Use of Anti-CD3 / CD20 bispecific antibody in preparation of drug for treating CD20 positive b-nhl

Abstract

The present invention provides a use of an anti-CD3 / CD20 bispecific antibody in the preparation of a drug for treating CD20 positive B-NHL. The use comprises: administering a type I anti-CD20 antibody prior to administering an anti-CD3 / CD20 bispecific antibody to a patient, wherein the anti-CD3 / CD20 bispecific antibody comprises a first binding site that specifically binds to CD20, a second binding site that specifically binds to CD3, and a third binding site that specifically binds to CD20, and the first and third binding sites that specifically bind to CD20 are both derived from a type II anti-CD20 antibody. The present invention can be used in the treatment of B-NHL.

Description

Use of anti-CD3 / CD20 bispecific antibody in the preparation of a medicament for treating CD20 positive B-NHL

[0001] This application claims priority to Chinese patent application 2024115295771 with the filing date of 2024 / 10 / 30. This application incorporates the entirety of the aforementioned Chinese patent application. TECHNICAL FIELD

[0002] The present application relates to the use of anti-CD3 / CD20 bispecific antibody in the preparation of a medicament for treating CD20 positive B-NHL. BACKGROUND

[0003] Bispecific antibodies that target CD3 and CD20 simultaneously can establish a physical connection between T cells and CD20 positive tumor cells, such as malignant B cells, leading to T cell activation and T cell-mediated killing of CD20 positive malignant B cell lymphoma.

[0004] However, the administration of such antibodies is accompanied by inevitable side effects, i.e. severe toxic reactions. While T cells are activated and kill tumor cells, the CD3 binding part in the bispecific antibody also activates the TCR complex signaling pathway of T cells, releasing cytokines such as IL-6, IL-10, IFN-r and TNF-a, and activating the proliferation and differentiation of T cells. The proliferation and differentiation of T cells are a double-edged sword in tumor treatment, on the one hand, they can produce more T cells to kill tumor cells, but on the other hand, they can also produce great toxic reactions in the body of the subject, i.e. cytokine release syndrome (“CRS”). In the clinic, side effects associated with CRS are described as fatigue, vomiting, tachycardia, hypertension, back pain, and can also be accompanied by central nervous system toxicity manifestations such as seizures, encephalopathy, brain edema, aseptic meningitis and headache, etc., which are more common in severe CRS or concurrent immune effector cell-related neurotoxicity syndrome (ICNS).

[0005] Therefore, there is an urgent need to address the CRS induced by anti-CD3 / CD20 antibodies during the treatment of diseases.

[0006] The citation of any document herein is not intended as an admission that such document is prior art to the present application. SUMMARY

[0007] To solve the problem of CRS induced by anti-CD3 / CD20 antibodies in the treatment of diseases in the prior art, the present application provides the use of an anti-CD3 / CD20 bispecific antibody in the preparation of a medicament for treating patients with CD20 positive B-cell non-Hodgkin lymphoma (B-Cell Non-Hodgkin Lymphoma, B-NHL).

[0008] For treating CD20-positive B-NHL, one aspect of the present application provides use of an anti-CD3 / CD20 bispecific antibody for treating a patient with CD20-positive B-NHL, the anti-CD3 / CD20 bispecific antibody comprising a first binding domain that specifically binds CD20, a second binding domain that specifically binds CD3, and a third binding domain that specifically binds CD20, and the first and third binding domains that specifically bind CD20 are both derived from a Type II anti-CD20 antibody.

[0009] Another aspect of the present application provides a method of treating CD20-positive B-NHL, the method comprising administering to a patient in need thereof an effective amount of an anti-CD3 / CD20 bispecific antibody, the anti-CD3 / CD20 bispecific antibody comprising a first binding domain that specifically binds CD20, a second binding domain that specifically binds CD3, and a third binding domain that specifically binds CD20, and the first and third binding domains that specifically bind CD20 are both derived from a Type II anti-CD20 antibody.

[0010] Another aspect of the present application relates to use of an anti-CD3 / CD20 bispecific antibody for the manufacture of a medicament for treating a patient with CD20-positive B-cell non-Hodgkin lymphoma (B-NHL); the anti-CD3 / CD20 bispecific antibody comprising a first binding domain that specifically binds CD20, a second binding domain that specifically binds CD3, and a third binding domain that specifically binds CD20, and the first and third binding domains that specifically bind CD20 are both derived from a Type II anti-CD20 antibody.

[0011] Another aspect of the present application provides an anti-CD3 / CD20 bispecific antibody for use in a method of treating a patient with CD20-positive B-cell non-Hodgkin lymphoma (B-NHL); the anti-CD3 / CD20 bispecific antibody comprising a first binding domain that specifically binds CD20, a second binding domain that specifically binds CD3, and a third binding domain that specifically binds CD20, and the first and third binding domains that specifically bind CD20 are both derived from a Type II anti-CD20 antibody.

[0012] In the present context, the two domains in an anti-CD3 / CD20 bispecific antibody that specifically bind CD20 are defined as the first and third binding domains, respectively, and the domain that specifically binds CD3 is defined as the second binding domain. It is specifically noted that the first and third binding domains, although both bind CD20, can exist as independent functional domains. The following description will use this nomenclature uniformly.

[0013] In some embodiments, the anti-CD3 / CD20 bispecific antibody is used for treating a CD20 positive B-NHL patient. Herein, non-Hodgkin's lymphoma is also referred to as NHL.

[0014] In some embodiments, the administration of the anti-CD3 / CD20 bispecific antibody comprises a first dosing cycle and a second dosing cycle, preferably further comprising a third dosing cycle or optionally more subsequent dosing cycles.

[0015] In some embodiments, the first dosing cycle comprises a first priming dose and a second priming dose of the bispecific antibody, wherein the first priming dose is 0.05-1.5 mg, preferably 1 mg; and the second priming dose is 0.15-6 mg, preferably 5 mg.

[0016] In some embodiments, the second dosing cycle comprises a therapeutic dose of the bispecific antibody of 15-45 mg.

[0017] In certain embodiments, the second dosing cycle comprises a therapeutic dose of the bispecific antibody of 15 mg, 20 mg, 25 mg, 30 mg, or 35 mg.

[0018] In certain specific embodiments, the second dosing cycle comprises a therapeutic dose of the bispecific antibody of 25 mg.

[0019] In some embodiments, the single dose of the third dosing cycle or subsequent dosing cycles is 15-45 mg, for example 15 mg, 20 mg, 25 mg, 30 mg, or 35 mg, preferably 25 mg.

[0020] In certain embodiments, the dosing cycle of the anti-CD3 / CD20 bispecific antibody comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17.

[0021] In certain embodiments, the dosing cycle of the anti-CD3 / CD20 bispecific antibody comprises 17 dosing cycles in total.

[0022] In some embodiments, the first dosing cycle is 14 days.

[0023] In some embodiments, the first dosing cycle further comprises a third priming dose, and when the first dosing cycle comprises a third priming dose, the first dosing cycle is 21 days.

[0024] In some embodiments, the second dosing cycle, the third dosing cycle, and optionally the subsequent dosing cycles are 21 days.

[0025] In certain embodiments, the first dosing cycle is 14 days, and the second, third, and optionally subsequent dosing cycles are 21 days.

[0026] In some embodiments, a first pre-priming dose is administered on day 1 of the first dosing cycle, and / or a second pre-priming dose is administered on day 8.

[0027] In some embodiments, the therapeutic dose of the therapeutic agent (bispecific antibody) is administered on day 1 of the second, third, and optionally subsequent dosing cycles.

[0028] In some embodiments, the CD20-positive B-cell non-Hodgkin lymphoma patient experiences Cytokine Release Syndrome (CRS) after administration of the anti-CD3 / CD20 bispecific antibody, without experiencing a grade ≥3 CRS (CRS grade assessed according to the 2019 version of the ASTCT consensus grading criteria).

[0029] In some embodiments, the CD20-positive B-cell non-Hodgkin lymphoma patient experiences an Objective Response Rate (ORR) of at least 60% after administration of the anti-CD3 / CD20 bispecific.

[0030] In some embodiments, the first binding portion and the second binding portion of the anti-CD3 / CD20 bispecific antibody are in Fab configuration, and the third binding portion is in scFv configuration.

[0031] In some embodiments, the first binding portion of the anti-CD3 / CD20 bispecific antibody comprises a heavy chain variable region (VH1) and a light chain variable region (VL1); the third binding portion comprises a heavy chain variable region (VH3) and a light chain variable region (VL3); and the second binding portion comprises a heavy chain variable region (VH2) and a light chain variable region (VL2).

[0032] In some embodiments, the first binding portion of the anti-CD3 / CD20 bispecific antibody further comprises a heavy chain constant region 1 (CH1-1) and a light chain constant region (CL-1); and the second binding portion further comprises a heavy chain constant region 1 (CH1-2) and a light chain constant region (CL-2).

[0033] In some embodiments, the anti-CD3 / CD20 bispecific antibody further comprises an Fc region, which is linked to the C-terminus of the first binding portion, and / or the C-terminus of the second binding portion.

[0034] In some embodiments, the first binding portion is directly linked to the second binding portion or linked via a linker.

[0035] In some embodiments, the third binding portion is bound at the N-terminus of the second binding portion, for example, via a second linker (L2).

[0036] In some embodiments, the CH1-1 is connected to VH1, and the CL-1 is connected to VL1 accordingly; the CH1-2 is connected to VH2, and the CL-2 is connected to VL2 accordingly.

[0037] In some embodiments, the scFv comprises a first linker (L1), and the structure of the scFv from N-terminus to C-terminus is VH3-L1-VL3.

[0038] In some embodiments, the anti-CD3 / CD20 bispecific antibody comprises a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein:

[0039] the first polypeptide chain has a structure from N-terminus to C-terminus of: VH1-CH1-1-Fc;

[0040] the second polypeptide chain has a structure from N-terminus to C-terminus of: VL1-CL-1;

[0041] the third polypeptide chain has a structure from N-terminus to C-terminus of: VH3-L1-VL3-L2-VH2-CH1-2-Fc; and,

[0042] the fourth polypeptide chain has a structure from N-terminus to C-terminus of: VL2-CL-2.

[0043] In some embodiments, the Fc region is selected from the Fc regions of IgG1, IgG2, IgG3, and IgG4; when the sequences of the two Fc chains are different, the two Fc chains are connected via a Knob-into-Hole structure, one Fc is called Fc Hole, and the other Fc is called Fc Knob.

[0044] In some embodiments, the Fc of the first polypeptide chain is called Fc Hole, and the Fc of the third polypeptide chain is called Fc Knob.

[0045] In some embodiments, the VH1 has the same amino acid sequence of the complementarity determining region (CDR) of VH3, and the VL1 has the same amino acid sequence of the complementarity determining region (CDR) of VL3.

[0046] In some embodiments, the VH1 or VH3 comprises a HCDR1 as set forth in SEQ ID NO: 11, a HCDR2 of the amino acid sequence as set forth in SEQ ID NO: 12, and a HCDR3 of the amino acid sequence as set forth in SEQ ID NO: 13, and the VL1 or VL3 comprises a LCDR1 as set forth in SEQ ID NO: 14, a LCDR2 of the amino acid sequence as set forth in SEQ ID NO: 15, and a LCDR3 of the amino acid sequence as set forth in SEQ ID NO: 16.

[0047] In some embodiments, the VH2 comprises a HCDR1 as set forth in SEQ ID NO: 21, a HCDR2 of the amino acid sequence as set forth in SEQ ID NO: 22, and a HCDR3 of the amino acid sequence as set forth in SEQ ID NO: 23, and the VL2 comprises a LCDR1 as set forth in SEQ ID NO: 24, a LCDR2 of the amino acid sequence as set forth in SEQ ID NO: 25, and a LCDR3 of the amino acid sequence as set forth in SEQ ID NO: 26.

[0048] In some embodiments, the amino acid sequence of the VH1 is identical to that of VH3.

[0049] In some embodiments, the amino acid sequence of the VH1 or VH3 is set forth in SEQ ID NO: 17, or has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17.

[0050] In some embodiments, the amino acid sequence of the VL1 is set forth in SEQ ID NO: 18, or has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18.

[0051] In some embodiments, the amino acid sequence of the VL3 is set forth in SEQ ID NO: 19, or has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 19.

[0052] In some embodiments, the amino acid sequence of the VH2 is set forth in SEQ ID NO: 27, or has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 27.

[0053] In some embodiments, the VL2 has an amino acid sequence of SEQ ID NO: 28, or at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 28.

[0054] In some embodiments, the amino acid sequence of the first linker and the second linker is (G n S) p As shown, n is, for example, 3 or 4, and p is, for example, 3, 4, or 5.

[0055] In some embodiments, the amino acid sequence of the first linker is set forth in SEQ ID NO: 20, and / or the amino acid sequence of the second linker is set forth in SEQ ID NO: 29.

[0056] In some embodiments, the anti-CD3 / CD20 bispecific antibody comprises:

[0057] a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 30;

[0058] a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 31;

[0059] a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 32; and,

[0060] a fourth polypeptide chain comprising an amino acid sequence of SEQ ID NO: 33.

[0061] In some embodiments, the B-cell non-Hodgkin lymphoma comprises relapsed or refractory B-NHL, such as Aggressive Non-Hodgkin Lymphoma (aNHL) and Indolent Non-Hodgkin Lymphoma (iNHL).

[0062] In some embodiments, the aggressive NHL comprises Diffuse Large B-Cell Lymphoma (DLBCL), High-Grade B-Cell Lymphoma (HGBCL), Follicular Lymphoma (FL) 3b, Mantle Cell Lymphoma (MCL), Primary Mediastinal Large B-Cell Lymphoma (PMBCL), Richter Transformation-Diffuse Large B-Cell Lymphoma (RT-DLBCL), or Transformed FL (trFL); and / or, the indolent NHL comprises FL 1-3a or Marginal Zone Lymphoma (MZL).

[0063] In some embodiments, the patient is a patient who is not a chronic lymphocytic leukemia, not a Burkitt's lymphoma, and not a lymphoplasmacytic lymphoma.

[0064] In some embodiments, the patient is between 18 years old and 71 years old, for example, between 27 years old and 71 years old.

[0065] Another aspect of the present application provides an antibody combination comprising an anti-CD3 / CD20 bispecific antibody and a type I anti-CD20 antibody; the anti-CD3 / CD20 bispecific antibody is an anti-CD3 / CD20 bispecific antibody as defined above.

[0066] In some embodiments, the type I anti-CD20 antibody comprises a heavy chain variable region comprising HCDR1 of the amino acid sequence as set forth in SEQ ID NO: 1, HCDR2 of the amino acid sequence as set forth in SEQ ID NO: 2, and HCDR3 of the amino acid sequence as set forth in SEQ ID NO: 3, and a light chain variable region comprising LCDR1 of the amino acid sequence as set forth in SEQ ID NO: 4, LCDR2 of the amino acid sequence as set forth in SEQ ID NO: 5, and LCDR3 of the amino acid sequence as set forth in SEQ ID NO: 6.

[0067] In some embodiments, the amino acid sequence of the heavy chain variable region of the type I anti-CD20 antibody is as shown in SEQ ID NO:7, or has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:7.

[0068] In some embodiments, the amino acid sequence of the light chain variable region of the type I anti-CD20 antibody is as shown in SEQ ID NO:8, or has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:8.

[0069] In some embodiments, the type I anti-CD20 antibody is rituximab, the heavy chain of which comprises the amino acid sequence shown in SEQ ID NO:9, and / or the light chain comprises the amino acid sequence shown in SEQ ID NO:10.

[0070] In some embodiments, the antibody combination comprises an anti-CD3 / CD20 bispecific antibody in a mass ratio of 15:100 to a type I anti-CD20 antibody.

[0071] In some implementations, the antibody combination is used to treat patients with CD20-positive B-cell non-Hodgkin lymphoma.

[0072] In another aspect, the present invention provides a nucleic acid that encodes an antibody combination provided by the present invention.

[0073] Another aspect of the present invention provides an expression vector comprising the nucleic acid provided by the present invention.

[0074] In some implementations, the expression vector is selected from prokaryotic expression vectors or eukaryotic expression vectors.

[0075] In another aspect, the present invention provides a transformant, wherein the transformant is a host cell containing the nucleic acid or expression vector provided by the present invention; preferably, the transformant is a non-animal variety and a non-plant variety.

[0076] In some embodiments, the host cell of the transformant is a prokaryotic cell or a eukaryotic cell.

[0077] In some embodiments, the eukaryotic cells are selected from insect cells, mammalian cells, or yeast cells; the mammalian cells are, for example, CHO or 293 cells.

[0078] Another aspect of the present invention provides a medicament for treating CD20-positive B-cell non-Hodgkin lymphoma, the medicament comprising the antibody combination provided by the present invention and a pharmaceutically acceptable carrier.

[0079] In some embodiments, the drug further comprises a corticosteroid; the corticosteroid being, for example, dexamethasone and / or methylprednisolone.

[0080] Another aspect of the present invention provides a reagent kit or pharmaceutical kit containing an antibody combination provided by the present invention; or, a drug provided by the present invention.

[0081] In some implementations, the kit or pharmaceutical kit also includes a device for administering the antibody or drug; and / or, instructions for administering the antibody or drug.

[0082] Another aspect of the present invention provides a method for treating CD20-positive B-cell non-Hodgkin lymphoma, the method comprising administering an effective amount of the antibody combination or drug provided by the present invention to a patient in need, or treating a patient in need using a kit or pharmacist kit provided by the present invention.

[0083] In some implementations, the method includes first administering an effective amount of the type I anti-CD20 antibody to a patient in need, and then administering an effective amount of the anti-CD3 / CD20 bispecific antibody to the patient in need.

[0084] In some embodiments, the method includes administering the type I anti-CD20 antibody before administering the anti-CD3 / CD20 bispecific antibody to the patient.

[0085] Another aspect of the present invention relates to the use of the antibody combination provided by the present invention in the preparation of a medicament or combination of medicaments for treating patients with CD20-positive B-cell non-Hodgkin lymphoma (B-NHL), wherein type I CD20 antibody is administered to the patient prior to administration of anti-CD3 / CD20 bispecific antibody.

[0086] Another aspect of the present invention relates to the use of the antibody combination therapy provided by the present invention for patients with CD20-positive B-cell non-Hodgkin lymphoma (B-NHL), wherein type I anti-CD20 antibody is administered to the patient before administering anti-CD3 / CD20 bispecific antibody.

[0087] Another aspect of the present invention relates to the use of a type I anti-CD20 antibody in the preparation of a medicament for reducing cytokine storm (CRS) formation in patients against an anti-CD3 / CD20 bispecific antibody, the method comprising administering a type I anti-CD20 antibody to the patient prior to administering the anti-CD3 / CD20 bispecific antibody; said anti-CD3 / CD20 bispecific antibody being the anti-CD3 / CD20 bispecific antibody as defined above in the present invention.

[0088] In some implementations, the patient's B-cell non-Hodgkin lymphoma includes relapsed or refractory B-NHL, such as aggressive non-Hodgkin lymphoma (aNHL) and indolent non-Hodgkin lymphoma (iNHL).

[0089] In some implementations, the patient is a patient with non-chronic lymphocytic leukemia, non-Burkitt lymphoma, or non-lymphoplasmacytic lymphoma.

[0090] In some embodiments, the aggressive NHL includes diffuse large B-cell lymphoma (DLBCL), high-grade B-cell lymphoma (HGBCL), follicular lymphoma (FL) 3b, mantle cell lymphoma (MCL), primary mediastinal large B-cell lymphoma (PMBCL), Richter's transformed DLBCL (RT-DLBCL), or transformed FL (trFL); and / or, the indolent NHL includes FL 1-3a or marginal zone lymphoma (MZL).

[0091] In some implementations, the patient's age is between 18 and 71 years old, for example, 27 to 71 years old.

[0092] In some embodiments, type I anti-CD20 antibody is administered to the patient on days 1 through 4 prior to administration of the anti-CD3 / CD20 bispecific antibody. In some embodiments, type I anti-CD20 antibody is administered to the patient on days 2 through 3 prior to administration of the anti-CD3 / CD20 bispecific antibody. In some embodiments, type I anti-CD20 antibody is administered to the patient on day 2 prior to administration of the anti-CD3 / CD20 bispecific antibody.

[0093] In this document, "administering type I anti-CD20 antibody on the nth day prior to administration of the anti-CD3 / CD20 bispecific antibody" means that the interval between the natural day of administration of the CD3 / CD20 bispecific antibody and the natural day of administration of the type I anti-CD20 antibody is n-1 natural days. A natural day refers to the period from 0:00 to 24:00 local time. For example, "administering type I anti-CD20 antibody on the 2nd day prior to administration of the anti-CD3 / CD20 bispecific antibody" means that the interval between the natural day of administration of the CD3 / CD20 bispecific antibody and the natural day of administration of the type I anti-CD20 antibody is 1 natural day. Optionally, the type I anti-CD20 antibody may be administered within a time window of 48 hours ± 24 hours prior to administration of the anti-CD3 / CD20 bispecific antibody. Preferably, this time window is 48 hours ± 12 hours, 48 ​​hours ± 8 hours, 48 ​​hours ± 6 hours, 48 ​​hours ± 4 hours, 48 ​​hours ± 2 hours, or 48 hours ± 1 hour prior to administration.

[0094] Specifically, in some implementation schemes, administering type I CD20 antibody on the second day before administering anti-CD3 / CD20 bispecific antibody to the patient means that the administration time of anti-CD3 / CD20 bispecific antibody and type I anti-CD20 antibody is at least one day apart, i.e. at least 24 hours apart, but not more than 72 hours apart.

[0095] In some embodiments, type I anti-CD20 antibody is administered at least 24 hours prior to administration of the anti-CD3 / CD20 bispecific antibody to the patient. In some embodiments, type I anti-CD20 antibody is administered at least 36 hours prior to administration of the anti-CD3 / CD20 bispecific antibody to the patient.

[0096] In some implementations, the timing of administering type I anti-CD20 antibody on the second day prior to administering anti-CD3 / CD20 bispecific antibody may be the same as or no more than 2 hours after administering anti-CD3 / CD20 bispecific antibody.

[0097] In some implementations, type I anti-CD20 antibody is administered once, twice, or multiple times.

[0098] In some implementations, type I anti-CD20 antibodies are administered subcutaneously or intravenously.

[0099] In some implementations, the target dose of type I anti-CD20 antibody is administered to achieve effective depletion of the patient's peripheral blood B cells.

[0100] In some implementations, effective depletion of peripheral blood B cells after administration of type I anti-CD20 antibody is defined as a CD19+ / CD20+ cell count of <5 cells / μL.

[0101] In some implementations, the dosage of type I anti-CD20 antibody is 375 mg / m². 2 .

[0102] In some implementations, administration of the anti-CD3 / CD20 bispecific antibody includes: a first dosing cycle and a second dosing cycle, preferably including a third dosing cycle or optionally more subsequent dosing cycles.

[0103] In some embodiments, the first dosing cycle includes a first pre-excitation dose and a second pre-excitation dose of the bispecific antibody, wherein the first pre-excitation dose is 0.05-1.5 mg, preferably 1 mg; and the second pre-excitation dose is 0.15-6 mg, preferably 5 mg.

[0104] In some implementations, the second dosing cycle includes a therapeutic dose of 15-45 mg of the bispecific antibody.

[0105] In some implementations, the second dosing cycle includes a therapeutic dose of the bispecific antibody of 15 mg, 20 mg, 25 mg, 30 mg, or 35 mg.

[0106] In some specific implementations, the second dosing cycle includes a therapeutic dose of 25 mg of the bispecific antibody.

[0107] In some embodiments, the single dose of the third or subsequent dosing cycle is 15-45 mg, for example 15 mg, 20 mg, 25 mg, 30 mg or 35 mg, preferably 25 mg.

[0108] In some embodiments, the dosing cycles of the CD3 / CD20 bispecific antibody include 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 cycles.

[0109] In some embodiments, the dosing cycle of the anti-CD3 / CD20 bispecific antibody includes a total of 17 dosing cycles.

[0110] In some implementations, the first dosing cycle is 14 days.

[0111] In some implementations, the first dosing cycle further includes a third pre-excitation dose, and when the first dosing cycle includes a third pre-excitation dose, the first dosing cycle is 21 days.

[0112] In some implementations, the second dosing cycle, the third dosing cycle, and optionally subsequent dosing cycles are 21 days.

[0113] In some implementations, the first dosing cycle is 14 days, and the second, third, and optionally subsequent dosing cycles are 21 days.

[0114] In some implementations, a first pre-excitation dose is administered on day 1 of the first dosing cycle, and / or a second pre-excitation dose is administered on day 8.

[0115] In some implementations, the therapeutic dose (of the bispecific antibody) is administered on day 1 of the second, third, and optionally subsequent dosing cycles.

[0116] In some implementations, cytokine release syndrome (CRS) occurred after administration of anti-CD3 / CD20 bispecific antibodies to patients with CD20-positive B-cell non-Hodgkin lymphoma, but grade ≥3 CRS (CRS grade assessed according to the 2019 ASTCT consensus rating criteria) did not occur.

[0117] In some implementation schemes, patients with CD20-positive B-cell non-Hodgkin lymphoma who receive anti-CD3 / CD20 bispecific therapy achieve an objective response rate (ORR) of at least 60%.

[0118] In some implementations, the administration of anti-CD3 / CD20 bispecific antibodies may also include the administration of anti-interleukin-6 receptor antibodies and / or corticosteroids in the event of CRS.

[0119] In some implementations, the anti-interleukin-6 receptor antibody is tocilizumab.

[0120] In some implementations, the corticosteroid is dexamethasone and / or methylprednisolone.

[0121] In some embodiments, the single dose of tocilizumab is 8 mg / kg, and ≤800 mg. Optionally, tocilizumab can be administered a maximum of 4 times in the event of a single CRS.

[0122] In some embodiments, the single dose of dexamethasone is 10-20 mg, and / or the single dose of methylprednisolone is 1000-2000 mg.

[0123] The positive and progressive effects of this invention are as follows:

[0124] The anti-CD3 / CD20 bispecific antibody provided by this invention can reduce the cytokine storm caused by the anti-CD3 / CD20 bispecific antibody during the treatment of CD20-positive B-cell non-Hodgkin lymphoma (B-NHL) and has a better therapeutic effect. Attached Figure Description

[0125] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention.

[0126] Figure 1 illustrates three dosing regimens for the anti-CD3 / CD20 bispecific antibody according to an embodiment of the present invention; BOIN = Bayesian optimal interval; CRS = cytokine release syndrome; DLT = dose-limiting toxicity; RTX = rituximab.

[0127] Figure 2 shows a comparison of the maximum release levels of cytokines IL-6, IL-2, IL-4, IL-10, TNF-α and IFN-r of the anti-CD3 / CD20 bispecific antibody in various dosing regimens according to embodiments of the present invention; ULN = upper limit of normal value.

[0128] Figure 3 shows the configuration of the anti-CD3 / CD20 bispecific antibody of the present invention. Detailed Implementation

[0129] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0130] To better understand this application, some terms are first defined. Other definitions are listed throughout the detailed implementation section.

[0131] The term "CD3" refers to differentiated cluster 3, which includes the γ chain, δ chain, ε chain, and ζ chain. The term "CD3ε" refers to the ε chain of CD3. This term includes variants, homologs, orthologs, and parallel homologs.

[0132] The term "human CD3ε" refers to a CD3ε protein with a human amino acid sequence, such as the CD3ε protein with the amino acid sequence NCBI reference number NP_000724.1 (Wipa P et al., (2020) Immunology 159(3):298-308). The term "monkey CD3ε" refers to a CD3ε protein with a monkey amino acid sequence, such as the CD3 protein with the amino acid sequence NCBI reference number NP_001244149.1 (Maudhoo MD et al., (2014) Gigascience 3:14).

[0133] The term "CD20" refers to a molecular marker expressed on the surface of B cells at all stages (excluding stem cells and plasma cells). "Human CD20" refers to the CD20 protein with a human amino acid sequence.

[0134] The term “antibody” as used in this article is intended to include full-length antibodies of IgG, IgA, IgD, IgE and IgM, as well as any antigen-binding fragments (i.e., antigen-binding fragments).

[0135] In “CnDm”, C represents the dosing cycle, D represents the day, n represents the nth dosing cycle, and m represents the mth day within the dosing cycle. For example, C1D1 represents the first day of the first dosing cycle, C1D8 represents the 8th day of the first dosing cycle, C1D15 represents the 15th day of the first dosing cycle, C2D1 represents the first day of the second dosing cycle, and so on.

[0136] Based on their binding epitopes, anti-CD20 antibodies are classified into type I and type II. Type I CD20 antibodies exhibit stronger complement-dependent cytotoxicity (CDC) activity, while type II CD20 antibodies demonstrate stronger antibody-dependent cell-mediated cytotoxicity (ADCC) and programmed cell death (PCD) compared to type I. Preclinical and clinical studies have shown that type II CD20 antibodies possess a stronger ability to clear B cells in vivo. Representative antibody drugs for type I CD20 include rituximab, while examples of type II CD20 antibodies include obinutuzumab.

[0137] To better illustrate the present invention, several specific embodiments are provided below as examples. These embodiments are merely illustrative and do not limit the scope of protection of the present invention.

[0138] Example 1: Introduction to Clinical Study Design

[0139] background

[0140] Injectable anti-CD3 / CD20 antibodies are bispecific antibodies that simultaneously target CD3 and CD20, belonging to the bispecific T-cell binder class. One end of the anti-CD3 / CD20 antibody binds to the CD20 antigen, and the other end binds to CD3 on the surface of T cells, bridging CD20-positive tumor cells and T cells. On one hand, it activates the CD3 signaling pathway by binding to TCR / CD3, thereby activating T cells; on the other hand, it forms an immune synapse with tumor cells at close range, leading to activated T cells specifically killing CD20-positive B cells. Anti-CD3 / CD20 antibodies can simultaneously exert antibody-induced direct apoptosis and T-cell-dependent cell killing mechanisms to achieve deeper B-cell clearance.

[0141] This clinical study of the anti-CD3 / CD20 antibody is the first human trial of an injectable anti-CD3 / CD20 antibody. It is a Phase I / II clinical study designed to evaluate the safety, tolerability, pharmacokinetic / pharmacodynamic (PK / PD) characteristics of intravenous monotherapy with the injectable anti-CD3 / CD20 antibody, and to preliminarily assess its efficacy. This invention will present comprehensive clinical data from dose escalation and dose expansion studies of the injectable anti-CD3 / CD20 antibody.

[0142] Research Objective

[0143] (1) Dose escalation study (Phase I)

[0144] Primary objective: To evaluate the safety and tolerability of anti-CD3 / CD20 antibodies in patients with relapsed / refractory CD20-positive B-cell non-Hodgkin lymphoma (B-NHL), to observe the dose-limiting toxicity (DLT) of anti-CD3 / CD20 antibodies, to determine the maximum tolerated dose (MTD) in humans (if any), and to determine the recommended phase II dose (RP2D) for subsequent clinical studies.

[0145] Secondary objectives: 1) To evaluate the pharmacokinetic (PK) characteristics of anti-CD3 / CD20 antibodies in relapsed / refractory CD20-positive B-NHL patients; 2) To evaluate the pharmacodynamic (PD) characteristics of anti-CD3 / CD20 antibodies in relapsed / refractory CD20-positive B-NHL patients; 3) To evaluate the immunogenicity of anti-CD3 / CD20 antibodies in relapsed / refractory CD20-positive B-NHL patients; 4) To preliminarily evaluate the efficacy of anti-CD3 / CD20 antibodies in relapsed / refractory CD20-positive B-NHL patients.

[0146] (2) Dosage expansion study (Phase II)

[0147] Primary objective: To evaluate the objective response rate (ORR) of anti-CD3 / CD20 antibodies in relapsed / refractory CD20-positive B-NHL patients.

[0148] Secondary objectives: 1) To evaluate the complete response rate (CRR), disease control rate (DCR), duration of response (DOR), time to response (TTR), progression-free survival (PFS), and overall survival (OS) of anti-CD3 / CD20 antibodies in relapsed / refractory CD20-positive B-NHL patients; 2) To evaluate the safety of anti-CD3 / CD20 antibodies in relapsed / refractory CD20-positive B-NHL patients; 3) To evaluate the pharmacokinetic (PK) characteristics of anti-CD3 / CD20 antibodies in relapsed / refractory CD20-positive B-NHL patients; 4) To evaluate the PD characteristics of anti-CD3 / CD20 antibodies in relapsed / refractory CD20-positive B-NHL patients; 5) To evaluate the immunogenicity of anti-CD3 / CD20 antibodies in relapsed / refractory CD20-positive B-NHL patients.

[0149] Study endpoints

[0150] (1) Dose escalation study (Phase I)

[0151] Primary study endpoint

[0152] Safety: 1) Adverse events (AEs), vital signs, physical examinations, laboratory tests, Eastern Cooperative Oncology Group (ECOG) scores, 12-lead electrocardiograms (ECGs), etc. during the study; 2) Incidence of DLT; 3) MTD (if any); 4) RP2D.

[0153] Secondary research endpoints

[0154] PK: including but not limited to the area under the plasma drug concentration-time curve (0-the concentration of the last test) (AUC) 0-t ), Area under the plasma drug concentration-time curve (0-∞) (AUC) 0-∞ Half-life 1 / 2 Peak Time (T) max Apparent Volume of Distribution (V) d ), plasma clearance (CL), and mean resident time (MRT).

[0155] PD: 1) Immune cells: B cells (CD19) + 1) T cells (CD3, CD4, CD8, CD25, CD69); 2) Immune cytokines: IL-2, IL-4, IL-6, IL-10, IFN-γ, TNF-α.

[0156] Immunogenicity: Anti-drug Antibody (ADA).

[0157] Therapeutic effects: ORR, CRR, DCR, DOR, TTR, PFS, OS.

[0158] (2) Dosage expansion study (Phase II)

[0159] Primary study endpoint

[0160] Therapeutic effect: ORR.

[0161] Secondary research endpoints

[0162] Other therapeutic effects: CRR, DCR, DOR, TTR, PFS, OS.

[0163] Safety: adverse events, vital signs, physical examination, laboratory tests, ECOG score, 12-lead ECG, etc. during the study.

[0164] PK: including but not limited to AUC 0-t AUC 0-∞ t 1 / 2 T max V d CL, MRT.

[0165] PD: 1) Immune cells: B cells (CD19) + 1) T cells (CD3, CD4, CD8, CD25, CD69); 2) Immune cytokines: IL-2, IL-4, IL-6, IL-10, IFN-γ, TNF-α.

[0166] Immunogenicity: ADA.

[0167] Research Design

[0168] This is a study in relapsed / refractory CD20 + This is an open-label, multicenter, dose-escalation and dose-expansion phase I / II clinical study in patients with B-NHL, designed to evaluate the safety, tolerability, PK / PD characteristics of intravenous monotherapy with injectable anti-CD3 / CD20 antibody, and to preliminarily assess the efficacy of injectable anti-CD3 / CD20 antibody.

[0169] This is the first human trial of an injectable anti-CD3 / CD20 antibody, including a dose escalation study (Phase I) and a dose expansion study (Phase II), enrolling histologically confirmed relapsed / refractory CD20-positive patients. + B-NHL patients, specifically as follows:

[0170] Dose escalation study (Phase I): Prior to receiving at least one systemic therapy containing an anti-CD20 monoclonal antibody; relapsed or refractory, or intolerant to previous treatment; diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) grades 1-3b, marginal zone lymphoma (MZL) (splenic, lymph node, extra-lymph node), mantle cell lymphoma (MCL), high-grade B-cell lymphoma (HGBCL), primary mediastinal large B-cell lymphoma (PMBCL), Richter's transformation, or transformed follicular lymphoma (DLBCL Arising from Follicular Lymphoma, trFL); or currently there is no suitable treatment option available to prolong survival.

[0171] Dose expansion study (Phase II):

[0172] The LBCL cohort includes DLBCL NOS, HGBCL, PMBCL, and trFL. Subjects were relapsed or refractory after receiving at least two prior systemic therapies (at least one immunochemotherapy regimen containing an anti-CD20 monoclonal antibody).

[0173] FL cohort: FL 1-3a; subjects who have relapsed or are refractory after receiving at least two prior systemic therapies and have previously received systemic therapy containing anti-CD20 monoclonal antibodies;

[0174] MCL cohort: Subjects who have relapsed or are refractory after receiving at least one prior systemic therapy and have previously received systemic therapy with a regimen containing an anti-CD20 monoclonal antibody and a BTK inhibitor.

[0175] The enrolled patients were histologically confirmed CD20-positive B-NHL patients.

[0176] Patients with chronic lymphocytic leukemia, Burkitt lymphoma, and lymphoplasmacytic lymphoma were excluded.

[0177] To reduce the risk of infusion-related reactions (IRR) or cytokine release syndrome (CRS), prophylactic administration is required before rituximab pretreatment and before each dose of anti-CD3 / CD20 antibody. Hospitalization for 72 hours is required after administration of the anti-CD3 / CD20 antibody pre-excitation dose and the first target dose.

[0178] Dose escalation studies

[0179] To minimize the number of patients receiving potentially lower than therapeutic doses, the Phase I dose exploration of the anti-CD3 / CD20 antibody began with an accelerated titration design, with one patient in each of the first and second dose groups, and multiple patients starting from the third dose group. A Bayesian Optimal Interval Design (BION) was employed, with a target toxicity rate set at 0.3. BION will guide dose escalation / de-escalation decisions for the anti-CD3 / CD20 antibody, while the final dose escalation / de-escalation decision will be based on recommendations from the DSMB.

[0180] A step-up dosing (SUD) regimen will be used, with a starting pre-stimulation dose of 0.05 mg. Based on preclinical pharmacokinetic studies of anti-CD3 / CD20 antibodies in cynomolgus monkeys indicating no accumulation, the regimen will be designed to escalate 3–4 doses within the same patient. The sponsor will analyze the obtained safety and tolerability data, and / or PK / PD data in real time. After discussion with the DSMB, appropriate adjustments may be made to the dosing strategy, dose group design, dosing regimen, dosing frequency, PK / PD blood collection points, and prophylactic medications. If the mean time to treatment (MTD) is not achieved by the 7th dose group, the sponsor and DSMB will discuss whether to continue escalating to higher doses, taking into account existing safety and tolerability, PK / PD, and preliminary efficacy data.

[0181] Rituximab was selected as the basis for pretreatment.

[0182] Preclinical in vivo and in vitro studies of anti-CD3 / CD20 antibodies [1] The results showed that anti-CD20 antibody pretreatment significantly cleared B cells from peripheral blood and secondary lymphoid organs, and reduced T cell activation and cytokine release in peripheral blood, thereby increasing the safety of anti-CD3 / CD20 antibody administration. Anti-CD20 antibody pretreatment was more effective and safer than the SUD regimen. (Phase I / II clinical trial (NCT03075696)) [2] This indicates that pretreatment with anti-CD20 antibody can effectively reduce the occurrence of severe CRS.

[0183] Rituximab can rapidly eliminate or reduce peripheral blood B cells to some extent. [3] This reduces the risk of CRS.

[0184] By consulting literature [3-5] It can be seen that rituximab has a better overall safety profile than oxotuzumab, and the incidence of grade 3-4 adverse events after administration is lower than that of oxotuzumab. See Tables 1 and 2 for details.

[0185] Table 1 compares the safety profiles of rituximab and octozumab as described in their product information.

[0186] *: Grade 3-5 thrombocytopenia.

[0187] Source: National Medical Products Administration. Rituximab Instructions for Use. May 15, 2024.

[0188] National Medical Products Administration. Oxtuzumab Instructions for Use. November 29, 2024.

[0189] Table 2. Meta-analysis of the safety profiles of rituximab and oxotuzumab. Abbreviations: CHOP = cyclophosphamide, doxorubicin, vincristine and prednisone; CLB = chlorambucil; G = oxotuzumab; IRR = infusion-related reaction; R = rituximab.

[0190] Source: Table 3 of reference 4.

[0191] Compared with oxotuzumab, rituximab has a good overall safety profile and a low incidence of hematologic toxicity, which can reduce the occurrence of delayed administration of anti-CD3 / CD20 antibodies in C1D1 and is more conducive to the implementation of C1D1 administration.

[0192] The basis for choosing rituximab pretreatment time as the day of administration (D-2) two days before the first dose of anti-CD3 / CD20 antibody.

[0193] Based on literature review, rituximab has a safety profile slightly better than or equal to that of oxutuzumab, and it rapidly clears or reduces peripheral blood B cells within 24 hours (<1 day) after administration. The favorable safety profile and rapid B-cell clearance of rituximab support its pretreatment on day D-2, reducing the risk of delayed administration of anti-CD3 / CD20 antibodies on day C1D1 and facilitating the first-dose administration of anti-CD3 / CD20 antibodies. Furthermore, compared to pretreatment with oxutuzumab on day D-7, pretreatment with rituximab on day D-2 reduces the number of hospital visits required by subjects, increasing clinical convenience and operability, and improving subject compliance. Therefore, rituximab was administered two days prior to the first dose of anti-CD3 / CD20 antibodies (day D-2).

[0194] Recovery after long-term delayed drug administration

[0195] If a subject delays anti-CD3 / CD20 antibody therapy for any reason (e.g., Covid-19 infection, pseudoprogression, or delayed remission), and the investigator and sponsor assess that the benefit outweighs the risk, treatment may be resumed. The SUD regimen should be restarted according to Table 4. If the subject has undergone rituximab pretreatment, rituximab pretreatment must be restarted. Hospitalization for 72 hours is recommended whenever possible when restarting the pre-excitation dose and the initial target dose.

[0196] Table 3. Dosage and infusion rate of anti-CD3 / CD20 antibodies * : Only for the dose escalation study group of scheme 2; # For subjects with high CRS risk, increased CRS risk, previous infusions that caused IRR / CRS, or subsequent doses that would increase the risk of IRR / CRS recurrence, the infusion time can be extended to 8h±15min. & If a subject experienced IRR / CRS during the previous dose, or if subsequent doses would increase the risk of IRR / CRS recurrence, the next infusion time may be extended to 4 h ± 15 min.

[0197] Abbreviations: CRS = Cytokine Release Syndrome; IRR = Infusion-Related Reaction; SUD = Incremental Dose.

[0198] Table 4. Recommendations for restarting treatment after delayed anti-CD3 / CD20 antibody therapy. Note: If a subject is given a new or direct dose of pre-excitation 1, pre-excitation 2, pre-excitation 3, or the first dose of C2D1, hospitalization for 72 hours is recommended. If a subject experienced IRR / CRS during the previous administration, hospitalization for 72 hours is also recommended for the next administration. * Prophylactic medication for IRR / CRS should be administered according to Table 5.

[0199] Poor efficacy due to insufficient dosage

[0200] During treatment, if the investigator determines that the subject's disease progression (PD) is confirmed by imaging examination or the treatment response is poor (according to the 2014 Lugano criteria), achieving only a partial response (PR) or less, but the safety is tolerable, the subject's subsequent treatment dose may be adjusted to the highest dose level that has been confirmed as safe by the completed DLT assessment, after discussion and confirmation with the investigator and the sponsor's medical monitor.

[0201] Selection criteria

[0202] Subjects who meet all of the following inclusion criteria are eligible to be enrolled in this study:

[0203] 1) Understand and voluntarily sign the written informed consent form (ICF), and are willing and able to comply with all trial requirements;

[0204] 2) Must be ≥18 years old and gender is not limited when signing the ICF;

[0205] 3) Histologically confirmed CD20-positive B-NHL, which must meet the following criteria depending on the stage of the study:

[0206] Dose escalation studies: Prior to receiving at least one systemic therapy containing an anti-CD20 monoclonal antibody; relapsed or refractory, or intolerant to previous treatment; DLBCL, FL grade 1-3b, MZL (splenic, lymph node, extra-lymph node), MCL, HGBCL, PMBCL, Richter's transformation, or trFL; or currently there is no suitable treatment option available to prolong survival.

[0207] Dose expansion studies:

[0208] The LBCL cohort includes DLBCL NOS, HGBCL, PMBCL, and trFL. Subjects must have relapsed or been refractory to disease after receiving at least two prior systemic therapies (at least one immunochemotherapy regimen containing an anti-CD20 monoclonal antibody). If available, results of all tissue examinations performed at the time of initial diagnosis must be provided, including but not limited to cell origin typing, BCL2 and MYC abnormalities; trFL subjects must also provide a pathology report at the time of disease transformation.

[0209] FL cohort: FL 1-3a; subjects who have relapsed or are refractory after receiving at least two prior systemic therapies and have previously received systemic therapy containing anti-CD20 monoclonal antibodies;

[0210] MCL cohort: Subjects who have relapsed or are refractory after receiving at least one prior systemic therapy and have previously received systemic therapy with a regimen containing an anti-CD20 monoclonal antibody and a BTK inhibitor.

[0211] 4) There is at least one two-dimensional measurable lesion, i.e., the longest diameter of the lymph node lesion is >1.5cm; the longest diameter of the extranodal lesion is >1cm;

[0212] 5) ECOG score: 0-1 point;

[0213] 6) Expected survival ≥ 3 months;

[0214] 7) Any toxicity caused by previous anti-tumor treatment must be restored to grade 1 of the Common Terminology Criteria for Adverse Events (CTCAE) v5.0 (excluding hair loss, fatigue, and loss of appetite);

[0215] 8) Left ventricular ejection fraction (LVEF) ≥ 50%;

[0216] 9) Laboratory tests conducted within 14 days prior to the screening test must meet the following criteria:

[0217] a) Hematological function: Without blood transfusion or other blood products, and without correction using granulocyte colony-stimulating factor (G-CSF) or other hematopoietic stimulating factors: Platelet count ≥75×10⁻⁶ 9 / L, neutrophil count (NEUT) ≥1.5×10 9 / L, hemoglobin ≥100g / L;

[0218] b) Liver function: Aspartate aminotransferase (AST) or alanine aminotransferase (ALT) ≤2.5 × Upper Limit of Normal (ULN); total bilirubin ≤1.5 × ULN (≤3 × ULN for subjects with a confirmed diagnosis of Gilbert's syndrome);

[0219] c) Renal function: Serum creatinine (SCr) ≤ 1.5 × ULN, or endogenous creatinine clearance (Ccr) ≥ 50 mL / min (calculated according to the Cockcroft-Gault formula);

[0220] d) Coagulation function: International Normalized Ratio (INR) or Prothrombin Time (PT) ≤ 1.5 × ULN; Activated Partial Thromboplastin Time (APTT) ≤ 1.5 × ULN.

[0221] Exclusion criteria

[0222] Subjects who meet any of the following criteria are ineligible for enrollment in this study:

[0223] 1) Patients with chronic lymphocytic leukemia, Burkitt lymphoma, and lymphoplasmacytic lymphoma;

[0224] 2) Current or past history of central nervous system (CNS) lymphoma;

[0225] 3) Having an active infection of grade ≥2 (CTCAE v5.0 standard) within 14 days prior to the first anti-CD3 / CD20 antibody infusion, including bacterial, viral, mycobacterial, parasitic or other infections (excluding nail bed fungal infection);

[0226] 4) Currently participating in other clinical trials or the time between the first anti-CD3 / CD20 antibody infusion and the end of the previous clinical trial (last treatment) is less than 28 days or 5 days since the drug in the clinical trial was administered. 1 / 2 (whichever is shorter), or plans to participate in other clinical trials concurrently with this study;

[0227] 5) History of immune-related adverse events occurring during previous immunotherapy treatments, as shown below:

[0228] Grade 3 or higher adverse events, excluding Grade 3 endocrine disorders treated with replacement therapy;

[0229] Grade 1 to 2 adverse events that did not reach baseline after discontinuation of medication.

[0230] 6) Received any of the following anti-tumor therapies prior to the first anti-CD3 / CD20 antibody infusion: ① within 28 days or 5 days after the first infusion of the drug. 1 / 2The following criteria apply to patients who have received anti-tumor treatment (including surgery, chemotherapy, targeted therapy, immunotherapy, radiotherapy, tumor embolization, etc.) within the past 14 days: ① received traditional Chinese medicine or proprietary Chinese medicine for anti-tumor treatment within the past 14 days; ③ received autologous stem cell transplantation or cell therapy within the past 100 days; ④ underwent allogeneic stem cell transplantation; ⑤ underwent solid organ transplantation; ⑥ used any anti-cancer vaccine.

[0231] 7) History of autoimmune diseases, including but not limited to myasthenia gravis, myositis, autoimmune hepatitis, systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, vascular thrombosis associated with antiphospholipid syndrome, Wegener's granulomatosis, Sjögren's syndrome, Guillain-Barré syndrome, multiple sclerosis, vasculitis, or glomerulonephritis; except for the following:

[0232] Patients with a history of autoimmune-related hypothyroidism who are currently receiving stable doses of thyroid replacement hormone therapy are eligible to be enrolled.

[0233] Patients with a history of disease-related immune thrombocytopenic purpura or autoimmune hemolytic anemia are eligible to enroll.

[0234] Patients with a history of type 1 diabetes and good control (defined as screening hemoglobin A1c <8% and no urinary ketoacidosis) are eligible to enroll.

[0235] 8) History of severe allergic reaction to monoclonal antibody therapy (CTCAE v5.0 criteria ≥ grade 3), or known allergy to any component of anti-CD3 / CD20 antibody or rituximab;

[0236] 9) Current or past history of CNS disease, such as stroke, epilepsy, CNS vasculitis, or neurodegenerative disease. Note: Based on the researcher's judgment, a history of stroke without a stroke or transient ischemic attack within the past 2 years and without residual neurological deficits is acceptable;

[0237] 10) Received (attenuated) live vaccine treatment within 28 days prior to the first anti-CD3 / CD20 antibody infusion, or is expected to receive (attenuated) live vaccine during the study period. Live vaccines include, but are not limited to: measles, mumps, rubella, varicella / shingles, yellow fever, rabies, Bacillus Calmette-Guerin (BCG), and typhoid vaccines. Injectable seasonal influenza vaccines are usually inactivated virus vaccines and are therefore permitted; however, intranasal influenza vaccines (e.g., This is a live attenuated vaccine, therefore its use is not permitted;

[0238] 11) Other serious, poorly controlled comorbidities that may affect trial protocol adherence or outcome interpretation, including but not limited to: clinically significant valvular heart disease; history of congestive heart failure; angina requiring medication or unstable angina; grade III or higher cardiac dysfunction (New York Heart Association, NYHA); QTc (calculated using the Fridricia correction formula QTcF = QT / (RR^0.33)) ≥470 ms (female) or ≥450 ms (male) or a history of congenital long QT syndrome; myocardial infarction, stroke, or intracranial hemorrhage within 6 months prior to screening; clinically significant arrhythmias (e.g., sustained ventricular tachycardia, ventricular fibrillation, torsades de pointes, severe atrioventricular block); poorly controlled hypertension. Good blood pressure (systolic blood pressure >160 mmHg or diastolic blood pressure >100 mmHg); poorly controlled diabetes (fasting blood glucose ≥10 mmol / L); clinically significant lung disease (e.g., obstructive pulmonary disease, history of bronchospasm, interstitial pneumonia, resting dyspnea or requiring oxygen therapy due to complications or comorbidities of malignant tumors, acute respiratory distress syndrome); presence of uncontrollable pleural effusion, pericardial effusion, or ascites requiring repeated drainage (once every 2 months or more) or medical intervention;

[0239] 12) Patients who are scheduled to undergo major surgery within 28 days prior to their first anti-CD3 / CD20 antibody infusion or during the study period, excluding diagnostic biopsy and central venous catheterization;

[0240] 13) History of any of the following diseases: ① Having another aggressive malignant tumor within the past 3 years, except for basal or squamous cell skin cancer that has been cured, or carcinoma in situ of the cervix, breast, or bladder; ② Progressive multifocal leukoencephalopathy (PML); ③ Severe bleeding disorders such as hemophilia A, hemophilia B, von Willebrand disease, or spontaneous bleeding requiring blood transfusions or other medical interventions;

[0241] 14) Systemic immunosuppressants (including but not limited to cyclophosphamide, azathioprine, methotrexate, thalidomide, and anti-tumor necrosis factor drugs) received within 2 weeks prior to the first anti-CD3 / CD20 antibody infusion are permitted. Corticosteroid therapy (<20 mg / day prednisone or equivalent) is permitted. Inhaled and topical steroids are permitted.

[0242] 15) Positive for Hepatitis B surface antigen (HBsAg) and / or Hepatitis B core antibody (HBcAb), and Hepatitis B virus deoxyribonucleic acid (HBV DNA) exceeding the normal range; HBsAg and / or HBcAb positive but do not agree to regular DNA testing, or do not receive antiviral prophylaxis; Positive for Hepatitis C virus (HCV) antibody and Hepatitis C virus ribonucleic acid (HCV RNA) exceeding the normal range; Positive for Human Immunodeficiency Virus (HIV) serological reaction;

[0243] 16) Pregnant and lactating women; for women of reproductive age who have not undergone sterilization: do not agree to use appropriate methods of contraception, such as oral contraceptives, intrauterine devices, barrier methods of contraception or spermicides, during treatment and for at least 12 months after the last dose of the study drug;

[0244] 17) For men who have not undergone sterilization: They do not agree to the use of barrier contraception during this treatment and for at least 12 months after the last dose of the study drug, and they do not agree to their partner using appropriate methods of contraception, such as oral contraceptives, intrauterine devices, barrier contraception, or spermicides.

[0245] 18) Other circumstances deemed unsuitable for participation in this study by the researchers.

[0246] Research period

[0247] The maximum treatment cycle for anti-CD3 / CD20 antibodies is 17 cycles (Q3W) until one of the following occurs: disease progression, intolerable toxicity, withdrawal of informed consent, death, loss to follow-up, initiation of new anti-tumor therapy, or other reasons requiring discontinuation of study treatment, whichever occurs first.

[0248] Safety assessment

[0249] All patients participating in this study will be assessed for safety during the screening period, weekly during cycles 1–2, every 3 weeks (±7 days) during cycles 3–6, and every 6 weeks (±7 days) during cycles 7–17, with a final assessment at the end of treatment or early withdrawal. Safety endpoints include adverse events (AEs), vital signs (respiration, temperature, pulse, blood pressure), physical examination, laboratory tests (complete blood count, blood biochemistry, urinalysis, coagulation function, serological virology, pregnancy tests), ECOG score, and 12-lead ECG. Adverse events are classified according to the Common Adverse Event Evaluation Criteria (CTCAE) v5.0.

[0250] In summary, the safety / tolerability results are evaluated as follows:

[0251] Dose escalation studies: Incidence and nature of DLT

[0252] Dose escalation and dose expansion studies: 1) Incidence and nature of adverse events (AEs); 2) Laboratory tests (complete blood count, blood biochemistry, urinalysis, coagulation function, serological virology); 3) Physical examination; 4) Vital signs (respiration, temperature, pulse, blood pressure); 5) 12-lead ECG; 6) ECOG score; 7) Pregnancy examination; 8) ADA test.

[0253] Therapeutic effect evaluation

[0254] During treatment, if the first cycle is 21 days, evaluations are performed every 6 weeks (±7 days); if the first cycle is 14 days, the initial evaluation of tumor efficacy is performed every 5 weeks (±7 days), followed by evaluations every 6 weeks (±7 days). Disease progression follow-up is performed every 9 weeks (±7 days), evaluating the following parameters.

[0255] ORR: Defined as the percentage of subjects who achieve CR and PR as the best therapeutic effect.

[0256] CRR: Defined as the percentage of subjects who achieve CR (complete response) as the best therapeutic effect.

[0257] DCR: Defined as the percentage of subjects who achieved CR, PR, or stable disease (SD) as their best response.

[0258] DOR: Defined as the time from the first tumor assessment of CR or PR to the first assessment of disease progression or death from any cause.

[0259] TTR: Defined as the time from the first administration of the investigational drug to the first assessment of CR or PR.

[0260] PFS: defined as the time from the first administration of the investigational drug to tumor progression or death from any cause.

[0261] OS: Defined as the time from the first administration of the experimental drug to death from any cause.

[0262] Researchers assessed tumor response using enhanced CT or MRI according to the 2014 Lugano criteria, ensuring consistency in the examination methods for each patient throughout the study. Tumor assessments were performed at baseline, after every two cycles, at the end of treatment (EOT), and every nine weeks before disease progression. During treatment, if investigators required confirmation of complete remission, they decided whether to add positron emission tomography / computed tomography (PET / CT). Adverse events (AEs) were assessed according to NCI CTCAE v5.0.

[0263] All enrolled patients signed the ICF (Independent Ethics Committee). Prior to the start of this study, each research center received review and approval from its Independent Ethics Committee (IEC) or Institutional Review Board (IRB). The study was conducted strictly in accordance with the protocol requirements, adhering to the Declaration of Helsinki, the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Guideline E6 for Clinical Trials, Good Clinical Practice (GCP), and the local laws and regulations of each research center.

[0264] investigational drugs

[0265] Anti-CD3 / CD20 antibody: will be administered via intravenous infusion at a body weight-independent dose. The dosage for each patient will depend on the dose level assigned to them. Each patient will receive the SUD regimen in cycle 1, followed by the target dose on day 1 of each subsequent cycle, with cycles 2 and thereafter occurring every 21 days.

[0266] Rituximab: If pretreatment is required, administer rituximab 375 mg / m² only on the day of first administration of the anti-CD3 / CD20 antibody (D-2) or at a time to be determined after discussion. 2 Or the dosage determined after discussion. Rituximab is a marketed drug; administer 375 mg / m² as per the instructions. 2The medication is administered only once, via intravenous infusion, based on baseline weight.

[0267] The intravenous infusion rate for the pre-excitation dose and the first target dose of anti-CD3 / CD20 antibody is 4 h ± 15 min. If no IRR / CRS occurs, the intravenous infusion rate for cycles 3 to 17 is 2 h ± 15 min. If CRS occurs and CRS-related symptoms appear during the infusion of the pre-excitation dose and the first target dose of CD3 / CD20 antibody, the administration of anti-CD3 / CD20 antibody must be stopped immediately. If the symptoms have subsided and persisted for 3 days, the next administration of anti-CD3 / CD20 antibody may be considered, and the next infusion time may be extended to 8 h ± 15 min. If, during cycles 3 to 17, the patient experienced IRR / CRS during the previous administration, or if subsequent treatment increases the risk of IRR / CRS recurrence, the infusion time may be extended to 4 h ± 15 min. See Table 3 for details.

[0268] Hospitalization for 72 hours is required for the pre-excitation dose and the first administration of the anti-CD3 / CD20 antibody. All dose groups should be administered for a maximum of 17 cycles until one of the following occurs: disease progression, intolerable toxicity, withdrawal of informed consent, death, loss to follow-up, initiation of new anti-tumor therapy, or other reasons for discontinuation of study treatment, whichever occurs first.

[0269] Tocilizumab

[0270] Anti-CD3 / CD20 antibodies may pose risks such as CRS (Compulsory Respiratory Syndrome). Common symptoms may include fever, hypotension, tachycardia, chills, hypoxia, and nausea. Anti-CD3 / CD20 antibodies should be administered in an environment where immediate transfer to an intensive care unit or similar facility is possible, along with the administration of tocilizumab (e.g., immediate resuscitation measures). Immediate access to steroids (dexamethasone 8–10 mg), epinephrine (1:1000 dilution), antihistamines (IV antihistamines), bronchodilators or equivalents, and oxygen therapy is essential.

[0271] Tocilizumab can be used as a rescue drug for the treatment of CRS. Tocilizumab can be provided by the sponsor, the research center, or procured locally by the research center for emergency purposes. Tocilizumab requires preparation, handling, and administration according to the instructions.

[0272] Preventive medication for IRR / CRS

[0273] To prevent IRR / CRS, prophylactic administration must be completed before rituximab or anti-CD3 / CD20 antibody infusion; the dosing regimen for preventing IRR / CRS is shown in Table 5.

[0274] Table 5. Dosage regimen for preventing IRR / CRS *: If pretreatment is required, administer rituximab 375 mg / m² intravenously to the subject on day 2 (D-2) before the first dose of anti-CD3 / CD20 antibody or at a time determined after discussion. 2 Or the dosage may be determined after discussion. Rituximab is a marketed drug; it is recommended to follow the instructions for use to complete the prophylactic administration. # : Only the dose escalation study group of scheme 2.

[0275] Abbreviations: IRR = Infusion-related reaction; CRS = Cytokine release syndrome.

[0276] Handling CRS

[0277] Based on non-clinical studies of anti-CD3 / CD20 antibodies and referencing clinical studies of anti-CD3 / CD20 bispecific antibodies, the earliest CRS events occurred 3–47 hours after the most recent administration (estimated median time to occurrence was 11 hours), primarily during the first cycle of treatment. The main manifestations of CRS include fever, tachypnea, headache, tachycardia, hypotension, rash, and / or hypoxia due to cytokine release. CRS should be assessed according to the 2019 American Society for Transplantation and Cellular Therapy (ASTCT) consensus grading criteria (Lee DW, et al. Biol Blood Marrow Transplant. 2019, 25(4):625-638.). The management of CRS also referenced the 2024 Chinese Society of Clinical Oncology (CSCO) Guidelines for the Management of Infections Related to CAR-T Cell Therapy for Hematologic Malignancies and Immunotherapy, and the 2023 Mayo Clinic Guidelines for the Management of CRS / Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS) with Dual Antibody Therapy. Treatment and infusion rate adjustments and / or treatment interruption / stopping for CRS depend primarily on severity. The management of CRS following anti-CD3 / CD20 antibody administration is outlined in the table below. To minimize the risk of CRS during the SUD dosing phase, aggressive intervention should be initiated early in the course of CRS to reduce the likelihood of progression to higher severity levels. See Table 6 for details.

[0278] Table 6. Management of CRS following administration of anti-CD3 / CD20 antibodies.

[0279] ASTCT = American Society for Transplantation and Cell Therapy; CRS = Cytokine Release Syndrome; G-CSF = Granulocyte Colony-Stimulating Factor; ICU = Intensive Care Unit; ICANS = Immune Effector Cell-Related Neurotoxicity Syndrome.

[0280] The management of different grades of CRS also referenced the 2024 Chinese Society of Clinical Oncology (CSCO) Guidelines for the Management of Infections Related to CAR-T Cell Therapy for Hematologic Malignancies and Immunotherapy, as well as the 2023 Mayo Clinic Guidelines for the Management of CRS / ICANS with Dual Antibody Therapy.

[0281] a Cytokine release syndrome should be assessed according to the 2019 ASTCT consensus rating criteria (Lee DW, et al, 2019).

[0282] b During a single CRS episode, tocilizumab should not be administered more than 4 times, with a single dose ≤800mg.

[0283] c Because subjects may receive corticosteroid prophylaxis, the febrile response may be attenuated. Therefore, all adverse events attributable to CD3 / CD20 antibodies that meet the diagnostic criteria for IRR or CRS and are associated with fever, hypotension, or hypoxemia (not attributable to any other cause) will be reported separately as CRS. CRS events presenting as hypotension and / or without fever should be graded according to the management required for hypotension and / or hypoxemia. These types of events correspond to a minimum ASTCT grade 2. Other adverse events occurring within 24 hours of CD3 / CD20 antibody administration should be reported as individual adverse events (e.g., headache or chills).

[0284] d For subjects receiving high-dose corticosteroid treatment for CRS, antifungal prophylaxis is strongly recommended.

[0285] e For subjects who demonstrate benefit and whose adverse events have completely resolved, resumption of CD3 / CD20 antibody therapy may be considered. CD3 / CD20 antibodies should only be re-administered after obtaining explicit approval from the investigator and sponsor. No further treatment should be given unless all of the following criteria are met:

[0286] The researchers' individual risk-benefit assessment of the subjects was favorable for continued treatment;

[0287] The subject's previous toxicity has been cured and his organ function is good, making him eligible for further treatment;

[0288] The subjects were fully informed of the risks associated with continuing treatment, and the subjects decided to continue receiving subsequent treatment;

[0289] The follow-up dosing plan was adequate, and preventative measures were taken, including dose reduction, mandatory hospitalization, and enhanced prophylactic dosing.

[0290] Handling ICANS occurrences

[0291] Neurological adverse events should be closely monitored during the trial. All subjects should undergo a baseline neurological examination as part of a physical examination before the first administration of anti-CD3 / CD20 antibody.

[0292] ICANS should be assessed according to the 2019 ASTCT consensus rating criteria (Lee DW, et al. Biol Blood Marrow Transplant. 2019, 25(4):625-638.). Management of ICANS also referenced the 2024 CSCO Guidelines for the Management of Infections Related to CAR-T Cell Therapy for Hematologic Malignancies and Immunotherapy, and the 2023 Mayo Clinic Guidelines for the Management of CRS / ICANS with Bispecific Antibody Therapy. Treatment and infusion rate adjustments and / or treatment interruptions / stops for ICANS depend primarily on severity, and the following table outlines the management of ICANS following anti-CD3 / CD20 antibody therapy. Based on experience with anti-CD3 / CD20 bispecific antibodies, neurological symptoms of ICANS may occur during CRS symptoms or are more common after a CRS response. The main manifestations include: aphasia, altered level of consciousness, cognitive impairment, motor weakness, seizures, and cerebral edema. Early intervention with ICANS is crucial to reduce the likelihood of progression to a higher level of illness. Consultations with specialists such as neurologists should be sought if necessary. See Table 7 for details.

[0293] Table 7. Management of ICANS following administration of anti-CD3 / CD20 antibodies. Note: Because tocilizumab binds to IL-6R, it increases serum free IL-6 levels, further increasing IL-6 concentration in cerebrospinal fluid, potentially exacerbating neurotoxicity. In ICANS management, corticosteroids are more important than tocilizumab. a: If the epilepsy is ICANS grade 3-4, anti-CD3 / CD20 antibody therapy should be permanently discontinued. b: Investigators should assess the overall benefit-risk of continuing anti-CD3 / CD20 antibody therapy and determine this in consultation with the sponsor.

[0294] Statistical analysis

[0295] This study is a dose escalation study and a dose expansion study. There are no formal research hypotheses. The sample size for the dose escalation study will be determined according to the BOIN design, with 7 dose groups and 17-42 participants. For the dose expansion study, the LBCL cohort, FL cohort, and MCL cohort will each have a maximum of 30 participants, with a maximum of about 90 participants in total, for a total of about 107-132 participants.

[0296] All patients receiving anti-CD3 / CD20 antibodies were analyzed by dose group and according to the Statistical Analysis Plan (SAP). The proportions of subjects with CR, PR, SD, PD, and Not Evaluated (NE) outcomes were presented and summarized according to the 2014 Lugano criteria. ORR, CRR, and DCR were calculated, and 95% confidence intervals (CIs) for these percentages were calculated using the Clopper-Pearson method. Descriptive analysis was performed on TTR. Median DOR, median PFS, median OS, and their 95% CIs were estimated using the Kaplan-Meier method, and survival curves were plotted.

[0297] Statistical analysis uses Calculations are performed using statistical analysis software version 9.4 or higher.

[0298] Example 2: Clinical study results of regimen 1 using two-step pre-excitation dosage

[0299] Test methods

[0300] Research Design

[0301] In regimen 1, dose groups 1-3 did not receive rituximab pretreatment. In cycle 1, anti-CD3 / CD20 antibody was administered via injection using a step-up dosing (SUD) regimen (see Figure 1). Pre-excitation dose 1 was administered at C1D1, and pre-excitation dose 2 was administered at C1D8. The target dose was administered at D1 in cycle 2 and subsequent cycles, for a maximum of 17 cycles. Cycle 1 was 14 days, and cycles 2-17 were 21 days each.

[0302] Clinical research results

[0303] patient population

[0304] Protocol 1 enrolled 5 patients with relapsed / relapsed b-negative neutrophils (R / R B-NHL) across three dose groups. Dose group 1 consisted of 0.05 / 0.15 / 0.45 mg (1 patient), dose group 2 consisted of 0.5 / 1.5 / 4.5 mg (1 patient), and dose group 3 consisted of 1 / 3 / 9 mg (3 patients). Protocol 1 did not involve pre-stimulation dose modification. In the target dose group of 9 mg, one of the 3 patients experienced a DLT event (grade 3 neutropenia, which did not recover to grade ≤2 within one week without growth factor support). However, clinical experts considered neutropenia a common adverse event in hematology, and treatment with granulocyte colony-stimulating factors could lead to rapid recovery. The definition of DLT was subsequently revised to allow growth factor treatment for neutropenia, and no subsequent enrolled patients experienced this DLT event.

[0305] The median age (minimum, maximum) of the 5 participants in scheme 1 was 56 (51, 68) years, 60% of whom were female, and the mean body mass index (BMI) was 22.83 kg / m². 2 80% of the participants had a US ECOG score of 1. The median number of prior lines of treatment was 3 (1, 4); 80% of the participants were refractory to previous anti-tumor therapy, 80% were refractory to previous anti-CD20 monoclonal antibody therapy, and 60% were in clinical stage III-IV. 40% of the participants had bone marrow involvement at baseline, 60% had extranodal lesions, and 40% had abnormally elevated lactate dehydrogenase at baseline. Four participants (80%) had invasive relapsed refractory B-NHL at baseline, all of whom had DLBCL, of which 3 (60%) had non-germinal center B-cell (non-GCB); one participant (20%) had indolent relapsed refractory B-NHL at baseline, which was FL.

[0306] Security

[0307] Five subjects across the three dosage groups received at least one dose of anti-CD3 / CD20 antibody and were included in the safety analysis set. The median treatment duration was 141 (15, 357) days, the mean number of treatment cycles (min, maximum) was 9.4 (2, 17), and the mean drug exposure (min, maximum) was 66.3 (3.35, 150.5) mg. Exposure adherence in all subjects ranged from 80% to 120%, indicating good adherence to anti-CD3 / CD20 antibody treatment in all subjects.

[0308] The incidence of TEAEs was 100% (5 cases); the incidence of adverse events (TRAEs) related to anti-CD3 / CD20 antibodies was 100% (5 cases); the incidence of grade ≥3 TEAEs was 80% (4 cases); the incidence of SAEs was 40% (2 cases); the incidence of SAEs related to anti-CD3 / CD20 antibodies was 40% (2 cases); the incidence of adverse events of particular concern was 60% (3 cases); the incidence of TEAEs leading to dose reduction was 20% (1 case); the incidence of TEAEs leading to discontinuation of treatment was 80% (4 cases); no TEAEs leading to death, reduction of infusion rate, permanent discontinuation of treatment, or withdrawal from the trial occurred. See Table 10 for details.

[0309] Common TRAEs (≥10%) included: neutrophil count decreased by 60% (3 cases), white blood cell count decreased by 60% (3 cases), platelet count decreased by 60% (3 cases), hypoalbuminemia by 60% (3 cases), lymphocyte count decreased by 40% (2 cases), cytokine levels increased by 40% (2 cases), serum bilirubin increased by 40% (2 cases), fever by 40% (2 cases), and anemia by 40% (2 cases); other conditions occurred in 1 case each (20%), including: cytokine release syndrome, decreased fibrinogen, elevated transaminases, hypokalemia, and asthenia. One case of CRS occurred, grade 2; no grade ≥3 CRS occurred. This occurred 3.7 hours after the first administration of anti-CD3 / CD20 antibody infusion, with symptoms of fever and hypoxemia. The patient recovered after symptomatic treatment with tocilizumab and did not discontinue medication due to CRS. The CRS resolved after 4 days (see Table 9 for details).

[0310] Four subjects (80%) experienced grade ≥3 TRAEs, including: a 40% decrease in neutrophil count (2 cases) and an increase in serum bilirubin by 40% (2 cases); the other cases were one in each (20%), including: a decrease in lymphocyte count, a decrease in white blood cell count, an increase in transaminase, herpes zoster, abdominal infection, and diarrhea.

[0311] Two subjects (40%) developed SAEs associated with anti-CD3 / CD20 antibodies, including herpes zoster, abdominal infection, and elevated transaminase levels, one of which (20%).

[0312] Validity

[0313] Five subjects from each of the three dosage groups were included in the efficacy analysis set. The median follow-up time for overall survival (OS) was 18.6 (range 17.9–20.1) months. The first enrolled subject achieved partial response (PR) with the target dose of only 0.45 mg, suggesting that the clinically effective dose is 0.45 mg. In the five patients with relapsed / refractory B-NHL, the complete response rate (CRR) was 40% (2 / 5), the objective response rate (ORR) was 60% (3 / 5), and the disease control rate (DCR) was 80% (4 / 5). In the four patients with relapsed / refractory DLBCL, the CRR was 25% (1 / 4), the ORR was 50% (2 / 4), and the DCR was 75% (3 / 4). In the one patient with relapsed / refractory FL, the CRR, ORR, and DCR were all 100% (1 / 1). In the target dose ≥9 mg group, the CRR and ORR were both 67% (2 / 3), and the DCR was 100% (3 / 3). See Table 8 for details.

[0314] Pharmacodynamics

[0315] Five subjects were included in the pharmacodynamic analysis set. Analysis of the peripheral blood B-cell percentage in the subjects showed a continuous downward trend after administration, with peripheral blood B cells essentially cleared and maintained in this state throughout subsequent dosing cycles. One subject had a baseline B-cell percentage of 36.01%, which gradually decreased to 0.1% after the first three doses of anti-CD3 / CD20 antibody. This subject experienced grade 2 CRS after administration of C1D1 and a DLT event after administration of C1D8 (although this DLT event is a common hematological toxicity that resolves quickly with treatment).

[0316] Analysis of cytokine changes before and after drug administration in 5 subjects across 3 dosage groups showed that the lower dose levels in dosage groups 1 and 2 resulted in milder or no significant changes in cytokine release. In dosage group 3, 2 out of 3 subjects showed higher levels of cytokine (IL-6, IL-10) release after the first cycle of administration. See Figure 2 for details.

[0317] Immunogenicity

[0318] Five subjects were included in the immunogenicity analysis set. All five subjects across the three dose groups showed negative results for ADA before and after anti-CD3 / CD20 antibody administration.

[0319] discuss

[0320] Preliminary clinical results for Protocol 1 indicate that the overall safety profile of the anti-CD3 / CD20 antibody in R / R B-NHL is manageable, with one subject experiencing a DLT event. The higher pre-excitation dose 1 (1 mg) in the third dose group led to higher levels of cytokine release in two subjects, resulting in a grade 2 CRS event in one of them. The subject recovered after treatment with glucocorticoids and tocilizumab; furthermore, the peripheral blood B cells in this subject were not rapidly cleared, requiring three doses of the anti-CD3 / CD20 antibody to achieve clearance.

[0321] Anti-CD3 / CD20 antibodies exhibit preliminary antitumor activity in relapsed / relapsed (R / R) B-NHL. Even before dose escalation to the recommended phase II dose (RP2D), efficacy results similar to those of similar drugs (RP2D) can be achieved. In the target dose group of 9 mg, the critical response rate (CRR) for B-NHL patients was 40%, and the objective response rate (ORR) was 60%. One R / R FL patient achieved 100% CRR, ORR, and disease control rate (DCR). The first enrolled patient achieved partial response (PR) at a target dose of only 0.45 mg.

[0322] Example 3: Clinical study results of regimen 2 using a 3-step pre-excitation dosage.

[0323] Test methods

[0324] Research Design

[0325] In Protocol 2, three subjects were enrolled in one dose group without rituximab pretreatment. An additional pre-excitation dose was added to the protocol 1 dose group to investigate whether increasing the number of pre-excitation doses improved subject safety and reduced the risk of CRS. In the first cycle of Protocol 2, the anti-CD3 / CD20 antibody was administered via the SUD regimen. Pre-excitation dose 1 was given at C1D1, pre-excitation dose 2 at C1D8, and pre-excitation dose 3 at C1D15. The target dose was given on day 1 of the second and subsequent cycles, for a maximum of 17 cycles, with one cycle every 21 days.

[0326] Clinical research results

[0327] patient population

[0328] In Protocol 2, three R / R B-NHL subjects were enrolled in one dose group, with doses of 0.5 / 1 / 3 / 9 mg. No DLT events occurred in this dose group, but two of the three subjects experienced grade 2 CRS, one of whom was treated with tocilizumab.

[0329] The median age (minimum, maximum) of the three participants in scheme 2 was 55 (54, 66) years, all of whom were female and had a BMI of 23.77 kg / m².2 67% of the participants had a US ECOG score of 1. The median number of prior lines of treatment was 2 (2, 4); 67% of the participants were refractory to previous anti-tumor therapy and refractory to previous anti-CD20 monoclonal antibody therapy, and 67% were in stage III-IV. 67% of the participants had no baseline bone marrow involvement, 67% had extranodal lesions, and 67% had abnormally elevated baseline lactate dehydrogenase. Two participants (67%) had invasive relapsed-refractory B-NHL with baseline pathological type (DLBCL non-GCB), and one participant (33%) had indolent relapsed-refractory B-NHL with baseline pathological type (FL 3a).

[0330] Security

[0331] All three subjects in one dose group received at least one dose of anti-CD3 / CD20 antibody and were included in the safety analysis set. The median treatment duration was 55 (37, 442) days, the mean number of treatment cycles (min, maximum) was 7.3 (2, 17), and the mean drug exposure (min, maximum) was 70.0 mg (25.5, 153.0). Exposure adherence in all subjects ranged from 80% to 120%, indicating good adherence to anti-CD3 / CD20 antibody treatment in all subjects.

[0332] The incidence of TEAE was 100% (3 cases), the incidence of TRAE was 100% (3 cases), the incidence of ≥ grade 3 TEAE was 100% (3 cases), the incidence of ≥ grade 3 TRAE was 67% (2 cases), the incidence of SAE was 67% (2 cases), the incidence of SAE related to anti-CD3 / CD20 antibody was 33% (1 case), the incidence of adverse events of particular concern was 100% (3 cases), the incidence of TEAE leading to discontinuation of treatment was 100% (3 cases), the incidence of TEAE leading to permanent discontinuation of treatment was 33% (1 case), the incidence of TEAE leading to withdrawal from the trial was 33% (1 case), and no TEAEs leading to death, dose reduction, or infusion rate reduction occurred. See Table 10 for details.

[0333] Common TRAEs (≥10%) included: decreased neutrophil count (67%) (2 cases), decreased white blood cell count (67%) (2 cases), cytokine release syndrome (67%) (2 cases), and decreased lymphocyte count (67%) (2 cases); other conditions occurred in 1 case each (33%), including: decreased platelet count, hypoalbuminemia, increased cytokines, decreased fibrinogen, fever, anemia, increased bilirubin, hypokalemia, increased transaminases, and weakness. Two cases of CRS occurred, both grade 2; no grade ≥3 CRS occurred. These occurred 9.5 h–116 h after the start of anti-CD3 / CD20 antibody infusion, with symptoms of fever and hypoxemia. One patient was treated with tocilizumab, and both recovered after symptomatic treatment without discontinuing medication due to CRS. CRS resolved within 4 h–5 days (see Table 9 for details).

[0334] Two subjects (67%) experienced grade ≥3 TRAEs, including a 67% decrease in neutrophil count (2 cases); the other cases were 1 case (33%), including septic shock and decreased lymphocyte count.

[0335] One patient (33%) developed SAE associated with anti-CD3 / CD20 antibodies, which was septic shock.

[0336] Validity

[0337] All three subjects in one dose group of regimen 2 were included in the efficacy analysis set. The median follow-up time for overall survival (OS) was 16.1 (NA, NA) months. The CRR was 33% (1 / 3) in the three relapsed / refractory B-NHL subjects, and the ORR and DCR were both 67% (2 / 3). The ORR and DCR were both 50% (1 / 2) in the two relapsed / refractory DLBCL subjects. The CRR, ORR, and DCR were all 100% (1 / 1) in the one relapsed / refractory FL subject. See Table 8 for details.

[0338] Pharmacodynamics

[0339] Three subjects in scheme 2 were included in the pharmacodynamic analysis set. Peripheral blood B cell analysis of the subjects showed that in two of the three subjects, the peripheral blood B cell count was higher after each administration of anti-CD3 / CD20 antibody during the DLT observation period, indicating that the peripheral blood B cells were not cleared.

[0340] Analysis of cytokine changes before and after drug administration in three subjects in Protocol 2 showed that two subjects had higher cytokine (IL-6, IL-10) release levels after the first cycle of drug administration, while one subject already had a higher cytokine (IL-10) release level before the first administration, indicating individual differences. See Figure 2 for details.

[0341] Immunogenicity

[0342] Two subjects were included in the immunogenicity analysis set. Two subjects in one dose group showed negative results for ADA both before and after anti-CD3 / CD20 antibody administration.

[0343] discuss

[0344] Preliminary clinical results from Protocol 2 indicate that the overall safety profile of the anti-CD3 / CD20 antibody in R / R B-NHL is manageable, with no DLT events occurring. Two cases of CRS occurred in this dose group. In one case, a high level of cytokine release occurred after administration of pre-excitation dose 1 (0.5 mg), leading to a grade 2 CRS event, which resolved after treatment with glucocorticoids and tocilizumab. In the other case, a high level of cytokine release was present before administration, and a grade 2 CRS event occurred after administration of pre-excitation dose 2 (1 mg), which resolved after treatment with glucocorticoids and oxygen therapy. This suggests that increasing the frequency of pre-excitation doses did not significantly clear peripheral blood B cells, did not significantly increase subject safety, and did not reduce the risk of CRS.

[0345] Anti-CD3 / CD20 antibodies exhibited preliminary antitumor activity in relapsed / relapsed (R / R) benign prostatic hyperplasia (B-NHL). Even without dose escalation to RP2D, efficacy results similar to those of similar drugs were achieved, with a CRR of 33% and an ORR of 67% in this dose group of B-NHL patients. One R / R FL patient achieved 100% CRR, ORR, and DCR.

[0346] Example 4: Clinical study results of regimen 3 using rituximab pretreatment

[0347] Test methods

[0348] Research Design

[0349] Given that increasing the number of pre-excitation doses in Option 2 did not significantly improve subject safety and tolerability, nor reduce the risk of CRS, Option 3 was adopted, administering rituximab 375 mg / m² on day D-2 before the first dose of anti-CD3 / CD20 antibody. 2 Pretreatment was performed to investigate whether rituximab pretreatment prior to the first dose of anti-CD3 / CD20 antibody to eliminate peripheral blood B cells increased subject safety and reduced the risk of CRS. In cycle 1, the injectable anti-CD3 / CD20 antibody was administered via the SUD regimen. Pre-stimulation dose 1 was given at C1D1, and pre-stimulation dose 2 at C1D8. The target dose was given on D1 of cycles 2 and subsequent cycles, for a maximum of 17 cycles. Cycle 1 was 14 days, and cycles 2–17 were administered every 21 days.

[0350] Clinical research results

[0351] patient population

[0352] Protocol 3 enrolled 14 patients with relapsed / relapsed b-NHL across three dose groups. Dose group 1 consisted of D-2 rituximab at doses of 1 / 5 / 15 mg, with 8 patients enrolled; dose group 2 consisted of D-2 rituximab at doses of 1 / 5 / 20 mg, with 3 patients enrolled; and dose group 3 consisted of D-2 rituximab at doses of 1 / 5 / 25 mg, with 3 patients enrolled.

[0353] The median age (minimum, maximum) of the 14 participants in scheme 3 was 61.5 (27, 71) years, with 57.1% being female and a BMI of 26.69 kg / m². 2 Of the participants, 36% had an ECOG score of 1. The median number of prior lines of treatment was 2 (1, 4); 64% were refractory to previous anti-tumor therapy, 64% were refractory to previous anti-CD20 monoclonal antibody therapy, and 79% were stage III-IV. 21% of participants had bone marrow involvement at baseline, 57% had extranodal lesions, and 36% had abnormally elevated lactate dehydrogenase at baseline. Six participants (43%) had aggressive relapsed-refractory B-NHL at baseline, including one case of primary mediastinal large B-cell lymphoma and five cases of DLBCL, of which one case (20%) was DLBCL non-GCB. Eight participants (57%) had indolent relapsed-refractory B-NHL at baseline, all of whom were FL grade 1-2.

[0354] Security

[0355] Fourteen subjects across the three dosage groups received at least one dose of anti-CD3 / CD20 antibody and were included in the safety analysis set. The median treatment duration was 120.0 (15, 349) days, the mean number of treatment cycles (min, maximum) was 7 (2, 17), and the mean drug exposure (min, maximum) was 115.0 (21.0, 246.0) mg. Exposure adherence in all subjects ranged from 80% to 120%, indicating good adherence to CD3 / CD20 antibody treatment in all subjects.

[0356] The incidence of TEAE was 86% (12 cases), the incidence of TRAE was 86% (12 cases), the incidence of ≥ grade 3 TEAE was 29% (4 cases), the incidence of SAE was 14% (2 cases), the incidence of SAE related to anti-CD3 / CD20 antibody was 14% (2 cases), the incidence of adverse events of particular concern was 50% (7 cases), the incidence of TEAE leading to discontinuation of treatment was 50% (7 cases), and no TEAEs leading to death, reduction of infusion rate, dose reduction, permanent discontinuation of treatment, or withdrawal from the trial occurred. See Table 10 for details.

[0357] Common TRAEs (≥10%) included: cytokine release syndrome (CRS) in 36% (5 cases), decreased neutrophil count in 29% (4 cases), decreased white blood cell count in 21% (3 cases), fever in 14% (2 cases), anemia in 14% (2 cases), and hypokalemia in 14% (2 cases). Five cases (36%) of six episodes of CRS occurred, the vast majority being grade 1, with only one episode being grade 2. These occurred 8.8 h to 42.4 h after the start of CD3 / CD20 antibody infusion, presenting with fever and hypotension. All cases resolved with symptomatic treatment, and no medication was discontinued due to CRS. CRS resolved within 0.7 to 16.4 days (see Table 9 for details). At target doses increased to near RP2D, the incidence and severity of CSR were lower than in regimen 2.

[0358] Four subjects (29%) experienced grade ≥3 TRAEs, including decreased neutrophil count, decreased lymphocyte count, infectious pneumonia, and upper respiratory tract infection, each of which was 1 case (7%).

[0359] Two subjects (14%) developed SAEs associated with anti-CD3 / CD20 antibodies, including infectious pneumonia and elevated transaminase levels, one of which (7%).

[0360] Validity

[0361] Fourteen subjects from the three dose groups in regimen 3 were included in the efficacy analysis set. The median follow-up time for overall survival (OS) was 5.3 (range 2.0–6.7) months. In the 14 evaluable patients with relapsed / refractory B-NHL, the objective response rate (ORR) was 64% (9 / 14), and the disease control rate (DCR) was 86% (12 / 14). In the 5 patients with relapsed / refractory DLBCL, the ORR was 40% (2 / 5), and the DCR was 80% (4 / 5). In the 8 patients with relapsed / refractory FL, the ORR was 75%, and the DCR was 88% (7 / 8). See Table 8 for details.

[0362] Pharmacodynamics

[0363] Fourteen subjects from the three dosage groups in Protocol 3 were included in the pharmacodynamic analysis set. Peripheral blood B cell analysis of the subjects showed that: in 10 of the 14 subjects, peripheral blood B cell levels were significantly reduced after D-2 rituximab pretreatment, and 8 of them achieved B cell clearance; after administration of anti-CD3 / CD20 antibody, peripheral blood B cells were further cleared, achieving deep clearance and maintaining clearance.

[0364] Analysis of cytokine changes before and after administration in 14 subjects across the three dose groups in Protocol 3 showed that: In all 14 subjects, cytokine release after D-2 rituximab pretreatment followed by anti-CD3 / CD20 antibody administration was relatively mild or showed no significant change. In some subjects, cytokine release was higher after the first target dose than after the pre-excitation dose. One subject (subject number: S08001) had a large tumor burden (Ann Arbor stage IV, two confluent lesions, the largest of which had a long diameter of 8.41 cm), placing them in a high-risk group for CRS. In this subject, the release levels of certain cytokines (IL-6, IL-10, IFN-γ) increased at different visits after the first cycle and the first target dose administration, but the release was relatively mild. See Figure 2 for details.

[0365] Immunogenicity

[0366] Thirteen subjects were included in the immunogenicity analysis set. All 13 subjects across the three dose groups showed negative results for ADA before and after anti-CD3 / CD20 antibody administration.

[0367] discuss

[0368] Preliminary clinical results from Protocol 3 indicate that the overall safety of anti-CD3 / CD20 antibodies in R / R B-NHL is manageable. Pretreatment with D-2 rituximab to eliminate peripheral blood B cells in all three dose groups resulted in generally mild or no significant changes in cytokine release levels after anti-CD3 / CD20 antibody administration. Although the target dose in Protocol 3 is close to the RP2D compared to the target doses in Protocols 1 and 2, only 5 subjects (36%) experienced CRS, and the vast majority (4 subjects) experienced grade 1 CRS, with only one subject experiencing a sub-grade 2 CRS. All CRS events resolved with symptomatic treatment. This suggests that pretreatment with D-2 rituximab can significantly reduce peripheral blood B cell levels and the magnitude of cytokine release after anti-CD3 / CD20 antibody administration, thereby increasing subject safety and significantly reducing the incidence and severity of CRS. This indicates that the pretreatment method before the first administration of anti-CD3 / CD20 antibody significantly improves the safety of anti-CD3 / CD20 antibody administration and is a powerful measure to reduce the risk of CRS. It enables the rapid escalation of anti-CD3 / CD20 antibody to the target dose. This pretreatment method supports dose escalation in dose escalation studies and the early attainment of RP2D of anti-CD3 / CD20 antibody.

[0369] Anti-CD3 / CD20 antibodies exhibit preliminary antitumor activity in relapsed / relapsed (R / R) benign prostatic hyperplasia (B-NHL). Even without dose escalation to RP2D, similar efficacy to RP2D drugs can be achieved. In overall regimen 3, the ORR for B-NHL patients in the target dose group (15–25 mg) was 64%, and the DCR was 86%. In 8 R / R FL patients, the CRR was 50%, the ORR was 75%, and the DCR was 88%.

[0370] Table 8 Summary of efficacy results for each dosage group

[0371] Table 9 Summary of subjects who experienced CRS in each dosage group Note: Cell clearance* refers to the clearance of peripheral blood B cells before the first administration of the anti-CD3 / CD20 antibody.

[0372] Table 10 Summary of adverse events for each dosage group

[0373] The exemplary sequence information of this application is as follows.

[0374] References

[0375] [1] Marina B, Sara Colombetti, Sylvia Herter, et al. CD20-TCB with Obinutuzumab Pretreatment as Next-Generation Treatment of Hematologic Malignancies[J]. Clin Cancer Res. 2018, 24(19): 4785-4797.

[0376] [2]Martin H, Franck M, Gloria I, et al. Glofitamab, a Novel, Bivalent CD20-Targeting T-Cell Engaging Bispecific Antibody, Induces Durable Complete Remissions in Relapsed / Refractory B-Cell Lymphoma: A Phase I Trial[J].J Clin Oncol.2021,39(18):1959-1970.

[0377] [3]Gilles S,Martin B,Robin F,et al.Rituximab in B-Cell Hematologic Malignancies:A Review of 20Years of Clinical Experience[J].Adv Ther.2017,34(10):2232-2273.

[0378] [4]Irina A, Anat GG, Liat SA, et al. Obinutuzumab-related adverse events: A systematic review and meta-analysis[J]. Hematol Oncol. 2021, 39(2): 215-221.

[0379] [5] National Medical Products Administration. Rituximab Instructions for Use. May 15, 2024.

[0380] [6] National Medical Products Administration. Oxtuzumab Instructions for Use. November 29, 2024.

[0381] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. The use of an anti-CD3 / CD20 bispecific antibody in the preparation of a medicament for the treatment of CD20-positive B-cell non-Hodgkin lymphoma (B-NHL), characterized in that, Before administering anti-CD3 / CD20 bispecific antibody to the patient, administer type I anti-CD20 antibody; the anti-CD3 / CD20 bispecific antibody includes a first binding portion that specifically binds to CD20, a second binding portion that specifically binds to CD3, and a third binding portion that specifically binds to CD20, and the first binding portion and the third binding portion that specifically binds to CD20 are both derived from type II anti-CD20 antibody; optionally, the first binding portion and the second binding portion are of Fab configuration, and the third binding portion is of scFv configuration.

2. Use of a combination of an anti-CD3 / CD20 bispecific antibody containing a type II anti-CD20 antibody and a type I anti-CD20 antibody in the preparation of a combination drug for treating patients with CD20-positive B-cell non-Hodgkin lymphoma; wherein the anti-CD3 / CD20 bispecific antibody comprises a first binding portion specifically binding to CD20, a second binding portion specifically binding to CD3, and a third binding portion specifically binding to CD20, and wherein the first binding portion and the third binding portion specifically binding to CD20 are both derived from the type II anti-CD20 antibody; optionally, the first binding portion and the second binding portion are of the Fab configuration, and the third binding portion is of the scFv configuration.

3. A combination of an anti-CD3 / CD20 bispecific antibody containing type II anti-CD20 antibody and a type I anti-CD20 antibody, for the treatment of CD20-positive B-cell non-Hodgkin lymphoma; wherein, The anti-CD3 / CD20 bispecific antibody includes a first binding portion that specifically binds to CD20, a second binding portion that specifically binds to CD3, and a third binding portion that specifically binds to CD20, wherein the first binding portion and the third binding portion that specifically binds to CD20 are both derived from type II anti-CD20 antibody; optionally, the first binding portion and the second binding portion are of Fab configuration, and the third binding portion is of scFv configuration.

4. The use according to claim 1 or 2, or the combination according to claim 3, characterized in that, The type I anti-CD20 antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1 with an amino acid sequence as shown in SEQ ID NO:1, HCDR2 with an amino acid sequence as shown in SEQ ID NO:2, and HCDR3 with an amino acid sequence as shown in SEQ ID NO:

3. The light chain variable region comprises LCDR1 with an amino acid sequence as shown in SEQ ID NO:4, LCDR2 with an amino acid sequence as shown in SEQ ID NO:5, and LCDR3 with an amino acid sequence as shown in SEQ ID NO:

6. Preferably, the heavy chain variable region of the type I anti-CD20 antibody includes the amino acid sequence shown in SEQ ID NO:7, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:8; and / or, the heavy chain of the type I anti-CD20 antibody includes the amino acid sequence shown in SEQ ID NO:9, and the light chain of the type I anti-CD20 antibody includes the amino acid sequence shown in SEQ ID NO:

10.

5. The use according to claim 1 or 2, or the combination according to claim 3, characterized in that, The first binding region of the anti-CD3 / CD20 bispecific antibody includes a heavy chain variable region (VH1) and a light chain variable region (VL1); the third binding region includes a heavy chain variable region (VH3) and a light chain variable region (VL3); and the second binding region includes a heavy chain variable region (VH2) and a light chain variable region (VL2). The amino acid sequences of the complementarity-determining regions (CDRs) of VH1 and VH3 are identical, and the amino acid sequences of the complementarity-determining regions (CDRs) of VL1 and VL3 are identical; VH1 or VH3 contains HCDR1 as shown in SEQ ID NO:11, HCDR2 as shown in SEQ ID NO:12, and HCDR3 as shown in SEQ ID NO:13; VL1 or VL3 contains LCDR1 as shown in SEQ ID NO:14, LCDR2 as shown in SEQ ID NO:15, and LCDR3 as shown in SEQ ID NO:

16. The VH2 comprises HCDR1 as shown in SEQ ID NO:21, HCDR2 as shown in SEQ ID NO:22, and HCDR3 as shown in SEQ ID NO:23; the VL2 comprises LCDR1 as shown in SEQ ID NO:24, LCDR2 as shown in SEQ ID NO:25, and LCDR3 as shown in SEQ ID NO:

26. Preferably, VH1 or VH3 contains the amino acid sequence shown in SEQ ID NO:17, VL1 contains the amino acid sequence shown in SEQ ID NO:18, VL3 contains the amino acid sequence shown in SEQ ID NO:19, and / or, VH2 contains the amino acid sequence shown in SEQ ID NO:27, and VL2 contains the amino acid sequence shown in SEQ ID NO:

28.

6. The use according to claim 1 or 2, or the combination according to claim 3, characterized in that, The anti-CD3 / CD20 bispecific antibody meets one or more of the following criteria: 1) The first binding region of the anti-CD3 / CD20 bispecific antibody further includes a heavy chain constant region 1 (CH1-1) and a light chain constant region (CL-1); the second binding region further includes a heavy chain constant region 1 (CH1-2) and a light chain constant region (CL-2); 2) The anti-CD3 / CD20 bispecific antibody further comprises an Fc region, wherein the Fc region is connected to the C-terminus of the first binding site, and / or, the Fc region is connected to the C-terminus of the second binding site; 3) The scFv contains a first linker (L1), and the structure of the scFv from the N-terminus to the C-terminus is VH3-L1-VL3; the amino acid sequence of L1 is, for example, (G n S) p As shown, n is, for example, 3 or 4, and p is, for example, 3, 4, or 5; the amino acid sequence of L1 is preferably as shown in SEQ ID NO:20; and, 4) The third binding portion is connected to the N-terminus of the second binding portion via a second linker (L2); the amino acid sequence of L2 is, for example, (G n S) p As shown, n is, for example, 3 or 4, and p is, for example, 3, 4 or 5; the amino acid sequence of L2 is preferably as shown in SEQ ID NO:29; Preferably, the anti-CD3 / CD20 bispecific antibody comprises a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein: The structure of the first polypeptide chain, from the N-terminus to the C-terminus, is: VH1-CH1-1-Fc; The second polypeptide chain, from the N-terminus to the C-terminus, has the structure: VL1-CL-1; The third polypeptide chain, from the N-terminus to the C-terminus, has the following structure: VH3-L1-VL3-L2-VH2-CH1-2-Fc; and, The fourth polypeptide chain, from the N-terminus to the C-terminus, has the structure: VL2-CL-2; The Fc of the first polypeptide chain is called the Fc mortar, and the Fc of the third polypeptide chain is called the Fc pestle. More preferably, the anti-CD3 / CD20 bispecific antibody comprises: The first polypeptide chain contains the amino acid sequence shown in SEQ ID NO:30; The second polypeptide chain contains the amino acid sequence shown in SEQ ID NO:31; A third polypeptide chain comprising the amino acid sequence shown in SEQ ID NO:32; and The fourth polypeptide chain contains the amino acid sequence shown in SEQ ID NO:

33.

7. The use according to claim 1 or 2, or the combination according to claim 3, characterized in that, Type I anti-CD20 antibody is used to clear or reduce peripheral blood B cells in patients; and / or, the target dose of type I anti-CD20 antibody achieves effective depletion of peripheral blood B cells in patients; Preferably, the effective depletion of peripheral blood B cells after administration of type I anti-CD20 antibody is CD19. + / CD20 + Cell count <5 cells / μL; More preferably, the type I anti-CD20 antibody is rituximab, and the dosage of rituximab is 375 mg / m². 2 .

8. The use according to claim 1 or 2, or the combination according to claim 3, characterized in that, The type I anti-CD20 antibody is administered to the patient on day 1 to day 4 before administration of the anti-CD3 / CD20 bispecific antibody, optionally on day 2 to day 3, preferably on day 2; Optionally, the type I anti-CD20 antibody may be administered once, twice, or multiple times; and / or, the type I anti-CD20 antibody may be administered subcutaneously or intravenously.

9. The use according to claim 1 or 2, or the combination according to claim 3, characterized in that, The dosing cycle of the anti-CD3 / CD20 bispecific antibody includes: The first dosing cycle includes a first pre-stimulation dose and a second pre-stimulation dose of the bispecific antibody, wherein the first pre-stimulation dose is 0.05-1.5 mg, preferably 1 mg; and the second pre-stimulation dose is 0.15-6 mg, preferably 5 mg. The second dosing cycle includes a therapeutic dose of the bispecific antibody of 15-45 mg, optionally 15 mg, 20 mg, 25 mg, 30 mg or 35 mg, and more preferably 25 mg. Optionally, the dosing cycle of the anti-CD3 / CD20 bispecific antibody further includes: The third or optional subsequent dosing cycle, wherein the single dose of the third or subsequent dosing cycle is 15-45 mg, optionally 15 mg, 20 mg, 25 mg, 30 mg or 35 mg, and further optionally 25 mg; Preferably, the first dosing cycle is 14 days; or, the first dosing cycle further includes a third pre-excitation dose, and when the first dosing cycle includes a third pre-excitation dose, the first dosing cycle is 21 days; the second dosing cycle, the third dosing cycle, and optionally subsequent dosing cycles are 21 days, and optionally, the first pre-excitation dose is administered on day 1 of the first dosing cycle, the second pre-excitation dose is administered on day 8, and the therapeutic dose is administered on day 1 of the second and third dosing cycles and optionally subsequent dosing cycles.

10. The use according to claim 1 or 2, or the combination according to claim 3, characterized in that, The CD20-positive B-NHL includes relapsed or refractory B-NHL, and optionally, the CD20-positive B-NHL includes aggressive non-Hodgkin Lymphoma (aNHL) and indolent non-Hodgkin Lymphoma (iNHL). Preferably, the aNHL includes diffuse large B-cell lymphoma (DLBCL), high-grade B-cell lymphoma (HGBCL), follicular lymphoma (FL) 3b, mantle cell lymphoma (MCL), primary mediastinal large B-cell lymphoma (PMBCL), Richter's transformed DLBCL (RT-DLBCL), or transformed FL (trFL); and / or, the iNHL includes FL 1-3a and marginal zone lymphoma (MZL).

11. The use according to claim 1 or 2, or the combination according to claim 3, characterized in that, A CD20-positive B-NHL patient developed cytokine release syndrome (CRS) after receiving anti-CD3 / CD20 bispecific antibodies, but did not develop grade ≥3 CRS, and / or, For CD20-positive B-NHL patients, anti-CD3 / CD20 bispecific therapy resulted in an objective response rate (ORR) of at least 60%.

12. The use according to claim 1 or 2, characterized in that, The method or application satisfies one or more of the following: 1) The patients described are those with non-chronic lymphocytic leukemia, non-Burkitt lymphoma, and non-lymphoplasmacytic lymphoma; 2) During the administration of the anti-CD3 / CD20 bispecific antibody, if CRS occurs, administration of an anti-interleukin-6 receptor antibody and / or corticosteroids may also be included; the anti-interleukin-6 receptor antibody is, for example, tocilizumab; the corticosteroids are, for example, dexamethasone and / or methylprednisolone; and, 3) The patients receiving treatment are between 18 and 71 years old, for example, 27 to 71 years old; Preferably, the single dose of tocilizumab is 8 mg / kg and ≤800 mg; the single dose of dexamethasone is 10-20 mg; and / or the single dose of methylprednisolone is 1000-2000 mg.

13. An antibody combination, characterized in that, The antibody combination comprises an anti-CD3 / CD20 bispecific antibody containing a type II anti-CD20 antibody and a type I anti-CD20 antibody; the anti-CD3 / CD20 bispecific antibody includes a first binding portion that specifically binds to CD20, a second binding portion that specifically binds to CD3, and a third binding portion that specifically binds to CD20, and the first binding portion and the third binding portion that specifically binds to CD20 are both derived from the type II anti-CD20 antibody; Optionally, the antibody combination may further comprise an anti-interleukin-6 receptor antibody, such as tocilizumab.

14. The antibody combination according to claim 13, characterized in that, The antibody combination is used to treat patients with CD20-positive B-cell non-Hodgkin lymphoma; Preferably, the patient meets one or more of the following criteria: 1) The B-cell non-Hodgkin lymphomas include relapsed or refractory B-NHL, such as aggressive non-Hodgkin lymphoma (aNHL) and indolent non-Hodgkin lymphoma (iNHL); 2) The patients mentioned are those who have been excluded from chronic lymphocytic leukemia, Burkitt lymphoma, and lymphoplasmacytic lymphoma; and, 3) The patient's age is between 18 and 71 years old, for example, 27 to 71 years old; More preferably, the aggressive NHL includes diffuse large B-cell lymphoma (DLBCL), high-grade B-cell lymphoma (HGBCL), follicular lymphoma (FL) 3b, mantle cell lymphoma (MCL), primary mediastinal large B-cell lymphoma (PMBCL), Richter's transformed DLBCL (RT-DLBCL), or transformed FL (trFL); and / or, the indolent NHL includes FL 1-3a or marginal zone lymphoma (MZL).

15. The antibody combination according to claim 13 or 14, characterized in that, The first binding region of the anti-CD3 / CD20 bispecific antibody comprises a heavy chain variable region (VH1) and a light chain variable region (VL1); the third binding region comprises a heavy chain variable region (VH3) and a light chain variable region (VL3); the second binding region comprises a heavy chain variable region (VH2) and a light chain variable region (VL2); the antibody combination satisfies one or more of the following: 1) The type I anti-CD20 antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1 with an amino acid sequence as shown in SEQ ID NO:1, HCDR2 with an amino acid sequence as shown in SEQ ID NO:2, and HCDR3 with an amino acid sequence as shown in SEQ ID NO:

3. The light chain variable region comprises LCDR1 with an amino acid sequence as shown in SEQ ID NO:4, LCDR2 with an amino acid sequence as shown in SEQ ID NO:5, and LCDR3 with an amino acid sequence as shown in SEQ ID NO:

6. 2) The amino acid sequences of the complementarity-determining regions (CDRs) of VH1 and VH3 are identical, and the amino acid sequences of the complementarity-determining regions (CDRs) of VL1 and VL3 are identical; VH1 or VH3 comprises HCDR1 as shown in SEQ ID NO:11, HCDR2 as shown in SEQ ID NO:12, and HCDR3 as shown in SEQ ID NO:13; VL1 or VL3 comprises LCDR1 as shown in SEQ ID NO:14, LCDR2 as shown in SEQ ID NO:15, and LCDR3 as shown in SEQ ID NO:16; and, 3) The VH2 comprises HCDR1 as shown in SEQ ID NO:21, HCDR2 as shown in SEQ ID NO:22, and HCDR3 as shown in SEQ ID NO:23; the VL2 comprises LCDR1 as shown in SEQ ID NO:24, LCDR2 as shown in SEQ ID NO:25, and LCDR3 as shown in SEQ ID NO:

26. Preferably, the antibody combination satisfies one or more of the following: 1) The amino acid sequence of the heavy chain variable region of the type I anti-CD20 antibody is as shown in SEQ ID NO:7, or has at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:7; 2) The amino acid sequence of the light chain variable region of the type I anti-CD20 antibody is as shown in SEQ ID NO:8, or has at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:8; 3) The amino acid sequence of VH1 or VH3 is as shown in SEQ ID NO:17, or has at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:17; 4) The amino acid sequence of the VL1 is as shown in SEQ ID NO:18, or has at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:18; 5) The amino acid sequence of the VL3 is as shown in SEQ ID NO:19, or has at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:19; 6) The amino acid sequence of the VH2 is as shown in SEQ ID NO:27, or has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:27; and, 7) The amino acid sequence of the VL2 is as shown in SEQ ID NO:28, or has at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:28; More preferably, the type I anti-CD20 antibody is rituximab, the heavy chain of which includes the amino acid sequence shown in SEQ ID NO:9, and / or the light chain of which includes the amino acid sequence shown in SEQ ID NO:

10.

16. The antibody combination according to claim 15, characterized in that, The anti-CD3 / CD20 bispecific antibody meets one or more of the following criteria: 1) The first binding region of the anti-CD3 / CD20 bispecific antibody further includes a heavy chain constant region 1 (CH1-1) and a light chain constant region (CL-1); the second binding region further includes a heavy chain constant region 1 (CH1-2) and a light chain constant region (CL-2); 2) The anti-CD3 / CD20 bispecific antibody further comprises an Fc region, wherein the Fc region is connected to the C-terminus of the first binding site, and / or, the Fc region is connected to the C-terminus of the second binding site; 3) The scFv contains a first linker (L1), and the structure of the scFv from the N-terminus to the C-terminus is VH3-L1-VL3; the amino acid sequence of L1 is, for example, (G n S) p As shown, n is, for example, 3 or 4, and p is, for example, 3, 4, or 5; the amino acid sequence of L1 is preferably as shown in SEQ ID NO:20; and, 4) The third binding portion is connected to the N-terminus of the second binding portion via a second linker (L2); the amino acid sequence of L2 is, for example, (G n S) p As shown, n is, for example, 3 or 4, and p is, for example, 3, 4 or 5; the amino acid sequence of L2 is preferably as shown in SEQ ID NO:29; Preferably, the anti-CD3 / CD20 bispecific antibody comprises a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein: The structure of the first polypeptide chain, from the N-terminus to the C-terminus, is: VH1-CH1-1-Fc; The second polypeptide chain, from the N-terminus to the C-terminus, has the structure: VL1-CL-1; The third polypeptide chain, from the N-terminus to the C-terminus, has the following structure: VH3-L1-VL3-L2-VH2-CH1-2-Fc; and, The fourth polypeptide chain, from the N-terminus to the C-terminus, has the structure: VL2-CL-2; The Fc of the first polypeptide chain is called the Fc mortar, and the Fc of the third polypeptide chain is called the Fc pestle. More preferably, the anti-CD3 / CD20 bispecific antibody comprises: The first polypeptide chain contains the amino acid sequence shown in SEQ ID NO:30; The second polypeptide chain contains the amino acid sequence shown in SEQ ID NO:31; A third polypeptide chain comprising the amino acid sequence shown in SEQ ID NO:32; and, The fourth polypeptide chain contains the amino acid sequence shown in SEQ ID NO:

33.

17. A nucleic acid, characterized in that, The nucleic acid encodes the antibody combination as described in any one of claims 13-16.

18. An expression carrier, characterized in that, The expression vector comprises the nucleic acid as described in claim 17; Preferably, the expression vector is selected from prokaryotic expression vectors or eukaryotic expression vectors; More preferably, the eukaryotic expression vector is selected, for example, from viral vectors or non-viral plasmid vectors.

19. A transformant, characterized in that, The transformant is a host cell containing the nucleic acid as described in claim 17 or the expression vector as described in claim 18; preferably, the transformant is a non-animal variety and a non-plant variety. Preferably, the host cell of the transformant is a prokaryotic cell or a eukaryotic cell; More preferably, the eukaryotic cells are selected from insect cells, mammalian cells, or yeast cells; the mammalian cells are, for example, CHO or 293 cells.

20. A drug for treating CD20-positive B-cell non-Hodgkin lymphoma, characterized in that, The drug comprises an antibody combination as described in any one of claims 13-16, and a pharmaceutically acceptable carrier; Preferably, the drug further comprises a corticosteroid; the corticosteroid being, for example, dexamethasone and / or methylprednisolone.

21. A reagent kit or medicine box, characterized in that, The kit or cassette contains an antibody combination as described in any one of claims 13-16; or, a drug as described in claim 20; Optionally, the kit or kit may also include a device for administering the antibody or drug; and / or instructions for use of the antibody or drug.

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