appreciate that the bifunctional fusion protein of this application is not limited to a two-arm form, but can also be a four-arm or other form, similar to the various configurations of bispecific antibodies in the art.

[0017] The term "antibody" as used herein broadly encompasses any form of molecule that can specifically bind to an antigen, including intact antibodies and antigen-binding fragments thereof ("antigen-binding portion") or single-chain forms. The term "antigen-binding portion" or "antigen-binding fragment" may particularly refer to antibody fragments, such as Fv, scFv, Fab, F(ab')2, Fab', VHH, etc.

[0018] In some embodiments, the functional portion / arm that blocks CD20 is in the form of an antibody to CD20, or an antigen-binding portion thereof.

[0019] In some embodiments, the functional part / arm that blocks CD47 is an antibody or antigen binding portion thereof to CD47 and / or a natural ligand of CD47 or a ligand-active portion derived from the natural ligand (wild type or mutant). In some embodiments, the natural ligand of CD47 is SIRPα protein. SIRPα protein is a transmembrane protein, so the main function of its ligand activity is the extracellular domain. Therefore, in some embodiments, the ligand-active portion of SIRPα protein comprises the complete extracellular domain of SIRPα protein. In some embodiments, the ligand-active portion of SIRPα protein is an extracellular truncation of SIRPα protein. In some embodiments, the ligand-active portion of the SIRPα protein is the extracellular D1 domain of the SIRPα protein (e.g., the amino acid sequence shown at positions 20-136 of SEQ ID NO: 6) or a non-high-affinity mutant thereof (e.g., the amino acid sequence shown at positions 20-136 of SEQ ID NO: 3, which comprises an N99A mutation relative to SEQ ID NO: 6 (i.e., an N80A mutation relative to the extracellular D1 domain itself)). In some embodiments, it is more ideal for the functional portion / arm that blocks CD47 to have a lower affinity for CD47, and thus a non-high-affinity mutant (i.e., a mutation that does not result in other desired effects of increased affinity) is desirable. In addition to the extracellular D1 domain of the SIRPα protein, SEQ ID NO: 3 also comprises a signal peptide and an Fc region for forming a dimer with SEQ ID NO: 1, as described in detail below.

[0020] In some embodiments of the two-armed CD47 / CD20 dual-target fusion protein, the first arm targets CD20 with high affinity, and the second arm blocks the interaction between CD47 and SIRPα with low affinity. In some specific embodiments, the first arm acts in the form of an antibody, and the second arm acts in the form of a natural ligand for CD47 or a ligand-active portion derived from the natural ligand, such as an extracellular truncation of SIRPα or a non-high-affinity mutant thereof. The "high affinity" and "low affinity" described herein describe the relative relationship between the affinity of the dual-target fusion protein of the present application for the CD47 protein and the CD20 protein. For example, the affinity level of the dual-target fusion protein for the CD47 protein and the CD20 protein can be measured at the protein level, for example, by measuring the Kd value, and the affinity is determined by comparing the Kd values. In some specific embodiments, the binding affinity of the CD47 / CD20 dual-target fusion protein for the CD20 protein is at least 6 times that of the CD47 / CD20 dual-target fusion protein for the CD47 protein. In some embodiments, the two-armed CD47 / CD20 dual-targeting fusion protein is in the form of left and right arms similar to a natural antibody (refer to the general antibody structure diagram, which is different from the possible upper and lower arm structures). Such embodiments are also described below using left and right arms. In some specific embodiments, the left arm targets CD20 with high affinity, and the right arm blocks the interaction between CD47 and SIRPα with low affinity.

[0021] In some embodiments, the first arm and / or the second arm is in the form of an immunoglobulin Fab or Fab'. In some embodiments, the first arm or left arm is in the form of an immunoglobulin Fab or Fab', and the second arm or right arm comprises an extracellular truncation of SIRPα or a non-high affinity mutant thereof. In some embodiments, the first arm or left arm comprises the amino acid sequence shown at positions 20-244 of SEQ ID NO: 1 and the amino acid sequence shown at positions 21-234 of SEQ ID NO: 2, and the second arm or right arm comprises the amino acid sequence shown at positions 20-136 of SEQ ID NO: 3 or 6. In some embodiments, the first arm or left arm comprises the amino acid sequence shown at positions 20-241 of SEQ ID NO: 4 and the amino acid sequence shown at positions 21-239 of SEQ ID NO: 5, and the second arm or right arm comprises the amino acid sequence shown at positions 20-136 of SEQ ID NO: 3 or 6. SEQ ID NOs: 1 and 2 respectively show the heavy chain and light chain amino acid sequences of an exemplary antibody targeting CD20 (each with a signal peptide). SEQ ID NOs: 4 and 5 show the heavy chain and light chain amino acid sequences, respectively, of another exemplary antibody targeting CD20 (with signal peptides, respectively).

[0022] In some embodiments, the bifunctional fusion protein further comprises an Fc region. In some embodiments, each of the two arms of the bifunctional fusion protein comprises an Fc region, and heterodimers are formed through the interaction of the Fc regions. In some embodiments, the Fc region is wild-type. In some embodiments, the Fc region comprises a knobs-into-holes mutation. Knobs-into-holes mutations in the Fc region are a commonly used technique in the art to improve dimer assembly. As an example, the Fc regions comprised by SEQ ID NO:1 and SEQ ID NO:3 each comprise an engineered knobs-into-holes mutation. In some embodiments, the first or left arm of the bifunctional fusion protein comprises the amino acid sequence set forth at positions 20-471 of SEQ ID NO:1 and the amino acid sequence set forth at positions 21-234 of SEQ ID NO:2, and the second or right arm comprises the amino acid sequence set forth at positions 20-363 of SEQ ID NO:3 or 6. In some embodiments, the first arm or left arm of the bifunctional fusion protein comprises the amino acid sequence shown at positions 20-468 of SEQ ID NO: 4 and the amino acid sequence shown at positions 21-239 of SEQ ID NO: 5, and the second arm comprises the amino acid sequence shown at positions 20-363 of SEQ ID NO: 3 or 6. As shown in the sequence structures of SEQ ID NO: 1-6 listed at the end of the specification, positions 1-19 of SEQ ID NO: 1, positions 1-20 of SEQ ID NO: 2, positions 1-19 of SEQ ID NO: 3, positions 1-19 of SEQ ID NO: 4, positions 1-20 of SEQ ID NO: 5, and positions 1-19 of SEQ ID NO: 6 are signal peptides, which can be replaced with signal peptides of other structures or omitted.

[0023] In some embodiments, the first or left arm comprises the amino acid sequence set forth in SEQ ID NO:1 and SEQ ID NO:2, and the second or right arm comprises the amino acid sequence set forth in SEQ ID NO:3.

[0024] The bifunctional alkylating agent described in this application is a bifunctional derivative of nitrogen mustard. In some embodiments, the bifunctional alkylating agent is bendamustine or a pharmaceutically acceptable salt, solvate or prodrug thereof, chlorambucil, or (L-rotatory) phenylalanine mustard. In some embodiments, the bifunctional alkylating agent is bendamustine hydrochloride.

[0025] The present application also provides the use of the above-mentioned CD20 / CD47 dual-blocking bifunctional fusion protein and bifunctional alkylating agent in the preparation of a pharmaceutical composition for treating cancer (preferably CD20-positive tumors).

[0026] The present application also provides a combination comprising the above-mentioned CD20 / CD47 dual-blocking bifunctional fusion protein and a bifunctional alkylating agent, wherein the combination is used for treating cancer (preferably CD20-positive tumors).

[0027] The present application also provides a method for treating cancer (preferably a CD20-positive tumor) in an individual, comprising administering to the individual in need thereof a therapeutically effective amount of the CD20 / CD47 dual-blocking bifunctional fusion protein and a bifunctional alkylating agent described above.

[0028] In some embodiments of the above-mentioned uses, combinations, or methods, the CD20 / CD47 dual-blocking bifunctional fusion protein and the bifunctional alkylating agent are formulated together or physically separate (e.g., as different units of a kit or pharmaceutical set). The CD20 / CD47 dual-blocking bifunctional fusion protein can be formulated for administration before, simultaneously with, or after the bifunctional alkylating agent.

[0029] In some embodiments, the bifunctional fusion protein is administered at a dosage of 0.3 mg / kg to 20 mg / kg, such as 0.5 mg / kg, 2 mg / kg, 5 mg / kg, 10 mg / kg, or 15 mg / kg. In some embodiments, the bifunctional fusion protein is administered at least once every two weeks, such as once every two weeks, once every three weeks, or once every four weeks, preferably once every two weeks or once every three weeks; or the administration frequency may be multiple times a week, such as twice a week.

[0030] In some embodiments, the bifunctional alkylating agent (e.g., bendamustine (including bendamustine hydrochloride)) is administered at a dosage of 5-40 mg / kg, for example, 5 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, or 40 mg / kg. In some embodiments, the bifunctional alkylating agent (e.g., bendamustine (including bendamustine hydrochloride)) is administered at least once every two weeks, for example, once every two weeks, once every three weeks, or once every four weeks, preferably once every two weeks or once every three weeks; or the administration frequency may be multiple times a week, for example, twice a week.

[0031] In some embodiments, the CD20 / CD47 dual-blocking bifunctional fusion protein and the bifunctional alkylating agent are independently administered by inhalation, intranasal, oral, intravenous, subcutaneous or intramuscular injection.

[0032] 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 full text of which is incorporated into this application by reference.

[0033] An example of a CD20 / CD47 dual-blocking bifunctional fusion protein described in the present application comprises a left arm consisting of the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 that targets CD20 with high affinity, and a right arm having an amino acid sequence as shown in SEQ ID NO: 3 that blocks the interaction between CD47 and SIRPα with low affinity. The CD20 / CD47 dual-blocking bifunctional fusion protein is referred to as JMT601 in the present application. Another example of a CD20 / CD47 dual-blocking bifunctional fusion protein described in the present application comprises a left arm consisting of the amino acid sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5 that targets CD20 with high affinity, and a right arm having an amino acid sequence as shown in SEQ ID NO: 3 or 6 that blocks the interaction between CD47 and SIRPα with low affinity.

[0034] 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.

[0035] BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1. Tumor volume change curves in each treatment group in the WSU-DLCL2 human lymphoma subcutaneous model.

[0037] Note: Data are expressed as mean ± standard error.

[0038] Figure 2. Changes in body weight of experimental mice in each treatment group over treatment time in the WSU-DLCL2 human lymphoma subcutaneous model.

[0039] Note: Data are expressed as mean ± standard error.

[0040] Figure 3. Schematic diagram of the structure of an embodiment of the CD20 / CD47 dual-blocking bifunctional fusion protein of the present application, a representative example of which is JMT601, wherein the left arm is a single-arm form of a natural antibody targeting CD20, and the Fc region contains a hole mutation; the right arm is the extracellular D1 domain of the SIRPα protein, and the Fc region contains a knob mutation. Example

[0041] Example 1: Pharmacodynamic Study of Combination Therapy in a WSU-DLCL2 Human Lymphoma Subcutaneously Transplanted NOD SCID Mouse Model

[0042] 1. Experimental Materials and Purpose

[0043] The main experimental materials used in the experiment are as follows:

[0044] Table 1: Main experimental materials and reagents used

[0045] This study used the WSU-DLCL2 human lymphoma subcutaneous xenograft model to evaluate the efficacy of test drug combinations in NOD SCID mice. The pharmacodynamic study design strategy is as follows:

[0046] Table 2: Dosage regimen Note: 1. BIW = twice a week, N = number of animals; 2. Dosing volume is 10 μl / g

[0047] 2. Experimental Methods

[0048] 2.1 Tumor inoculation and grouping

[0049] WSU-DLCL2 cells (human diffuse large B-cell lymphoma cells) (Cell No. CL-00648, Manufacturer: Crownbio) were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum. WSU-DLCL2 cells were harvested during the exponential growth phase, resuspended in PBS to an appropriate concentration, and then used for subcutaneous tumor inoculation in mice.

[0050] 1×10 7 WSU-DLCL2 cells were resuspended in PBS and Matrigel (1:1) (0.1 ml / mouse) and inoculated subcutaneously in experimental mice. The inoculation date was May 20, 2020. When the average tumor volume reached 150.53 mm 3 Patients were randomly divided into groups according to tumor size (see Table 2 for details).

[0051] 2.2 Preparation of test and control drugs

[0052] Table 3: Drug preparation method and storage conditions

[0053] 2.3 Observation

[0054] All animal experiment protocols for this study were reviewed and approved by the CrownBio IACUC Committee. Animal experimental procedures were performed in accordance with AAALAC guidelines. Following tumor inoculation, routine monitoring included monitoring of tumor growth and the effects of treatment on normal animal behavior, including activity, food and water intake, weight gain or loss, and any abnormalities of the eyes, coat, or other areas. Any clinical signs observed during the experiment were recorded in the original data.

[0055] The data were collected using StudyDirector™ (version 3.1.399.19, provided by Studylog System, Inc., San Francisco, CA, USA), including the measurement of the long and short diameters of the tumor and the weighing of the animal body weight.

[0056] The experiment was ended 21 days after drug administration. The tumor-bearing mice were photographed, and the tumors were weighed and photographed.

[0057] 2.4 Data Analysis

[0058] To compare different treatment groups, one-way ANOVA was performed while confirming homogeneity of variance. If homogeneity of variance was confirmed, pairwise comparisons were performed using the Dunnnett's test. If heterogeneity of variance was confirmed, one-way ANOVA was performed using the nonparametric Kruskal-Wallis test, while pairwise comparisons were performed using the Dunn's test. All statistical analyses and graphics were performed using GraphPad Prism 8.4.2, and p-values ​​less than 0.05 were considered statistically significant.

[0059] 3. Experimental Results

[0060] The average tumor volume of mice in the control group (IgG1 10 mg / kg group) was 1629.39 mm2 21 days after administration. 3 The tumor volume doubling time was 6 days. The 21st day after group administration was used as the evaluation point for drug treatment effect, and the relative tumor volume of the test group and the control group were compared to see if there was a significant difference. 21 days after group administration, the average tumor volume of mice in the rituximab 10mg / kg group was 1121.67mm 3 The tumor inhibition rate was 33.39%, which was not significantly different from the control group (p value>0.9999), and the tumor volume doubling time was 8 days. The average tumor volume of mice in the JMT601 3.3mg / kg group was 825.07mm 3 The tumor inhibition rate was 50.00%, which was not significantly different from the control group (p value>0.9999), and the tumor volume doubling time was 9 days. The average tumor volume of mice in the JMT601 10mg / kg group was 264.67mm 3 The tumor inhibition rate was 84.69%, which was significantly different from the control group (p value was 0.0002). The tumor volume doubling time was 27 days (calculated from existing data). The average tumor volume of mice in the bendamustine 30mg / kg group was 945.10mm 3The tumor inhibition rate was 44.00%, which was not significantly different from the control group (p value>0.9999), and the tumor volume doubling time was 8 days. The average tumor volume of mice in the bendamustine 30mg / kg combined with JMT601 3.3mg / kg group was 178.85mm 3 The tumor inhibition rate was 89.02%, which was significantly different from the control group (p value <0.0001), and the tumor volume doubling time was 88 days (calculated from existing data).

[0061] The analysis results of tumor weight were basically consistent with those of relative tumor volume.

[0062] The tumor growth inhibition of mice in each treatment group and control group is shown in Table 4 and Figure 1.

[0063]

[0064] Example 2: Safety Study Results and Discussion of Test Drugs in the WSU-DLCL2 Human Lymphoma Subcutaneous Transplant Model

[0065] In this study, one mouse (#4174) in the JMT601 3.3 mg / kg plus bendamustine 30 mg / kg group experienced a body weight loss greater than 20% on days 15, 18, and 19 after dosing, reaching the humane endpoint and requiring euthanasia. The weight loss in all mice was found to be due to decreased appetite. Autopsy results revealed no significant abnormalities, suggesting that the weight loss was due to individual differences in the mice. All other mice in each group tolerated all tested drugs well.

[0066] The changes in body weight of the treatment group and the control group after administration are shown in Table 5 and Figure 2.

[0067] Table 5: Medication effect data

[0068] The above results show that tumor-bearing mice can tolerate treatment with rituximab (10 mg / kg), JMT601 (3.3 mg / kg and 10 mg / kg), and bendamustine (30 mg / kg). There is no significant difference in tolerability between the combination therapy and the single drug, reflecting the good safety of the combination therapy.

[0069] The fusion protein sequences involved in this application are as follows:

[0070] SEQ ID NO.1

[0071] SEQ ID NO.2

[0072] SEQ ID NO.3 (the underlined and bold "A" indicates the position of the N80A mutation)

[0073] SEQ ID NO.4

[0074] SEQ ID NO.5

[0075] SEQ ID NO.6 (underlined and bold "N" represents the wild-type D1 domain residue) Note: The underlined part is the signal peptide sequence, which can be replaced as needed

[0076] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that, based on all the teachings disclosed, various modifications and substitutions can be made to those details, and these changes are all within the scope of protection of the present application. The full scope of the present application is given by the appended claims and any equivalents thereof.

Claims

1. A pharmaceutical composition for treating cancer, comprising a CD20 / CD47 dual-blocking bifunctional fusion protein and a bifunctional alkylating agent.

2. The pharmaceutical composition according to claim 1, wherein the functional part of the CD20-blocking bifunctional fusion protein of CD20 / CD47 dual blocking is an antibody against CD20 or an antigen-binding portion thereof; and / or The functional portion that blocks CD47 in the CD20 / CD47 dual-blocking bifunctional fusion protein is an antibody against CD47 or an antigen-binding portion thereof and / or a natural ligand of CD47 (e.g., SIRPα protein) or a ligand-active portion derived from the natural ligand (e.g., SIRPα protein); Optionally, the functional part that blocks CD20 and the functional part that blocks CD47 each comprise an Fc region, such as a wild-type Fc region or an Fc region comprising mutations based on Knobs-into-holes technology. 3 . The pharmaceutical composition according to claim 2 , wherein the ligand active portion is the complete extracellular domain, extracellular truncation or non-high affinity mutant of SIRPα protein.

4. A pharmaceutical composition as described in any one of claims 1 to 3, wherein the CD20 / CD47 dual-blocking bifunctional fusion protein is a fusion protein containing a first arm that targets CD20 with high affinity (e.g., a first arm in the form of an antibody) and a second arm that blocks the interaction between CD47 and SIRPα with low affinity (e.g., a CD47 natural ligand or a second arm in the form of a ligand-active portion derived from the natural ligand); preferably, the binding affinity of the fusion protein to the CD20 protein is at least 6 times that to the CD47 protein.

5. The pharmaceutical composition of claim 4, wherein The first arm comprises the amino acid sequence shown at positions 20-471 of SEQ ID NO: 1 and the amino acid sequence shown at positions 21-234 of SEQ ID NO: 2, and the second arm comprises the amino acid sequence shown at positions 20-363 of SEQ ID NO: 3 or 6; or The first arm comprises the amino acid sequence shown at positions 20-244 of SEQ ID NO: 1 and the amino acid sequence shown at positions 21-234 of SEQ ID NO: 2, and the second arm comprises the amino acid sequence shown at positions 20-136 of SEQ ID NO: 3 or 6; or The first arm comprises the amino acid sequence shown at positions 20-468 of SEQ ID NO:4 and the amino acid sequence shown at positions 21-239 of SEQ ID NO:5, and the second arm comprises the amino acid sequence shown at positions 20-363 of SEQ ID NO:3 or 6; or The first arm comprises the amino acid sequence shown at positions 20-241 of SEQ ID NO:4 and the amino acid sequence shown at positions 21-239 of SEQ ID NO:5, and the second arm comprises the amino acid sequence shown at positions 20-136 of SEQ ID NO:3 or 6.

6. The pharmaceutical composition of claim 4, wherein the first arm comprises the amino acid sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2, and the second arm comprises the amino acid sequence shown in SEQ ID NO:

3.

7. The pharmaceutical composition of claim 4, wherein the first arm consists of the amino acid sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2, and the second arm consists of the amino acid sequence shown in SEQ ID NO:

3.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the bifunctional alkylating agent is a bifunctional derivative of a nitrogen mustard, for example, bendamustine or a pharmaceutically acceptable salt, solvate or prodrug thereof, chlorambucil, or (L-rotatory)phenylalanine mustard; preferably bendamustine or a pharmaceutically acceptable salt, solvate or prodrug thereof.

9. The pharmaceutical composition of claim 8, wherein the bifunctional alkylating agent is bendamustine hydrochloride.

10. The pharmaceutical composition of any one of claims 1 to 9, wherein the cancer is selected from lymphoma, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, esophageal cancer, endometrial cancer, ovarian cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, myeloma, leukemia, thyroid cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, cutaneous fibrosarcoma protrusion, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma or myelodysplastic syndrome; preferably lymphoma, which includes: Hodgkin's lymphoma and non-Hodgkin's lymphoma; more preferably non-Hodgkin's lymphoma, which includes diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, mucosa-associated lymphoid tissue lymphoma; most preferably diffuse large B-cell lymphoma.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the cancer is a CD20-positive cancer, such as a CD20-positive non-Hodgkin's lymphoma (such as diffuse large B-cell lymphoma).

12. Use of a CD20 / CD47 dual-blocking bifunctional fusion protein and a bifunctional alkylating agent in the preparation of a pharmaceutical composition for treating cancer.

13. A combination of a CD20 / CD47 dual-blocking bifunctional fusion protein and a bifunctional alkylating agent for use in treating cancer.

14. A method of treating cancer in an individual, the method comprising administering to an individual in need thereof a therapeutically effective amount of a CD20 / CD47 dual-blocking bifunctional fusion protein and a bifunctional alkylating agent.

15. The use according to claim 12 or the combination of uses according to claim 13 or the method according to claim 14, wherein the bifunctional alkylating agent is a bifunctional derivative of a nitrogen mustard, for example, bendamustine or a pharmaceutically acceptable salt, solvate or prodrug thereof, chlorambucil, or (L-rotatory) phenylalanine mustard; preferably bendamustine or a pharmaceutically acceptable salt, solvate or prodrug thereof; more preferably bendamustine hydrochloride.

16. The use or combination or method according to any one of claims 12 to 15, wherein the functional part that blocks CD20 in the CD20 / CD47 dual-blocking bifunctional fusion protein is an antibody or an antigen-binding part thereof against CD20; and / or The functional portion that blocks CD47 in the CD20 / CD47 dual-blocking bifunctional fusion protein is an antibody against CD47 or an antigen-binding portion thereof and / or a natural ligand of CD47 (e.g., SIRPα protein) or a ligand-active portion derived from the natural ligand (e.g., SIRPα protein); Optionally, the functional part that blocks CD20 and the functional part that blocks CD47 each comprise an Fc region, such as a wild-type Fc region or an Fc region comprising mutations based on Knobs-into-holes technology.

17. The use or combination or method according to claim 16, wherein the ligand active portion is the complete extracellular domain, extracellular truncation or non-high affinity mutant of SIRPα protein.

18. The use or combination or method according to any one of claims 12 to 17, wherein the CD20 / CD47 dual-blocking bifunctional fusion protein is a fusion protein comprising a first arm that targets CD20 with high affinity (e.g., a first arm in the form of an antibody) and a second arm that blocks the interaction between CD47 and SIRPα with low affinity (e.g., a CD47 natural ligand or a second arm in the form of a ligand-active portion derived from the natural ligand); preferably, the binding affinity of the fusion protein to the CD20 protein is at least 6 times that to the CD47 protein.

19. The use or combination or method of claim 18, wherein The first arm comprises the amino acid sequence shown at positions 20-471 of SEQ ID NO: 1 and the amino acid sequence shown at positions 21-234 of SEQ ID NO: 2, and the second arm comprises the amino acid sequence shown at positions 20-363 of SEQ ID NO: 3 or 6; or The first arm comprises the amino acid sequence shown at positions 20-244 of SEQ ID NO: 1 and the amino acid sequence shown at positions 21-234 of SEQ ID NO: 2, and the second arm comprises the amino acid sequence shown at positions 20-136 of SEQ ID NO: 3 or 6; or The first arm comprises the amino acid sequence shown at positions 20-468 of SEQ ID NO:4 and the amino acid sequence shown at positions 21-239 of SEQ ID NO:5, and the second arm comprises the amino acid sequence shown at positions 20-363 of SEQ ID NO:3 or 6; or The first arm comprises the amino acid sequence shown at positions 20-241 of SEQ ID NO:4 and the amino acid sequence shown at positions 21-239 of SEQ ID NO:5, and the second arm comprises the amino acid sequence shown at positions 20-136 of SEQ ID NO:3 or 6.

20. The use or combination or method of claim 18, wherein the first arm comprises the amino acid sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2, and the second arm comprises the amino acid sequence shown in SEQ ID NO:

3.

21. The use or combination or method of claim 18, wherein the first arm consists of the amino acid sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2, and the second arm consists of the amino acid sequence shown in SEQ ID NO:

3.

22. The use or combination or method of any one of claims 12-21, wherein the cancer is selected from lymphoma, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, esophageal cancer, endometrial cancer, ovarian cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, myeloma, leukemia, thyroid cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, cutaneous fibrosarcoma protrusion, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma or myelodysplastic syndrome; preferably lymphoma, which includes: Hodgkin's lymphoma and non-Hodgkin's lymphoma; more preferably non-Hodgkin's lymphoma, which includes diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, mucosa-associated lymphoid tissue lymphoma; most preferably diffuse large B-cell lymphoma.

23. The use or combination or method of any one of claims 12 to 22, wherein the cancer is a CD20-positive cancer, such as a CD20-positive non-Hodgkin's lymphoma (such as diffuse large B-cell lymphoma).

24. The use or combination or method according to any one of claims 12 to 23, wherein the CD20 / CD47 dual-blocking bifunctional fusion protein and the bifunctional alkylating agent are formulated together or exist physically separately.

25. The use or combination or method according to any one of claims 12 to 23, wherein the CD20 / CD47 dual-blocking bifunctional fusion protein is administered before, simultaneously with or after the bifunctional alkylating agent.

26. The use or combination or method according to any one of claims 12 to 25, wherein the bifunctional fusion protein is administered at a dose of 0.3 mg / kg to 20 mg / kg, such as 0.5 mg / kg, 2 mg / kg, 5 mg / kg, 10 mg / kg or 15 mg / kg.

27. The use or combination or method of any one of claims 12 to 26, wherein the bifunctional alkylating agent is administered at a dose of 5-40 mg / kg, for example, 5 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg or 40 mg / kg.

28. The use or combination or method according to any one of claims 12 to 27, wherein the CD20 / CD47 dual-blocking bifunctional fusion protein and the bifunctional alkylating agent are independently administered at least once every 2 weeks, such as once every 2 weeks, once every 3 weeks or once every 4 weeks, preferably once every 2 weeks or once every 3 weeks; or the administration frequency is independently more than once a week, such as twice a week.

29. The use or combination or method according to any one of claims 12 to 28, wherein the CD20 / CD47 dual-blocking bifunctional fusion protein and the bifunctional alkylating agent are independently administered by inhalation, intranasal, oral, intravenous, subcutaneous or intramuscular injection.

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