Combination medicaments comprising CD20 / CD47 blocking bifunctional fusion proteins and uses thereof

Abstract

The present invention relates to a method and use of a CD20 / CD47 dual blocking bifunctional fusion protein in combination with a bifunctional alkylating agent to treat cancer, in particular to a method and use of a CD20 / CD47 dual blocking bifunctional fusion protein in combination with bendamustine or a salt thereof to treat cancer (in particular non-Hodgkin's lymphoma).

Description

[Technical Field]

[0001] The present invention relates to the field of biopharmaceuticals. More specifically, the present invention relates to a method for treating cancer by combining a CD20 / CD47 dual-blocking bifunctional fusion protein (bifunctional fusion protein) with a bifunctional alkylating agent (bifunctional alkylating agent), and related uses, in particular to a method for treating cancer (particularly non-Hodgkin's lymphoma) by combining a CD20 / CD47 dual-blocking bifunctional fusion protein with bendamustine or a salt thereof, and related uses.

[0002] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to Chinese Patent Application No. 202310194436.8, filed on March 1, 2023, entitled "Combined Medicine Comprising a CD20 / CD47 Blocking Bifunctional Fusion Protein and Use Thereof," which is incorporated herein by reference in its entirety for all purposes. [Background technology]

[0003] Non-Hodgkin's lymphoma (NHL) is primarily a B-cell type, accounting for 70% to 85% of all cases. There are four main types of B-cell lymphoma: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MC), and mucosa-associated lymphoid tissue lymphoma (MAL). Among these, diffuse large B-cell lymphoma is the most common. Clinically, lymphadenopathy is the most common localized clinical symptom. The incidence of lymphoma in China has gradually increased in recent years, and the current incidence rate is approximately 7 cases per 100,000 people per year.

[0004] Currently, R-CHOP (R: Rituxan (rituximab injection), C: cyclophosphamide, H: doxorubicin, O: vincristine, and P: prednisone) is a five-drug combination therapy that is the first choice for treating patients with non-Hodgkin's lymphoma, including diffuse large B-cell lymphoma. Compared with the conventional CHOP regimen, the R-CHOP regimen with the addition of rituximab increased the 5-year median overall survival from 45% to 58%, demonstrating that immunochemotherapy regimens can further improve patient outcomes compared to conventional chemotherapy regimens.

[0005] 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 an important role.

[0006] The causes of drug resistance or relapse of non-Hodgkin's lymphoma after R-CHOP treatment may be related to either the four chemotherapy drugs in CHOP or rituximab.

[0007] Bendamustine is a bifunctional alkylating agent that can overcome the cross-resistance of conventional alkylating agents (e.g., cyclophosphamide). Bendamustine has been developed in combination with rituximab and approved as a second-line treatment for non-Hodgkin's lymphoma (NHL), demonstrating favorable therapeutic effects.

[0008] At the same time, this study has identified CD47 as a novel immune checkpoint that binds to SIRPα on macrophages, thereby preventing macrophage phagocytosis. Blocking the binding of CD47 to SIRPα can promote macrophage phagocytosis. Several anti-CD47 monoclonal antibodies have been developed. Research has further shown that combining anti-CD47 monoclonal antibodies with rituximab can further enhance macrophage phagocytosis. However, existing treatment regimens still cannot meet the therapeutic needs of relevant patients. Summary of the Invention

[0009] In previous studies, the inventors developed a new generation of CD20 / CD47 dual-blocking bifunctional fusion protein that is significantly more effective than SIRPα-Fc fusion proteins in blocking the interaction between CD47 on tumor cells and SIRPα expressed on macrophages. Further studies by the inventors demonstrated that the CD20 / CD47 dual-blocking bifunctional fusion protein enhanced macrophage phagocytic activity (promoted phagocytosis) than rituximab, SIRPα fusion proteins, and ofatumumab, and also exhibited significant therapeutic effects in a rituximab-resistant diffuse large B-cell lymphoma (DLBCL) xenograft model.

[0010] Based on this, the present inventors, 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 the use of the combination drug in cancer treatment and in the manufacture of a medicament for cancer treatment. In particular, it has been found that in the treatment of lymphoma, particularly diffuse large B-cell lymphoma, administration of a combination (combination drug) of a CD20 / CD47 dual-blocking bifunctional fusion protein and a bifunctional alkylating agent, particularly the bifunctional alkylating agent bendamustine (including bendamustine hydrochloride), shows a very significant synergistic effect.

[0011] Specifically, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising a CD20 / CD47 dual-blocking bifunctional fusion protein and a bifunctional alkylating agent.

[0012] As used herein, the terms "pharmaceutical composition," "combined drug," "pharmaceutical combination," "combined drug," "combined drug," and the like are used interchangeably to refer to a combination of at least one drug and, optionally, a pharmaceutically acceptable carrier or excipient (auxiliary material) to achieve a specific purpose. In some embodiments, the pharmaceutical composition includes a combination separated in time and / or space, as long as they can act together to achieve the purpose of the present invention. For example, the components included in the pharmaceutical composition (e.g., the fusion protein and the bifunctional alkylating agent of the present invention) may be administered to a subject (individual) together or separately. When each component included in the pharmaceutical composition is administered separately to an individual (subject / subject), the components may be administered to the individual simultaneously or sequentially. For example, pharmaceutically acceptable carriers include water, aqueous buffer solutions, isotonic saline solutions (e.g., PBS (phosphate buffer solution)), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerin, hyaluronic acid, ethanol, or polyalkylene glycols such as polypropylene glycol, triglycerides, etc. The type of pharmaceutically acceptable carrier used will depend, inter alia, on whether the composition of the present invention is formulated for administration by inhalation, nasal administration, oral administration, intravenous injection, subcutaneous injection, or intramuscular injection. The composition of the present invention may contain a wetting agent, emulsifier, or buffer substance as an additive.

[0013] Cancers / tumors treatable with the present invention 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, renal cancer, cervical cancer, myeloma, leukemia, thyroid cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal carcinoma, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, 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 lymphoma. In some embodiments, the cancer is 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.

[0014] The CD20 / CD47 dual-blocking bifunctional fusion proteins described herein comprise two functional moieties, each capable of blocking CD20 and CD47. In some examples of the present invention, the two functional moieties are also referred to as two "arms" that can form heterodimers in the form of homodimeric structures similar to natural antibodies. Those skilled in the art will appreciate that the bifunctional fusion proteins of the present invention are not limited to a two-arm configuration, but may also be in a four-arm configuration or other configurations, similar to the various configurations of bispecific antibodies in the art.

[0015] As used herein, the term "antibody" broadly encompasses any form of molecule capable of specifically binding to an antigen, and can include intact antibodies and antigen-binding fragments ("antigen-binding portion") or single-chain forms thereof. The term "antigen-binding portion" or "antigen-binding fragment" can refer, inter alia, to antibody fragments such as Fv, scFv, Fab, F(ab')2, Fab', and VHH.

[0016] In some embodiments, the CD20 blocking functional moiety / arm is in the form of an antibody to CD20 or an antigen-binding portion thereof.

[0017] In some embodiments, the CD47-blocking functional portion / arm is an antibody against CD47 or an antigen-binding portion thereof, and / or a natural ligand of CD47 or a ligand active portion (wild-type or mutant) derived from the natural ligand. In some embodiments, the natural ligand of CD47 is a SIRPα protein. Because the SIRPα protein is a transmembrane protein, its ligand active function is mainly carried out by the extracellular domain. Thus, in some embodiments, the ligand active portion of the SIRPα protein comprises the entire extracellular domain of the SIRPα protein. In some embodiments, the ligand active portion of the SIRPα protein is an extracellular truncation of the 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 set forth at positions 20-136 of SEQ ID NO:6) or a non-high-affinity mutant thereof (e.g., the amino acid sequence set forth at positions 20-136 of SEQ ID NO:3, which contains 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 desirable for the CD47-blocking functional portion / arm to have a relatively low affinity for CD47, and therefore a non-high affinity mutant (i.e., other intended mutations that do not result in increased affinity) is desirable. In addition to the extracellular D1 domain of the SIRPα protein, SEQ ID NO:3 further comprises a signal peptide and an Fc region for dimerization with SEQ ID NO:1, as described in detail below.

[0018] In some embodiments of the two-armed CD47 / CD20 dual-targeting fusion protein, the first arm targets CD20 with high affinity, and the second arm blocks the interaction of CD47 with SIRPα with low affinity. In certain embodiments, the first arm functions as an antibody, and the second arm functions as a natural ligand of CD47 or a ligand-active portion derived from the natural ligand (e.g., an extracellular truncation of SIRPα protein or a non-high-affinity mutant thereof). The terms "high affinity" and "low affinity" described herein describe the relative relationship between the affinity of the dual-targeting fusion protein of the present invention for the CD47 protein and the CD20 protein. For example, the affinity level of the dual-targeting fusion protein for the CD47 protein and the CD20 protein can be measured at the protein level, e.g., by determining the Kd value, and the affinity level can be determined by comparing the Kd values. In certain embodiments, the binding affinity of the CD47 / CD20 dual-targeting fusion protein for the CD20 protein is at least six times higher than the binding affinity of the CD47 / CD20 dual-targeting fusion protein for the CD47 protein. In some embodiments, the two-armed CD47 / CD20 dual-targeting fusion protein is in the form of a left arm and a right arm similar to a natural antibody (see schematic diagram of a typical antibody structure and distinguish from possible upper and lower arm structures), and the terms left arm and right arm are also used below to describe such embodiments. In some embodiments, the left arm targets CD20 with high affinity, and the right arm blocks the interaction of CD47 with SIRPα with low affinity.

[0019] 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 set forth in positions 20-244 of SEQ ID NO:1 and the amino acid sequence set forth in positions 21-234 of SEQ ID NO:2, and the second arm or right arm comprises the amino acid sequence set forth in positions 20-136 of SEQ ID NO:3 or 6. In some embodiments, the first arm or left arm comprises the amino acid sequence set forth in positions 20-241 of SEQ ID NO:4 and the amino acid sequence set forth in positions 21-239 of SEQ ID NO:5, and the second arm or right arm comprises the amino acid sequence set forth in positions 20-136 of SEQ ID NO:3 or 6. SEQ ID NOs: 1 and 2 show the heavy and light chain amino acid sequences (each including a signal peptide) of one exemplary antibody that targets CD20, respectively. SEQ ID NOs: 4 and 5 show the heavy and light chain amino acid sequences (each including a signal peptide) of another exemplary antibody that targets CD20, respectively.

[0020] In some embodiments, the bifunctional fusion protein further comprises an Fc region. In some embodiments, the two arms of the bifunctional fusion protein each comprise an Fc region, and the Fc regions interact to form a heterodimer. In some embodiments, the Fc region is wild-type. In some embodiments, the Fc region has a knobs-into-holes mutation. Knobs-into-holes mutation in an Fc region is a common technique in the field for improving dimer assembly. As an example, the Fc regions contained in each of SEQ ID NO:1 and SEQ ID NO:3 contain engineered modifications to achieve knobs-into-holes mutation. In some embodiments, the first arm or left arm of the bifunctional fusion protein comprises the amino acid sequence set forth at positions 20 to 471 of SEQ ID NO:1 and the amino acid sequence set forth at positions 21 to 234 of SEQ ID NO:2, and the second arm or right arm comprises the amino acid sequence set forth at positions 20 to 363 of SEQ ID NO:3 or 6. In some embodiments, the first or left arm of the bifunctional fusion protein comprises the amino acid sequence set forth at positions 20-468 of SEQ ID NO:4 and the amino acid sequence set forth at positions 21-239 of SEQ ID NO:5, and the second arm comprises the amino acid sequence set forth at positions 20-363 of SEQ ID NO:3 or 6. As shown in the sequence structures of SEQ ID NOs:1-6 listed at the end of this 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 may be replaced by signal peptides of other structures or may be omitted.

[0021] In some embodiments, the first arm or left arm comprises the amino acid sequence set forth in SEQ ID NO:1 and SEQ ID NO:2, and the second arm or right arm comprises the amino acid sequence set forth in SEQ ID NO:3.

[0022] The bifunctional alkylating agent described in this application is a bifunctional derivative (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-phenylalanine mustard. In some embodiments, the bifunctional alkylating agent is bendamustine hydrochloride.

[0023] The present invention further provides the use of the above-described CD20 / CD47 dual blocking bifunctional fusion protein and bifunctional alkylating agent in the manufacture of a pharmaceutical composition for treating cancer (preferably, CD20-positive tumors).

[0024] The present invention further provides a combination comprising a CD20 / CD47 dual blocking bifunctional fusion protein as described above and a bifunctional alkylating agent for use in the treatment of cancer (preferably CD20-positive tumors).

[0025] The present invention further provides a method of treating cancer (preferably, a CD20-positive tumor) in an individual, the method comprising administering to an individual in need thereof a therapeutically effective amount of the above-described CD20 / CD47 dual-blocking bifunctional fusion protein and a bifunctional alkylating agent.

[0026] In some embodiments of the above uses, combinations, or methods, the CD20 / CD47 dual blocking bifunctional fusion protein and the bifunctional alkylating agent are formulated together or are physically separate (e.g., as separate units of a kit or medical kit). The CD20 / CD47 dual blocking bifunctional fusion protein can be formulated for administration before, simultaneously with, or after the bifunctional alkylating agent.

[0027] In some embodiments, the bifunctional fusion protein is administered at a dose of 0.3 mg / kg to 20 mg / kg, e.g., 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, e.g., 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 can be multiple times per week, e.g., twice per week.

[0028] In some embodiments, the bifunctional alkylating agent (e.g., bendamustine (including bendamustine hydrochloride)) is administered at a dose of 5 mg / kg to 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 a frequency of 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.

[0029] In some embodiments, the CD20 / CD47 dual blocking bifunctional fusion protein and the bifunctional alkylating agent are independently administered by inhalation, intranasal administration, oral administration, intravenous injection, subcutaneous injection, or intramuscular injection.

[0030] Examples of the CD20 / CD47 dual-blocking bifunctional fusion proteins described herein are described in Chinese Patent Application No. 201810430371.1 (CN108864290B), the entire contents of which are incorporated herein by reference.

[0031] One example of a CD20 / CD47 double-blocking bifunctional fusion protein described herein comprises a left arm consisting of the amino acid sequence set forth in SEQ ID NO:1 and SEQ ID NO:2, which targets CD20 with high affinity, and a right arm having the amino acid sequence set forth in SEQ ID NO:3, which blocks the interaction between CD47 and SIRPα with low affinity. This CD20 / CD47 double-blocking bifunctional fusion protein is referred to herein as JMT601. Another example of a CD20 / CD47 double-blocking bifunctional fusion protein described herein comprises a left arm consisting of the amino acid sequence set forth in SEQ ID NO:4 and SEQ ID NO:5, which targets CD20 with high affinity, and a right arm having the amino acid sequence set forth in SEQ ID NO:3 or 6, which blocks the interaction between CD47 and SIRPα with low affinity.

[0032] For the preparation of JMT601, reference can be made to the preparation methods disclosed in Example 1 and Example 2 of CN108864290B, or it can be prepared by other methods in the prior art. [Brief explanation of the drawings]

[0033] [Figure 1] Figure 1 shows the tumor volume change curves for each treatment group in the WSU-DLCL2 human lymphoma subcutaneous model. Note: Data are expressed as "mean ± standard error." [Figure 2] Figure 1 shows the change in body weight of experimental mice over time during treatment in each treatment group in the WSU-DLCL2 human lymphoma subcutaneous model. Note: Data are expressed as "mean ± standard error." [Figure 3] 1 shows a schematic structural diagram of one embodiment of the CD20 / CD47 dual-blocking bifunctional fusion protein of the present invention (a representative example of which is JMT601), in which the left arm is the single-arm form of a natural antibody targeting CD20, the Fc region of which contains a hole mutation, and the right arm is the extracellular D1 domain of the SIRPα protein, the Fc region of which contains a knob mutation. [Example]

[0034] Example 1: Pharmacodynamic study of drug combinations in a NOD SCID mouse model with subcutaneously implanted WSU-DLCL2 human lymphoma 1. Experimental materials and objectives The main experimental materials used during the experiment are as follows:

[0035] [Table 1]

[0036] In this study, we evaluated the efficacy of the test drug combination in NOD SCID mice using a WSU-DLCL2 human lymphoma subcutaneous xenograft model. The pharmacodynamics study was designed as follows:

[0037] [Table 2]

[0038] 2. Experimental Method 2.1 Tumor inoculation and group allocation 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 harvested and resuspended in PBS to an appropriate concentration before being used for subcutaneous tumor inoculation in mice.

[0039] 1 x 10 WSU-DLCL2 cells in a 1:1 ratio of PBS to Matrigel. 7 The cells were resuspended and subcutaneously inoculated into experimental mice (0.1 mL / mouse). The inoculation date was May 20, 2020. The average tumor volume was 150.53 mm. 3 Once tumor size reached 100 mg / kg, the patients were randomly assigned to groups according to tumor size (see Table 2 for details).

[0040] 2.2 Preparation of test and control drugs

[0041] [Table 3]

[0042] 2.3 Observation The experimental protocol for this study was reviewed and approved by the CrownBio IACUC Committee. Animal experiments were conducted in accordance with AAALAC requirements. After tumor inoculation, regular monitoring included the effects of tumor growth and treatment on the animals' normal behavior, including activity, feeding and drinking, weight gain and loss, and eye, hair, and other abnormalities. Clinical symptoms observed during the study were recorded in the raw data.

[0043] In the experiment, data such as measurements of tumor length and width and animal weight were collected using StudyDirector™ software (version: 3.1.399.19, supplier: Studylog System, Inc., San Francisco, CA, USA).

[0044] The experiment was terminated after 21 days of treatment in groups. The tumor-bearing mice were photographed, and the tumors were weighed and photographed.

[0045] 2.4 Data analysis Different treatment groups were compared and one-way ANOVA was performed to confirm homogeneity of variance. If homogeneity of variance was confirmed, pairwise comparisons were subsequently performed using the Dunnnett test. If unequal variance was confirmed, the nonparametric Kruskal Wallis test was used instead of one-way ANOVA, and Dunn's test was used for pairwise comparisons. All statistical analyses and graphing were performed using GraphPad Prism 8.4.2, and p values ​​less than 0.05 were considered statistically significant.

[0046] 3. Experimental Results The average tumor volume of mice in the control IgG1 (10 mg / kg) group was 1629.39 mm on the 21st day after group administration. 3 The tumor volume doubling time was 6 days. The efficacy of drug treatment was evaluated on the 21st day after group administration, and the relative tumor volumes of mice in the test and control groups were compared to verify whether there was a significant difference. On the 21st day after group administration, the average tumor volume of mice in the rituximab 10 mg / kg group was 1121.67 mm 3 The tumor inhibition rate was 33.39%, which was not significantly different from the control group (p-value > 0.9999). The tumor volume doubling time was 8 days. The mean tumor volume in mice treated with JMT601 (3.3 mg / kg) was 825.07 mm. 3 The tumor inhibition rate was 50.00%, which was not significantly different from the control group (p-value > 0.9999). The tumor volume doubling time was 9 days. The mean tumor volume in mice treated with JMT601 (10 mg / kg) was 264.67 mm. 3 The tumor inhibition rate was 84.69%, which was significantly different from the control group (p-value 0.0002). The tumor volume doubling time was 27 days (calculated from existing data). The mean tumor volume in mice treated with bendamustine (30 mg / kg) was 945.10 mm 3 The tumor inhibition rate was 44.00%, which was not significantly different from the control group (p-value > 0.9999). The tumor volume doubling time was 8 days. The mean tumor volume in mice treated with the combination of bendamustine (30 mg / kg) and JMT601 (3.3 mg / kg) was 178.85 mm. 3 The tumor inhibition rate was 89.02%, which was significantly different from the control group (p<0.0001), and the tumor volume doubling time was 88 days.

[0047] The results of the tumor weight analysis were essentially consistent with the results of the relative tumor volume analysis. The tumor growth inhibition in mice in each treatment group and the control group is shown in Table 4 and Figure 1.

[0048] [Table 4]

[0049] The above experimental results showed that compared with the use of JMT601 (3.3 mg / kg) and bendamustine (10 mg / kg) alone, the combined use of JMT601 (3.3 mg / kg) and bendamustine (10 mg / kg) significantly improved the tumor inhibition rate, had a significant synergistic effect, and achieved an unexpected technical effect.

[0050] Example 2: Safety study results and discussion of the test drug in the WSU-DLCL2 human lymphoma subcutaneous transplant model In this study, one mouse (4174#) in the JMT601 (3.3 mg / kg) and bendamustine (30 mg / kg) co-administration group experienced a weight loss of over 20% on days 15, 18, and 19 after administration, reaching the humane endpoint, and was euthanized. The weight loss was due to a loss of appetite, and no obvious abnormalities were found in the autopsy results. It is speculated that this weight loss was due to individual differences in the mice. All test drugs were well tolerated by the other mice in each group. The changes in body weight in the treatment and control groups after administration are shown in Table 5 and Figure 2.

[0051] [Table 5]

[0052] The results above show that tumor-bearing mice tolerated rituximab (10 mg / kg), JMT601 (3.3 mg / kg and 10 mg / kg), and bendamustine (30 mg / kg), respectively. There was no significant difference in the tolerability between the combination therapy and monotherapy, reflecting the favorable safety of the combination drug therapy. The fusion protein related sequences according to the invention are as follows:

[0053] [ka]

[0054] [ka]

[0055] [ka]

[0056] [ka]

[0057] [ka]

[0058] [ka]

[0059] 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 changes and modifications can be made to these details, and these changes will fall within the protection scope of the present invention. The full scope of the present application is defined by the appended claims and their equivalents.

Claims

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

2. the CD20-blocking functional moiety in said CD20 / CD47 double-blocking bifunctional fusion protein is an antibody against CD20 or an antigen-binding portion thereof; and / or the CD47-blocking functional portion 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., a SIRPα protein) or a ligand-active portion derived from the natural ligand (e.g., a SIRPα protein); Optionally, the CD20-blocking functional moiety and the CD47-blocking functional moiety each comprise an Fc region, e.g., a wild-type Fc region, or an Fc region containing a mutation based on Knobs-into-holes technology; The pharmaceutical composition of claim 1.

3. 3. The pharmaceutical composition of claim 2, wherein the ligand active portion is an intact extracellular domain of a SIRPα protein, an extracellular truncation, or a non-high affinity variant thereof.

4. The CD20 / CD47 dual blocking bifunctional fusion protein is a fusion protein comprising a first arm (e.g., a first arm in the form of an antibody) that targets CD20 with high affinity and a second arm (e.g., a second arm in the form of a natural ligand of CD47 or a ligand active portion derived from said natural ligand) that blocks the interaction of CD47 with SIRPα with low affinity, and preferably the binding affinity of the fusion protein to the CD20 protein is at least 6-fold higher than the binding affinity of the fusion protein to the CD47 protein. The pharmaceutical composition according to any one of claims 1 to 3.

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

6. 5. 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. 5. 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 of any one of claims 1 to 7, wherein the bifunctional alkylating agent is a bifunctional derivative of a nitrogen mustard, such as bendamustine or a pharmaceutically acceptable salt, solvate or prodrug thereof, chlorambucil, or L-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 cancer is selected from the group consisting of lymphoma, breast cancer, colon 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 carcinoma, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma or myelodysplastic syndrome, preferably lymphoma including Hodgkin's lymphoma and non-Hodgkin's lymphoma, preferably non-Hodgkin's lymphoma including diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma and mucosa-associated lymphoid tissue lymphoma, and most preferably diffuse large B-cell lymphoma; The pharmaceutical composition according to any one of claims 1 to 9.

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 (e.g., diffuse large B-cell lymphoma).

12. Use of a CD20 / CD47 dual-blocking bifunctional fusion protein and a bifunctional alkylating agent in the manufacture 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, comprising administering to the individual in need thereof a therapeutically effective amount of a CD20 / CD47 dual blocking bifunctional fusion protein and a bifunctional alkylating agent.

15. The bifunctional alkylating agent is a bifunctional derivative of a nitrogen mustard, such as bendamustine or a pharmaceutically acceptable salt, solvate or prodrug thereof, chlorambucil, or L-phenylalanine mustard, preferably bendamustine or a pharmaceutically acceptable salt, solvate or prodrug thereof, more preferably bendamustine hydrochloride.

15. The use according to claim 12, the combination according to claim 13, or the method according to claim 14.

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

17. 17. The use, combination, or method of claim 16, wherein the ligand active portion is an intact extracellular domain of a SIRPα protein, an extracellular truncation of a SIRPα protein, or a non-high affinity variant thereof.

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

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

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

3.

21. 19. The use, 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 cancer is selected from the group consisting of lymphoma, breast cancer, colon 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 carcinoma, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma or myelodysplastic syndrome, preferably lymphoma including Hodgkin's lymphoma and non-Hodgkin's lymphoma, preferably non-Hodgkin's lymphoma including diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma and mucosa-associated lymphoid tissue lymphoma, and most preferably diffuse large B-cell lymphoma; 22. The use, combination or method according to any one of claims 12 to 21.

23. 23. The use, 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 (e.g. diffuse large B-cell lymphoma).

24. The use, combination or method of 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 are physically separate.

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

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

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

28. 28. The use, combination or method of any one of claims 12 to 27, wherein the administration frequency of said CD20 / CD47 dual blocking bifunctional fusion protein and said bifunctional alkylating agent is independently 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 is independently more than once per week, such as twice per week.

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

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