Antigen binding molecule specifically binding to CD19, CD20 and CD3, and pharmaceutical use thereof

Abstract

Provided are an antigen binding molecule specifically binding to CD19, CD20 and CD3, and a pharmaceutical use thereof. Further provided is a use of a trispecific antibody specifically binding to CD19, CD20 and CD3 in the preparation of a drug for treating a tumor and an autoimmune disease.

Description

Antigen-binding molecules that specifically bind to CD19, CD20, and CD3 and their pharmaceutical uses

[0001] This application claims priority to Chinese patent application CN 202510042644.5, filed on January 10, 2025. Technical Field

[0002] This disclosure pertains to the field of biotechnology, and more specifically, to antigen-binding molecules that specifically bind to CD19, CD20, and CD3 and their pharmaceutical uses. Background Technology

[0003] The statements herein are provided only as background information in connection with this disclosure and do not necessarily constitute prior art.

[0004] Non-Hodgkin's lymphoma (NHL) is the most common hematologic malignancy worldwide, accounting for approximately 60% of all lymphomas. Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma in adults, accounting for approximately 30-40% of newly diagnosed lymphomas. The most common clinical symptom of DLBCL is painless, progressive lymphadenopathy. Due to its aggressive nature, the five-year survival rate is only 60%-70%, indicating a significant unmet medical need. Some patients can be cured after receiving first-line standard chemotherapy regimen R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). Relapsed / refractory patients account for approximately 40%, with a five-year survival rate of only about 25% after relapse. While significant progress has been made clinically in treatments targeting CD19 or CD20, such as CAR-T cell therapy, TCE bispecific antibodies, ADC therapy, and monoclonal antibody-chemotherapy combinations, there is still room for improvement in efficacy, and many patients still develop drug resistance after treatment. Loss of CD19 or CD20 targets (approximately 20%–30%) is one of the main causes of drug resistance and relapse. Currently, the clinical need for antibody drugs to treat non-Hodgkin's lymphoma remains unmet. Summary of the Invention

[0005] This disclosure relates to a T-cell connector that simultaneously targets CD19 and CD20. The CD19 and CD20 ends are independent and both have strong killing functions, which can address the loss or downregulation of expression of either antigen, better cope with drug resistance, increase population coverage and heterogeneous tumor cell killing, and achieve better response and cure rates.

[0006] This disclosure provides an antigen-binding molecule that specifically binds to CD19, CD20 and CD3, and an antigen-binding molecule that specifically binds to CD3.

[0007] In one aspect, this disclosure provides an antigen-binding molecule that specifically binds to CD19, CD20, and CD3, comprising a first antigen-binding domain that specifically binds to CD19, a second antigen-binding domain that specifically binds to CD20, and a third antigen-binding domain that specifically binds to CD3.

[0008] Regarding the first antigen-binding domain that specifically binds to CD19:

[0009] In some embodiments, as described in any of the preceding embodiments, an antigen-binding molecule that specifically binds to CD19, CD20, and CD3, wherein the first antigen-binding domain that specifically binds to CD19 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0010] The VH contains one, two, or three HCDR amino acid sequences from SEQ ID NO: 19 or 21; and / or the VL contains one, two, or three LCDR amino acid sequences from SEQ ID NO: 20 or 22.

[0011] In some embodiments, antigen-binding molecules that specifically bind to CD19, CD20, and CD3, as described in any of the preceding embodiments, wherein:

[0012] The VH contains one, two, or three HCDR amino acid sequences from SEQ ID NO: 19; and the VL contains one, two, or three LCDR amino acid sequences from SEQ ID NO: 20; or

[0013] The VH contains one, two, or three HCDR amino acid sequences from the sequence SEQ ID NO: 21; and the VL contains one, two, or three LCDR amino acid sequences from the sequence SEQ ID NO: 22.

[0014] In some embodiments, antigen-binding molecules that specifically bind to CD19, CD20, and CD3, as described in any of the preceding embodiments, wherein:

[0015] The VH contains HCDR1, HCDR2, and HCDR3 from the sequence SEQ ID NO: 19; and the VL contains LCDR1, LCDR2, and LCDR3 from the sequence SEQ ID NO: 20; or

[0016] The VH contains HCDR1, HCDR2 and HCDR3 in the sequence SEQ ID NO: 21; and the VL contains LCDR1, LCDR2 and LCDR3 in the sequence SEQ ID NO: 22.

[0017] In some embodiments, antigen-binding molecules that specifically bind to CD19, CD20, and CD3, as described in any of the preceding embodiments, wherein:

[0018] The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 that specifically bind to the first antigen-binding domain of CD19 are defined according to a numbering rule selected from Kabat, IMGT, Chothia, AbM, and Contact. In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 that specifically bind to the first antigen-binding domain of CD19 are defined according to the Kabat numbering rule. In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 that specifically bind to the first antigen-binding domain of CD19 are defined according to the IMGT numbering rule. In some embodiments, the antigen-binding molecules that specifically bind CD19, CD20, and CD3 as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, LCDR3 that specifically bind the first antigen-binding domain of CD19 are defined according to the Chothia numbering rules. In some embodiments, the antigen-binding molecules that specifically bind CD19, CD20, and CD3 as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, LCDR3 that specifically bind the first antigen-binding domain of CD19 are defined according to the AbM numbering rules. In some embodiments, the antigen-binding molecules that specifically bind CD19, CD20, and CD3 as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, LCDR3 that specifically bind the first antigen-binding domain of CD19 are defined according to the Contact numbering rules.

[0019] In some embodiments, antigen-binding molecules that specifically bind to CD19, CD20, and CD3, as described in any of the preceding embodiments, wherein:

[0020] The first antigen-binding domain that specifically binds to CD19 comprises a heavy chain variable region (VH) and a light chain variable region (VL). The VH comprises HCDR1, HCDR2, and HCDR3. HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 3. The VL comprises LCDR1, LCDR2, and LCDR3. LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6.

[0021] In some embodiments, the antigen-binding molecules that specifically bind to CD19, CD20, and CD3 as described in any of the preceding embodiments, wherein the heavy chain variable regions HCDR1, HCDR2, and HCDR3 and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to the Kabat numbering rules.

[0022] In some embodiments, antigen-binding molecules that specifically bind to CD19, CD20, and CD3, as described in any of the preceding embodiments, wherein:

[0023] The first antigen-binding domain that specifically binds to CD19 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises SEQ ID NO: 19, 21, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and the VL comprises SEQ ID NO: 20, 22, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0024] In some embodiments, antigen-binding molecules that specifically bind to CD19, CD20, and CD3, as described in any of the preceding embodiments, wherein:

[0025] The first antigen-binding domain that specifically binds to CD19 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 19 and the VL comprises the amino acid sequence of SEQ ID NO: 20.

[0026] Regarding the second antigen-binding domain that specifically binds to CD20:

[0027] In some embodiments, as described in any of the preceding embodiments, an antigen-binding molecule that specifically binds to CD19, CD20, and CD3, wherein the second antigen-binding domain that specifically binds to CD20 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0028] The VH contains one, two, or three HCDR amino acid sequences from the sequence SEQ ID NO: 23; and / or the VL contains one, two, or three LCDR amino acid sequences from the sequence SEQ ID NO: 24.

[0029] In some embodiments, the antigen-binding molecules that specifically bind to CD19, CD20, and CD3 as described in any of the preceding embodiments, wherein the VH comprises HCDR1, HCDR2, and HCDR3 of the sequence SEQ ID NO: 23; and the VL comprises LCDR1, LCDR2, and LCDR3 of the sequence SEQ ID NO: 24.

[0030] In some embodiments, antigen-binding molecules that specifically bind to CD19, CD20, and CD3, as described in any of the preceding embodiments, wherein:

[0031] The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 of the second antigen-binding domain that specifically binds to CD20 are defined according to the numbering rules selected from Kabat, IMGT, Chothia, AbM, and Contact. In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 of the second antigen-binding domain that specifically binds to CD20 are defined according to the Kabat numbering rules. In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 of the second antigen-binding domain that specifically binds to CD20 are defined according to the IMGT numbering rules. In some embodiments, the antigen-binding molecules that specifically bind CD19, CD20, and CD3 as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, LCDR3 of the second antigen-binding domain specifically binding CD20 are defined according to the Chothia numbering rules. In some embodiments, the antigen-binding molecules that specifically bind CD19, CD20, and CD3 as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, LCDR3 of the second antigen-binding domain specifically binding CD20 are defined according to the AbM numbering rules. In some embodiments, the antigen-binding molecules that specifically bind CD19, CD20, and CD3 as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, LCDR3 of the second antigen-binding domain specifically binding CD20 are defined according to the Contact numbering rules.

[0032] In some embodiments, antigen-binding molecules that specifically bind to CD19, CD20, and CD3, as described in any of the preceding embodiments, wherein:

[0033] The second antigen-binding domain that specifically binds to CD20 comprises a heavy chain variable region (VH) and a light chain variable region (VL). The VH comprises HCDR1, HCDR2, and HCDR3. HCDR1 comprises the amino acid sequence of SEQ ID NO: 7, HCDR2 comprises the amino acid sequence of SEQ ID NO: 8, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 9. The VL comprises LCDR1, LCDR2, and LCDR3. LCDR1 comprises the amino acid sequence of SEQ ID NO: 10, LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 12.

[0034] In some embodiments, the antigen-binding molecules that specifically bind to CD19, CD20, and CD3 as described in any of the preceding embodiments, wherein the heavy chain variable regions HCDR1, HCDR2, and HCDR3 and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to the Kabat numbering rules.

[0035] In some embodiments, antigen-binding molecules that specifically bind to CD19, CD20, and CD3, as described in any of the preceding embodiments, wherein:

[0036] The second antigen-binding domain that specifically binds to CD20 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises SEQ ID NO: 23, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith, and the VL comprises SEQ ID NO: 24, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith.

[0037] In some embodiments, antigen-binding molecules that specifically bind to CD19, CD20, and CD3, as described in any of the preceding embodiments, wherein:

[0038] The second antigen-binding domain that specifically binds to CD20 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 23 and the VL comprises the amino acid sequence of SEQ ID NO: 24.

[0039] Regarding the third antigen-binding domain that specifically binds to CD3:

[0040] In some embodiments, as described in any of the preceding embodiments, an antigen-binding molecule that specifically binds to CD19, CD20, and CD3, wherein the third antigen-binding domain that specifically binds to CD3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0041] The VH contains one, two, or three HCDR amino acid sequences from the sequence SEQ ID NO: 25; and / or the VL contains one, two, or three LCDR amino acid sequences from the sequence SEQ ID NO: 26.

[0042] In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20 and CD3 as described in any of the preceding embodiments includes a heavy chain variable region, wherein the heavy chain variable region includes the HCDR3 amino acid sequence in SEQ ID NO: 25.

[0043] In some embodiments, the antigen-binding molecules that specifically bind to CD19, CD20, and CD3 as described in any of the preceding embodiments, wherein the VH comprises HCDR1, HCDR2, and HCDR3 of the sequence SEQ ID NO: 25; and the VL comprises LCDR1, LCDR2, and LCDR3 of the sequence SEQ ID NO: 26.

[0044] In some embodiments, antigen-binding molecules that specifically bind to CD19, CD20, and CD3, as described in any of the preceding embodiments, wherein:

[0045] The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 that specifically bind to the third antigen-binding domain of CD3 are defined according to a numbering rule selected from Kabat, IMGT, Chothia, AbM, and Contact. In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 that specifically bind to the third antigen-binding domain of CD3 are defined according to the Kabat numbering rule. In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 that specifically bind to the third antigen-binding domain of CD3 are defined according to the IMGT numbering rule. In some embodiments, the antigen-binding molecules that specifically bind CD19, CD20, and CD3 as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, LCDR3 of the third antigen-binding domain specifically binding CD3 are defined according to the Chothia numbering rules. In some embodiments, the antigen-binding molecules that specifically bind CD19, CD20, and CD3 as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, LCDR3 of the third antigen-binding domain specifically binding CD3 are defined according to the AbM numbering rules. In some embodiments, the antigen-binding molecules that specifically bind CD19, CD20, and CD3 as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, LCDR3 of the third antigen-binding domain specifically binding CD3 are defined according to the Contact numbering rules.

[0046] In some embodiments, antigen-binding molecules that specifically bind to CD19, CD20, and CD3, as described in any of the preceding embodiments, wherein:

[0047] The third antigen-binding domain that specifically binds to CD3 comprises a heavy chain variable region (VH) and a light chain variable region (VL). The VH comprises HCDR1, HCDR2, and HCDR3. HCDR1 comprises the amino acid sequence of SEQ ID NO: 13, HCDR2 comprises the amino acid sequence of SEQ ID NO: 14, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 15. The VL comprises LCDR1, LCDR2, and LCDR3. LCDR1 comprises the amino acid sequence of SEQ ID NO: 16, LCDR2 comprises the amino acid sequence of SEQ ID NO: 17, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 18.

[0048] In some embodiments, the antigen-binding molecules that specifically bind to CD19, CD20, and CD3 as described in any of the preceding embodiments, wherein the heavy chain variable regions HCDR1, HCDR2, and HCDR3 and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to the Kabat numbering rules.

[0049] In some embodiments, antigen-binding molecules that specifically bind to CD19, CD20, and CD3, as described in any of the preceding embodiments, wherein:

[0050] The third antigen-binding domain that specifically binds to CD3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises SEQ ID NO: 25, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith, and the VL comprises SEQ ID NO: 26, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith.

[0051] In some embodiments, antigen-binding molecules that specifically bind to CD19, CD20, and CD3, as described in any of the preceding embodiments, wherein:

[0052] The VH contains the amino acid sequence of SEQ ID NO: 25, and the VL contains the amino acid sequence of SEQ ID NO: 26.

[0053] Regarding antigen-binding molecules that specifically bind to CD19, CD20, and CD3:

[0054] In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments comprises: at least one (e.g., 1, 2, 3, 4, 5, 6) first antigen-binding domain that specifically binds to CD19, at least one (e.g., 1, 2, 3, 4, 5, 6) second antigen-binding domain that specifically binds to CD20, and at least one (e.g., 1, 2, 3, 4, 5, 6) third antigen-binding domain that specifically binds to CD3.

[0055] In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20 and CD3 as described in any of the preceding embodiments comprises a first antigen-binding domain that specifically binds to CD19, a second antigen-binding domain that specifically binds to CD20 and a third antigen-binding domain that specifically binds to CD3.

[0056] In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments comprises:

[0057] The first antigen-binding domain that specifically binds to CD19 is Fab or a substituted Fab.

[0058] A second antigen-binding domain that specifically binds to CD20, wherein the second antigen-binding domain that specifically binds to CD20 is Fab or a substituted Fab; and

[0059] The third antigen-binding domain that specifically binds to CD3 is scFv.

[0060] In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments comprises:

[0061] The first antigen-binding domain that specifically binds to CD19 is Fab.

[0062] A second antigen-binding domain that specifically binds to CD20, wherein the second antigen-binding domain that specifically binds to CD20 is a substituted Fab; and

[0063] A third antigen-binding domain that specifically binds to CD3, wherein the third antigen-binding domain that specifically binds to CD3 is scFv; or

[0064] The antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding claims comprises:

[0065] The first antigen-binding domain that specifically binds to CD19 is Fab.

[0066] A second antigen-binding domain that specifically binds to CD20, wherein the second antigen-binding domain that specifically binds to CD20 is Fab; and

[0067] The third antigen-binding domain that specifically binds to CD3 is scFv.

[0068] In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3, as described in any of the preceding embodiments, is an antibody. In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 is a multispecific antibody. In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 is a trispecific antibody. In some embodiments, the antibody is a trivalent trispecific antibody that specifically binds to CD19, CD20, and CD3.

[0069] In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments comprises:

[0070] The first half antibody comprises a replaced Fab and Fc1 that bind to CD20;

[0071] The second half-antibody comprises a Fab that binds to CD19, an scFv that binds to CD3, and an Fc2, wherein the first half-antibody and the second half-antibody are associated with each other via Fc1 and Fc2; or

[0072] The antigen-binding molecules that specifically bind to CD19, CD20, and CD3 include:

[0073] The first half antibody comprises Fab and Fc1 that bind to CD20;

[0074] The second half antibody comprises a Fab that binds to CD19, an scFv that binds to CD3, and an Fc1, wherein the first half antibody and the second half antibody are associated with each other via Fc1 and Fc2.

[0075] In some embodiments, antigen-binding molecules that specifically bind to CD19, CD20, and CD3, as described in any of the preceding embodiments, wherein:

[0076] The antigen-binding molecule that specifically binds to CD19, CD20, and CD3 is a (trivalent) trispecific antibody and comprises:

[0077] The first half antibody comprises a replaced Fab and Fc1 that bind to CD20;

[0078] The second half-antibody comprises a Fab that binds to CD19, an scFv that binds to CD3, and an Fc2, wherein the first half-antibody and the second half-antibody are associated with each other via Fc1 and Fc2; or

[0079] The antigen-binding molecule that specifically binds to CD19, CD20, and CD3 is a (trivalent) trispecific antibody and comprises:

[0080] The first half antibody comprises Fab and Fc1 that bind to CD20;

[0081] The second half antibody comprises a Fab that binds to CD19, an scFv that binds to CD3, and an Fc2, wherein the first half antibody and the second half antibody are associated with each other via Fc1 and Fc2.

[0082] In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments is a (trivalent) trispecific antibody and comprises:

[0083] The first hapten, comprising, from the N-terminus to the C-terminus, substituted Fab and Fc1 that bind CD20;

[0084] The second hemibody, comprising, from the N-terminus to the C-terminus, a Fab that binds CD19, an scFv that binds CD3, and an Fc2, wherein the first and second hemibody are associated with each other via Fc1 and Fc2; or

[0085] The antigen-binding molecule that specifically binds to CD19, CD20, and CD3 is a (trivalent) trispecific antibody and comprises:

[0086] The first half antibody contains Fab and Fc1 binding to CD20 from the N-terminus to the C-terminus.

[0087] The second half antibody includes, from the N-terminus to the C-terminus, a Fab that binds CD19, an scFv that binds CD3, and an Fc2, wherein the first half antibody and the second half antibody are associated with each other via Fc1 and Fc2.

[0088] In some embodiments, antigen-binding molecules that specifically bind to CD19, CD20, and CD3, as described in any of the preceding embodiments, wherein:

[0089] The antigen-binding molecule that specifically binds to CD19, CD20, and CD3 is a (trivalent) trispecific antibody and comprises:

[0090] The first hapten, comprising, from the N-terminus to the C-terminus, substituted Fab and Fc1 that bind CD20;

[0091] The second half antibody includes, from the N-terminus to the C-terminus, a Fab that binds CD19, an scFv that binds CD3, and an Fc2, wherein the first half antibody and the second half antibody are associated with each other via Fc1 and Fc2.

[0092] In some embodiments, antigen-binding molecules that specifically bind to CD19, CD20, and CD3, as described in any of the preceding embodiments, wherein:

[0093] The antigen-binding molecule that specifically binds to CD19, CD20, and CD3 is a (trivalent) trispecific antibody and comprises:

[0094] The first hapten includes, from the N-terminus to the C-terminus, a CD20-binding replaced Fab and an Fc1, wherein the C-terminus of the CD20-binding replaced Fab is operatively connected to the N-terminus of the Fc1.

[0095] The second half antibody includes, from the N-terminus to the C-terminus, a Fab that binds CD19, a scFv that binds CD3, and an Fc2, wherein the N-terminus of the scFv that binds CD3 is operatively connected to the C-terminus of the Fab that binds CD19, and the C-terminus of the scFv that binds CD3 is operatively connected to the N-terminus of the Fc2.

[0096] Furthermore, the first and second halves of the antibody are associated with each other via Fc1 and Fc2.

[0097] In some embodiments, antigen-binding molecules that specifically bind to CD19, CD20, and CD3, as described in any of the preceding embodiments, wherein:

[0098] The antigen-binding molecule that specifically binds to CD19, CD20, and CD3 is a (trivalent) trispecific antibody and comprises:

[0099] The first hapten includes, from the N-terminus to the C-terminus, a CD20-binding replaced Fab and an Fc1, wherein the C-terminus of the CD20-binding replaced Fab is operatively connected to the N-terminus of the Fc1.

[0100] The second half antibody contains, from the N-terminus to the C-terminus, a Fab that binds to CD19, an scFv that binds to CD3, and an Fc2.

[0101] The Fab comprises a light chain variable region (VL), a light chain constant region (CL), a heavy chain variable region (VH), and a heavy chain constant region 1 (CH1);

[0102] The N-terminus of the CD3-bonded scFv is operably connected to the C-terminus of the CH1 of the CD19-bonded Fab, and the C-terminus of the CD3-bonded scFv is operably connected to the N-terminus of the Fc2.

[0103] Furthermore, the first and second halves of the antibody are associated with each other via Fc1 and Fc2.

[0104] In some embodiments, antigen-binding molecules that specifically bind to CD19, CD20, and CD3, as described in any of the preceding embodiments, wherein:

[0105] The antigen-binding molecule that specifically binds to CD19, CD20, and CD3 is a (trivalent) trispecific antibody and comprises:

[0106] The first hapten includes, from the N-terminus to the C-terminus, a CD20-binding replaced Fab and an Fc1, wherein the C-terminus of the CD20-binding replaced Fab is operatively connected to the N-terminus of the Fc1.

[0107] The second half antibody contains, from the N-terminus to the C-terminus, a Fab that binds to CD19, an scFv that binds to CD3, and an Fc2.

[0108] The Fab comprises a light chain variable region (VL), a light chain constant region (CL), a heavy chain variable region (VH), and a heavy chain constant region 1 (CH1);

[0109] The N-terminus of the scFv of CD3 is operatively connected to the C-terminus of the CL of the Fab of CD19.

[0110] The C-terminus of CH1 of the Fab combined with CD19 is operatively connected to the N-terminus of Fc2.

[0111] Furthermore, the first and second halves of the antibody are associated with each other via Fc1 and Fc2.

[0112] In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments, wherein the replaced Fab comprises a light chain variable region (VL), a light chain constant region (CL), a heavy chain variable region (VH), and a heavy chain constant region 1 (CH1), and comprises any of the following substitutions:

[0113] (i) VL and VH are interchangeable;

[0114] (ii) CL and CH1 are interchangeable; or

[0115] (iii) VL and CL, VH and CH1 are interchangeable.

[0116] In some implementations, the replaced Fab comprises CL and CH1 interchangeably.

[0117] In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments, wherein the replaced Fab of the second antigen-binding domain specifically binding to CD20 comprises CL and CH1 interchangeably, or the replaced Fab of the second antigen-binding domain specifically binding to CD20 comprises VL and VH interchangeably, or the replaced Fab of the second antigen-binding domain specifically binding to CD20 comprises VL and CL, VH and CH1 interchangeably. In some embodiments, CL and CH1 of the replaced Fab of the second antigen-binding domain specifically binding to CD20 are interchanged.

[0118] In some embodiments, as described in any of the preceding embodiments, the antigen-binding molecule specifically binding to CD19, CD20, and CD3, wherein the Fab comprises a light chain variable region (VL), a light chain constant region (CL), a heavy chain variable region (VH), and a heavy chain constant region 1 (CH1), and the amino acid at position 126 of CH1 is cysteine, and the amino acid at position 121 of CL is cysteine, numbered according to the EU index. In some embodiments, as described in any of the preceding embodiments, the antigen-binding molecule specifically binding to CD19, CD20, and CD3, wherein the Fab of the first antigen-binding domain specifically binding to CD19 comprises a light chain variable region (VL), a light chain constant region (CL), a heavy chain variable region (VH), and a heavy chain constant region 1 (CH1), and the amino acid at position 126 of CH1 is cysteine, and the amino acid at position 121 of CL is cysteine, numbered according to the EU index.

[0119] In some embodiments, as described in any of the preceding embodiments, the antigen-binding molecule specifically binds to CD19, CD20, and CD3, wherein the heavy chain constant region CH1 of the first antigen-binding domain specifically binding to CD19 is the heavy chain constant region CH1 of human IgG1, and the amino acid at position 126 is cysteine, and the light chain constant region CL is the light chain constant region CL of human kappa, and the amino acid at position 121 is cysteine, numbered according to the EU index. In some embodiments, as described in any of the preceding embodiments, the antigen-binding molecule specifically binds to CD19, CD20, and CD3, wherein the heavy chain constant region CH1 of the first antigen-binding domain specifically binding to CD19 is the heavy chain constant region CH1 of human IgG1, and the amino acid at position 126 is cysteine, the amino acid at position 220 is non-cysteine, and the light chain constant region CL is the light chain constant region CL of human kappa, and the amino acid at position 121 is cysteine, the amino acid at position 214 is non-cysteine, numbered according to the EU index.

[0120] In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments comprises:

[0121] A first chain having the structure shown in equation (a), a second chain having the structure shown in equation (b), a third chain having the structure shown in equation (c), and a fourth chain having the structure shown in equation (d), wherein the structures shown in equations (a), (b), (c), and (d) are arranged from the N end to the C end:

[0122] (a) [CD20-VL]-[Connector 1]-[CH1-1];

[0123] (b)[CD20-VH]-[CL1]-[Fc1];

[0124] (c)[CD19-VH]-[CH1-2]-[Connector 2]-[CD3-VH]-[Connector 3]-[CD3-VL]-[Connector 4]-[Fc2];

[0125] (d)[CD19-VL]-[CL2];

[0126] The linkers 1, 2, 3 and 4 are the same or different peptide linkers, or linkers 1, 2, 3 and / or 4 are absent;

[0127] Fc1 and Fc2 are interchangeable;

[0128] The first and second chains combine with each other, the third and fourth chains combine with each other, and the second and third chains combine with each other to form antigen-binding molecules that specifically bind to CD19, CD20 and CD3.

[0129] In some embodiments, the structure of the antigen-binding molecule that specifically binds to CD19, CD20 and CD3, as described in any of the preceding embodiments, is shown in Format A of Figure 1.

[0130] In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments comprises:

[0131] A first chain having the structure shown in equation (e), a second chain having the structure shown in equation (f), a third chain having the structure shown in equation (g), and a fourth chain having the structure shown in equation (h), wherein the structures shown in equations (e), (f), (g), and (h) are arranged from the N end to the C end:

[0132] (e)[CD20-VL]-[Connector 1]-[CH1-1];

[0133] (f)[CD20-VH]-[CL1]-[Fc1];

[0134] (g)[CD19-VH]-[CH1-3]-[Fc2];

[0135] (h)[CD19-VL]-[CL2]-[Connector 5]-[CD3-VH]-[Connector 3]-[CD3-VL];

[0136] The linkers 1, 3, and 5 may be the same or different peptide linkers, or linkers 1, 3, and / or 5 may not exist;

[0137] Fc1 and Fc2 are interchangeable;

[0138] The first and second chains combine with each other, the third and fourth chains combine with each other, and the second and third chains combine with each other to form antigen-binding molecules that specifically bind to CD19, CD20 and CD3.

[0139] In some embodiments, the structure of the antigen-binding molecule that specifically binds to CD19, CD20 and CD3, as described in any of the preceding embodiments, is shown in Format B of Figure 1.

[0140] In this disclosure, CH1-1, CH1-2, and CH1-3 are used only to distinguish the position of CH1 and are not used to define a specific sequence; CL1 and CL2 are used only to distinguish the position of CL and are not used to define a specific sequence.

[0141] In some embodiments, the linker is a peptide linker in the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments. In some embodiments, linkers 1, 2, 3, 4, and 5 are peptide linkers in the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments. In this disclosure, linkers 1, 2, 3, 4, and 5 are used only to distinguish linker positions and are not intended to limit the linker sequences. In some embodiments, the peptide linker may be a flexible peptide containing 1-50 or 1-20 amino acid residues. In some embodiments, each of the peptide linkers independently has an L1-(GGGGS)n-L2 structure, wherein L1 is a bond, A, G, GS, GGG, GGS, GGGS, or GGGG (SEQ ID NO: 66), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and L2 is a bond, G, GG, GGG, or GGGG (SEQ ID NO: 66), and the peptide linker is not a bond. In some embodiments, the peptide linker is 2-20 amino acid residues in length. In some embodiments, the peptide linker is represented by the following formula: (GS) a (GGS) b (GGGS) c (GGGGS) d (GGGGG) ea, b, c, d, and e are independent integers greater than or equal to 0; or the peptide linker is selected from: (EAAAK)3 (SEQ ID NO: 68), (EAAAR)3 (SEQ ID NO: 69), (EGGGK)3 (SEQ ID NO: 70), (EGGGR)3 (SEQ ID NO: 71), (DAAAR)3 (SEQ ID NO: 72), (DAAAK)3 (SEQ ID NO: 73), (DGGGR)3 (SEQ ID NO: 74) or (DGGGK)3 (SEQ ID NO: 75), SS (SEQ ID NO: 39), GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 41), EPKSS (SEQ ID NO: 42), RTVAA (SEQ ID NO: 44), ASEPKSS (SEQ ID NO: 45); or the peptide linker is (GxS)y, where x is selected from integers 1-5, and y is selected from integers 1-6, including but not limited to GGGS (SEQ ID NO: 68). (SEQ ID NO: 76), (GGGGS)2 (SEQ ID NO: 40), (GGGGS)2 (SEQ ID NO: 43), (GGGGS)3 (SEQ ID NO: 77); or the peptide linker is GGGGG (SEQ ID NO: 78). In some embodiments, the amino acid sequence of the peptide linker is as shown in SEQ ID NO: 39, 40, 41, 42, 43, 44 or 45. In some embodiments, the amino acid sequence of the peptide linker is as shown in SEQ ID NO: 39, 40 or 41.

[0142] In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments comprises a heavy chain constant region CH1 and a light chain constant region CL; wherein the heavy chain constant region CH1 is derived from human IgG1, IgG2, IgG3, or IgG4, and the light chain constant region CL is derived from human kappa or lambda. In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments, wherein the heavy chain constant region CH1 is derived from human IgG1, and the light chain constant region CL is derived from human kappa.

[0143] In some embodiments, an antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments, wherein the heavy chain constant region CH1 comprises the amino acid sequence of SEQ ID NO: 33, 34, or 35, and the light chain constant region CL comprises the amino acid sequence of SEQ ID NO: 36, 37, or 38.

[0144] In some embodiments, as described in any of the preceding embodiments, the antigen-binding molecules that specifically bind to CD19, CD20, and CD3, wherein the heavy chain constant region CH1 and the light chain constant region CL are selected from one or both of the following groups:

[0145] The heavy chain constant region CH1 contains the amino acid sequence of SEQ ID NO: 33, and the light chain constant region CL contains the amino acid sequence of SEQ ID NO: 36; or

[0146] The heavy chain constant region CH1 contains the amino acid sequence of SEQ ID NO: 34, and the light chain constant region CL contains the amino acid sequence of SEQ ID NO: 37; or

[0147] The heavy chain constant region CH1 contains the amino acid sequence of SEQ ID NO: 35, and the light chain constant region CL contains the amino acid sequence of SEQ ID NO: 37; or

[0148] The heavy chain constant region CH1 comprises the amino acid sequence of SEQ ID NO: 33, and the light chain constant region CL comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, as described in any of the preceding embodiments, the heavy chain constant region CH1 and the light chain constant region CL are selected from one or both of the following groups:

[0149] The heavy chain constant region CH1 contains the amino acid sequence of SEQ ID NO: 33, and the light chain constant region CL contains the amino acid sequence of SEQ ID NO: 36; or

[0150] The heavy chain constant region CH1 contains the amino acid sequence of SEQ ID NO: 34, and the light chain constant region CL contains the amino acid sequence of SEQ ID NO: 37.

[0151] In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20 and CD3 as described in any of the preceding embodiments, wherein the amino acid sequence of the heavy chain constant region CH1 of the second antigen-binding domain that specifically binds to CD20 is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain constant region CL of the second antigen-binding domain that specifically binds to CD20 is shown in SEQ ID NO: 36.

[0152] In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20 and CD3 as described in any of the preceding embodiments, wherein the amino acid sequence of the heavy chain constant region CH1 of the first antigen-binding domain that specifically binds to CD19 is shown in SEQ ID NO: 34, and the amino acid sequence of the light chain constant region CL of the first antigen-binding domain that specifically binds to CD19 is shown in SEQ ID NO: 37.

[0153] In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments further comprises an Fc region. In some embodiments, the Fc region is an Fc region derived from IgG. In some embodiments, the Fc region is an Fc region derived from IgG1. In some embodiments, the Fc region comprises one or more amino acid substitutions capable of reducing the binding of the Fc region to the Fcγ receptor. In some embodiments, the Fc region is the human IgG1 Fc region, with amino acids A at positions 234 and 235 and S at position 265; or the Fc region is the human IgG1 Fc region, with amino acids A at positions 234 and 235, numbered according to the EU index. In some embodiments, the Fc region is the human IgG1 Fc region, with amino acids A at positions 234 and 235 and S at position 265, numbered according to the EU index.

[0154] In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments includes an Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein Fc1 and Fc2 each independently have one or more amino acid substitutions that reduce homodimerization of the Fc region. In some embodiments, Fc1 includes a protruding structure according to a mortar and pestle technique, and Fc2 includes a porous structure according to a mortar and pestle technique. In some embodiments, the amino acid at position 366 of Fc1 is W; and the amino acid at position 366 of Fc2 is S, the amino acid at position 368 is A, and the amino acid at position 407 is V, numbered according to the EU index.

[0155] In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments includes an Fc region containing disulfide bond modifications. In some embodiments, the amino acid at position 354 of Fc1 is C; and the amino acid at position 349 of Fc2 is C, numbered according to the EU index.

[0156] In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments includes an IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other; wherein Fc1 has a C at position 354 and an amino acid W at position 366; and wherein Fc2 has a C at position 349, an amino acid S at position 366, an amino acid A at position 368, and an amino acid V at position 407, numbered according to the EU index.

[0157] In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments includes an Fc region containing one or more amino acid substitutions capable of eliminating the binding of the Fc region to Protein A. In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments includes an Fc region, wherein the Fc region is the human IgG Fc region, and the amino acid at position 435 is R and the amino acid at position 436 is F, numbered according to the EU index. In some embodiments, Fc1 or Fc2 is the human IgG1 Fc region, and the amino acid at position 435 is R and the amino acid at position 436 is F, numbered according to the EU index.

[0158] In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments comprises Fc1 and Fc2, wherein Fc1 is the human IgG1 Fc region and has amino acid A at positions 234 and 235, amino acid S at position 265, amino acid C at position 354, and amino acid W at position 366; and Fc2 is the human IgG1 Fc region and has amino acid A at positions 234 and 235, amino acid S at position 265, amino acid C at position 349, amino acid S at position 366, amino acid A at position 368, amino acid V at position 407, amino acid R at position 435, and amino acid F at position 436, numbered according to the EU index. In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20 and CD3 as described in any of the preceding embodiments, wherein Fc1 comprises the amino acid sequence of SEQ ID NO: 27 and Fc2 comprises the amino acid sequence of SEQ ID NO: 30.

[0159] In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding claims comprises Fc1 and Fc2, wherein Fc1 is the human IgG1 Fc region, and amino acids at positions 234 and 235 are A, at position 354 is C, and at position 366 is W; and Fc2 is the human IgG1 Fc region, and amino acids at positions 234 and 235 are A, at position 349 is C, at position 366 is S, at position 368 is A, at position 407 is V, at position 435 is R, and at position 436 is F, numbered according to the EU index. In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding claims, wherein Fc1 comprises the amino acid sequence of SEQ ID NO: 28, and Fc2 comprises the amino acid sequence of SEQ ID NO: 31.

[0160] In some embodiments, Fc1 comprises the amino acid sequence of SEQ ID NO: 27, 28 or 29; and Fc2 comprises the amino acid sequence of SEQ ID NO: 30, 31 or 32.

[0161] In some embodiments, as described in any of the preceding embodiments, antigen-binding molecules that specifically bind to CD19, CD20, and CD3, wherein Fc1 and Fc2 are selected from any of the following:

[0162] Fc1 contains the amino acid sequence of SEQ ID NO: 27 and Fc2 contains the amino acid sequence of SEQ ID NO: 30; or

[0163] Fc1 contains the amino acid sequence SEQ ID NO: 28 and Fc2 contains the amino acid sequence SEQ ID NO: 31; or

[0164] Fc1 contains the amino acid sequence SEQ ID NO: 29 and Fc2 contains the amino acid sequence SEQ ID NO: 32; or

[0165] Fc1 contains the amino acid sequence of SEQ ID NO: 27 and Fc2 contains the amino acid sequence of SEQ ID NO: 31; or

[0166] Fc1 contains the amino acid sequence of SEQ ID NO: 28 and Fc2 contains the amino acid sequence of SEQ ID NO: 30.

[0167] In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20 and CD3 as described in any of the preceding embodiments, wherein Fc1 comprises the amino acid sequence of SEQ ID NO: 27 and Fc2 comprises the amino acid sequence of SEQ ID NO: 30.

[0168] In this disclosure, Fc1 and Fc2 are used only to distinguish the position of Fc, and are not intended to limit the specific sequence.

[0169] In some embodiments, antigen-binding molecules that specifically bind to CD19, CD20, and CD3, as described in any of the preceding embodiments, wherein:

[0170] The first antigen-binding domain that specifically binds to CD19 comprises a heavy chain variable region (VH) and a light chain variable region (VL). The VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; the VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6; and

[0171] The second antigen-binding domain that specifically binds to CD20 comprises a heavy chain variable region (VH) and a light chain variable region (VL). The VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 7, HCDR2 comprises the amino acid sequence of SEQ ID NO: 8, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 9; the VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 10, LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 12; and

[0172] The third antigen-binding domain that specifically binds to CD3 comprises a heavy chain variable region (VH) and a light chain variable region (VL). The VH comprises HCDR1, HCDR2, and HCDR3. HCDR1 comprises the amino acid sequence of SEQ ID NO: 13, HCDR2 comprises the amino acid sequence of SEQ ID NO: 14, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 15. The VL comprises LCDR1, LCDR2, and LCDR3. LCDR1 comprises the amino acid sequence of SEQ ID NO: 16, LCDR2 comprises the amino acid sequence of SEQ ID NO: 17, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 18.

[0173] In some embodiments, antigen-binding molecules that specifically bind to CD19, CD20, and CD3, as described in any of the preceding embodiments, wherein:

[0174] The first antigen-binding domain that specifically binds to CD19 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises SEQ ID NO: 19, 21, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith, and the VL comprises SEQ ID NO: 20, 22, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; and

[0175] The second antigen-binding domain that specifically binds to CD20 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises SEQ ID NO: 23, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith, and the VL comprises SEQ ID NO: 24, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; and

[0176] The third antigen-binding domain that specifically binds to CD3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises SEQ ID NO: 25, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith, and the VL comprises SEQ ID NO: 26, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith.

[0177] In some embodiments, antigen-binding molecules that specifically bind to CD19, CD20, and CD3, as described in any of the preceding embodiments, wherein:

[0178] The first antigen-binding domain that specifically binds to CD19 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 19, and the VL comprises the amino acid sequence of SEQ ID NO: 20; and

[0179] The second antigen-binding domain that specifically binds to CD20 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 23, and the VL comprises the amino acid sequence of SEQ ID NO: 24; and

[0180] The third antigen-binding domain that specifically binds to CD3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 25 and the VL comprises the amino acid sequence of SEQ ID NO: 26.

[0181] In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments comprises:

[0182] i) A first strand containing SEQ ID NO: 46, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; a second strand containing SEQ ID NO: 47, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; a third strand containing SEQ ID NO: 48, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and a third strand containing SEQ ID NO: 48. NO: 49, or the fourth strand of an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; or

[0183] ii) A first strand containing SEQ ID NO: 50, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; a second strand containing SEQ ID NO: 51, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; a third strand containing SEQ ID NO: 52, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; and a third strand containing SEQ ID NO: 51. NO: 53, or the fourth strand of an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; or

[0184] iii) A first strand containing SEQ ID NO: 54, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; a second strand containing SEQ ID NO: 55, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; a third strand containing SEQ ID NO: 56, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; and a third strand containing SEQ ID NO: 56. NO: 57, or the fourth strand having an amino acid sequence that is at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0185] In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments comprises:

[0186] i) A first strand containing the amino acid sequence of SEQ ID NO: 46, a second strand containing the amino acid sequence of SEQ ID NO: 47, a third strand containing the amino acid sequence of SEQ ID NO: 48, and a fourth strand containing the amino acid sequence of SEQ ID NO: 49.

[0187] ii) A first strand containing the amino acid sequence of SEQ ID NO: 50, a second strand containing the amino acid sequence of SEQ ID NO: 51, a third strand containing the amino acid sequence of SEQ ID NO: 52, and a fourth strand containing the amino acid sequence of SEQ ID NO: 53.

[0188] In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20 and CD3 as described in any of the preceding embodiments comprises a first chain containing the amino acid sequence of SEQ ID NO: 46, a second chain containing the amino acid sequence of SEQ ID NO: 47, a third chain containing the amino acid sequence of SEQ ID NO: 48 and a fourth chain containing the amino acid sequence of SEQ ID NO: 49.

[0189] On the other hand, this disclosure provides an antigen-binding molecule that specifically binds to CD19, CD20, and CD3, comprising a first antigen-binding domain that specifically binds to CD19, a second antigen-binding domain that specifically binds to CD20, and a third antigen-binding domain that specifically binds to CD3, wherein:

[0190] The first antigen-binding domain that specifically binds to CD19 comprises a heavy chain variable region (VH) and a light chain variable region (VL). The VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; the VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6; and

[0191] The second antigen-binding domain that specifically binds to CD20 comprises a heavy chain variable region (VH) and a light chain variable region (VL). The VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 7, HCDR2 comprises the amino acid sequence of SEQ ID NO: 8, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 9; the VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 10, LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 12; and

[0192] The third antigen-binding domain that specifically binds to CD3 comprises a heavy chain variable region (VH) and a light chain variable region (VL). The VH comprises HCDR1, HCDR2, and HCDR3. HCDR1 comprises the amino acid sequence of SEQ ID NO: 13, HCDR2 comprises the amino acid sequence of SEQ ID NO: 14, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 15. The VL comprises LCDR1, LCDR2, and LCDR3. LCDR1 comprises the amino acid sequence of SEQ ID NO: 16, LCDR2 comprises the amino acid sequence of SEQ ID NO: 17, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 18.

[0193] On the other hand, this disclosure provides an antigen-binding molecule that specifically binds to CD19, CD20, and CD3, comprising a first antigen-binding domain that specifically binds to CD19, a second antigen-binding domain that specifically binds to CD20, and a third antigen-binding domain that specifically binds to CD3, wherein:

[0194] The first antigen-binding domain that specifically binds to CD19 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 19, and the VL comprises the amino acid sequence of SEQ ID NO: 20; and

[0195] The second antigen-binding domain that specifically binds to CD20 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 23, and the VL comprises the amino acid sequence of SEQ ID NO: 24; and

[0196] The third antigen-binding domain that specifically binds to CD3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 25 and the VL comprises the amino acid sequence of SEQ ID NO: 26.

[0197] In some embodiments, the antigen-binding molecules that specifically bind to CD19, CD20, and CD3 provided herein can specifically bind to human antigens (including human CD19, CD20, and CD3 antigens) and have good cross-binding ability with monkey antigens (including monkey CD19, CD20, and CD3 antigens).

[0198] In some embodiments, the antigen-binding molecules that specifically bind to CD19, CD20 and CD3 provided in this disclosure can effectively bind to cell lines with different antigen expression levels (the expression levels of human CD19 antigen and human CD20 antigen on the cell surface are different).

[0199] In some embodiments, the antigen-binding molecules specifically binding to CD19, CD20, and CD3 provided in this disclosure are effective against both cell lines expressing only human CD19 antigen and cell lines expressing only CD20 antigen. In some embodiments, the antigen-binding molecules specifically binding to CD19, CD20, and CD3 provided in this disclosure are effective not only against cells with high CD19 expression but also against cells with low CD19 expression or those expressing only CD20 after CD19 loss. In some embodiments, the antigen-binding molecules specifically binding to CD19, CD20, and CD3 provided in this disclosure are effective not only against cells with high CD20 expression but also against cells with low CD20 expression or those expressing only CD19 after CD20 loss. The antibodies disclosed herein can cover a wider range of patient populations.

[0200] In some implementations, the antigen-binding molecules that specifically bind to CD19, CD20, and CD3 provided in this disclosure have good killing ability against various tumor cells (CD19 and CD20 expression covers high, medium, and low levels of expression).

[0201] In some implementations, the antigen-binding molecules that specifically bind to CD19, CD20, and CD3 provided in this disclosure still exhibit good killing effects on tumor cells under different E / T ratios.

[0202] In some implementations, the antigen-binding molecules that specifically bind to CD19, CD20, and CD3 provided in this disclosure exhibit good killing ability against both Raji and JeKo-1 tumor cells.

[0203] In some embodiments, the antigen-binding molecules that specifically bind to CD19, CD20, and CD3 provided in this disclosure have smaller EC50 when rituximab or tancituzumab are used in combination. 50 Shift window. In some implementations, the antigen-binding molecules that specifically bind to CD19, CD20, and CD3 provided in this disclosure exhibit better tumor-killing activity when used in combination with rituximab or tancituzumab.

[0204] Regarding antigen-binding molecules that specifically bind to CD3:

[0205] On the other hand, this disclosure also provides an antigen-binding molecule that specifically binds to CD3, comprising a heavy chain variable region, wherein the heavy chain variable region comprises the HCDR3 amino acid sequence in SEQ ID NO: 25.

[0206] In some embodiments, the antigen-binding molecule that specifically binds to CD3 as described in any of the preceding embodiments, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the sequence SEQ ID NO: 25; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the sequence SEQ ID NO: 26.

[0207] In some embodiments, the heavy chain variable regions HCDR1, HCDR2, and HCDR3 and the light chain variable regions LCDR1, LCDR2, and LCDR3 of the antigen-binding molecule that specifically binds to CD3 as described in any of the preceding embodiments are defined according to a numbering rule selected from Kabat, IMGT, Chothia, AbM, and Contact. In some embodiments, the heavy chain variable regions HCDR1, HCDR2, and HCDR3 and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to the Kabat numbering rule. In some embodiments, the heavy chain variable regions HCDR1, HCDR2, and HCDR3 and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to the IMGT numbering rule. In some embodiments, the heavy chain variable regions HCDR1, HCDR2, and HCDR3 and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to the Chothia numbering rule. In some embodiments, the heavy chain variable regions HCDR1, HCDR2, and HCDR3, and the light chain variable regions LCDR1, LCDR2, and LCDR3, are defined according to the AbM numbering rules. In some embodiments, the heavy chain variable regions HCDR1, HCDR2, and HCDR3, and the light chain variable regions LCDR1, LCDR2, and LCDR3, are defined according to the Contact numbering rules.

[0208] In some embodiments, an antigen-binding molecule that specifically binds to CD3, as described in the preceding one, wherein:

[0209] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 13, HCDR2 comprises the amino acid sequence of SEQ ID NO: 14, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 15; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 16, LCDR2 comprises the amino acid sequence of SEQ ID NO: 17, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 18.

[0210] In some embodiments, the antigen-binding molecules that specifically bind to CD3 as described in any of the preceding embodiments, wherein the heavy chain variable regions HCDR1, HCDR2, and HCDR3 and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to the Kabat numbering rules.

[0211] In some embodiments, an antigen-binding molecule that specifically binds to CD3, as described in the preceding one, wherein:

[0212] The heavy chain variable region comprises SEQ ID NO: 25, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and the light chain variable region comprises SEQ ID NO: 26, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0213] In some embodiments, the antigen-binding molecule that specifically binds to CD3 as described in any of the preceding embodiments, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 25, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 26.

[0214] In some embodiments, the CD3-specific antigen-binding molecule as described in any of the preceding claims further comprises a heavy chain constant region CH1 and a light chain constant region CL; wherein the heavy chain constant region CH1 is derived from human IgG1, IgG2, IgG3, or IgG4, and the light chain constant region CL is derived from human kappa or lambda. In some embodiments, the CD3-specific antigen-binding molecule as described in any of the preceding claims, wherein the heavy chain constant region CH1 is derived from human IgG1, and the light chain constant region CL is derived from human kappa. In some embodiments, the CD3-specific antigen-binding molecule as described in any of the preceding claims, wherein the heavy chain constant region CH1 comprises the amino acid sequence SEQ ID NO: 33, 34, or 35, and the light chain constant region CL comprises the amino acid sequence SEQ ID NO: 36, 37, or 38.

[0215] In some embodiments, the antigen-binding molecule that specifically binds to CD3 as described in any of the preceding embodiments includes an Fc region. In some embodiments, the Fc region is an IgG Fc region. In some embodiments, the Fc region is an Fc region derived from human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is an Fc region derived from human IgG1. In some embodiments, the Fc region contains the amino acid sequence of SEQ ID NO: 27, 28, 29, 30, 31, or 32.

[0216] In some implementations, the antigen-binding molecule that specifically binds to CD3, as described in any of the preceding embodiments, is an anti-CD3 antibody.

[0217] In another aspect, this disclosure also provides a pharmaceutical composition comprising a therapeutically effective amount of an antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding claims, or an antigen-binding molecule that specifically binds to CD3 as described in any of the preceding claims, and one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients. In some embodiments, the pharmaceutical composition further comprises at least one second therapeutic agent. In some embodiments, the second therapeutic agent comprises rituximab and / or tafasitamab.

[0218] In some embodiments, the pharmaceutical composition is used to treat a disease or condition.

[0219] In some embodiments, based on the total weight of the composition, the pharmaceutical composition contains 0.01-99.99% of antigen-binding molecules that specifically bind CD19, CD20, and CD3 as described in any of the preceding embodiments, or antigen-binding molecules that specifically bind CD3 as described in any of the preceding embodiments. In some embodiments, the pharmaceutical composition contains 0.1-99.9% of antigen-binding molecules that specifically bind CD19, CD20, and CD3 as described in any of the preceding embodiments, or antigen-binding molecules that specifically bind CD3 as described in any of the preceding embodiments. In some embodiments, the pharmaceutical composition contains 0.5%-99.5% of antigen-binding molecules that specifically bind CD19, CD20, and CD3 as described in any of the preceding embodiments, or antigen-binding molecules that specifically bind CD3 as described in any of the preceding embodiments. In some embodiments, the pharmaceutical composition contains 1%-99% of antigen-binding molecules that specifically bind CD19, CD20, and CD3 as described in any of the preceding embodiments, or antigen-binding molecules that specifically bind CD3 as described in any of the preceding embodiments. In some embodiments, the pharmaceutical composition contains 2%-98% of an antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments, or an antigen-binding molecule that specifically binds to CD3 as described in any of the preceding embodiments. In some embodiments, based on the total weight of the composition, the pharmaceutical composition contains 0.01%-99.99% of a pharmaceutically acceptable carrier, diluent, buffer, or excipient. In some embodiments, the pharmaceutical composition contains 0.1%-99.9% of a pharmaceutically acceptable carrier, diluent, buffer, or excipient. In some embodiments, the pharmaceutical composition contains 0.5%-99.5% of a pharmaceutically acceptable carrier, diluent, buffer, or excipient. In some embodiments, the pharmaceutical composition contains 1%-99% of a pharmaceutically acceptable carrier, diluent, buffer, or excipient. In some embodiments, the pharmaceutical composition contains 2%-98% of a pharmaceutically acceptable carrier, diluent, buffer, or excipient.

[0220] In another aspect, this disclosure also provides one or more nucleic acids (e.g., isolated nucleic acids) that encode antigen-binding molecules that specifically bind CD19, CD20 and CD3 as described in any of the preceding claims, or antigen-binding molecules that specifically bind CD3 as described in any of the preceding claims.

[0221] On the other hand, this disclosure also provides a carrier that contains one or more nucleic acids (e.g., isolated nucleic acids) as described above.

[0222] In another aspect, this disclosure also provides a host cell comprising one or more nucleic acids (e.g., isolated nucleic acids) as described in any of the preceding claims, or a vector as described in any of the preceding claims. In some embodiments, the host cell is a non-human host cell. In some embodiments, the host cell is a bacterium, yeast, or mammalian cell. In some embodiments, the host cell is a mammalian cell.

[0223] In another aspect, this disclosure provides a method for preparing an antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding claims, or an antigen-binding molecule that specifically binds to CD3 as described in any of the preceding claims, the method comprising expressing one or more nucleic acids (e.g., isolated nucleic acids) as described in any of the preceding claims, or culturing host cells as described in any of the preceding claims to produce the antigen-binding molecule that specifically binds to CD19, CD20, and CD3, or the antigen-binding molecule that specifically binds to CD3.

[0224] On the other hand, this disclosure provides antigen-binding molecules that specifically bind CD19, CD20, and CD3 as described in any of the preceding claims, or antigen-binding molecules that specifically bind CD3 as described in any of the preceding claims, or pharmaceutical compositions as described in any of the preceding claims. In some embodiments, the pharmaceutical remedy is a remedy for treating a disease or condition.

[0225] In another aspect, this disclosure also provides the use of antigen-binding molecules that specifically bind CD19, CD20 and CD3 as described in any of the preceding claims, or antigen-binding molecules that specifically bind CD3 as described in any of the preceding claims, or pharmaceutical compositions as described in any of the preceding claims in the preparation of medicaments for treating a disease or condition.

[0226] In another aspect, this disclosure also provides a method for treating a disease or condition, the method comprising administering to a subject an antigen-binding molecule that specifically binds to CD19, CD20 and CD3 as described in any of the preceding claims, or an antigen-binding molecule that specifically binds to CD3 as described in any of the preceding claims, or a pharmaceutical composition as described in any of the preceding claims.

[0227] In some embodiments, the disease or condition as described in any of the preceding embodiments is a tumor or an autoimmune disease. In some embodiments, the tumor is a hematologic malignancy. In some embodiments, the tumor is lymphoma or leukemia. In some embodiments, the tumor is non-Hodgkin lymphoma or acute lymphoblastic leukemia. In some embodiments, the tumor is B-cell non-Hodgkin lymphoma (B-NHL). In some embodiments, the tumor is diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, Burkitt lymphoma, or follicular lymphoma. In some embodiments, the tumor is diffuse large B-cell lymphoma (DLBCL). In some embodiments, the autoimmune disease is systemic lupus erythematosus (SLE) or lupus nephritis (LN). In some embodiments, the disease or condition is a tumor expressing CD19 and / or expressing CD20.

[0228] In some embodiments, the method further includes administering a second therapeutic agent; the antigen-binding molecule that specifically binds to CD19, CD20 and CD3, or the antigen-binding molecule that specifically binds to CD3 as described in any of the preceding embodiments, and the second therapeutic agent are administered simultaneously, sequentially or separately.

[0229] In some embodiments, the second therapeutic agent is selected from any of the following: antitumor agents, radiotherapy, antibody drug conjugates, or combinations thereof.

[0230] In some implementations, the second therapeutic agent comprises rituximab and / or tafasitamab. Attached Figure Description

[0231] Figure 1 shows a schematic diagram of trispecific antibodies with different structures. Detailed Implementation

[0232] Terminology (Definition)

[0233] To facilitate understanding of this disclosure, certain technical and scientific terms are described below. Unless otherwise expressly defined in this disclosure, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0234] The singular forms “a,” “an,” and “the” used in the specification and claims include plural references unless the context clearly indicates otherwise.

[0235] Unless the context clearly requires otherwise, the words “comprising,” “having,” “including,” etc., in the patent specification and claims should be understood as “including but not limited to,” rather than as exclusive or exhaustive.

[0236] The term "and / or" implies both "and" and "or". For example, the phrase "A, B and / or C" is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0237] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem., 243, p3558 (1968).

[0238] The term "CD3" refers to an antigen expressed on T cells as a portion of the multimolecular T cell receptor (TCR), composed of homodimers or heterodimers formed from two of the following four receptor chains: CD3-ε (CD3E or CD3 epsilon), CD3-δ (CD3D), CD3-ζ, and CD3-γ. Human CD3-ε (hCD3ε) contains the amino acid sequence described in UniProtKB / Swiss-Prot: P07766.2. Human CD3-δ (hCD3δ) contains the amino acid sequence described in UniProtKB / Swiss-Prot: P04234.1. Therefore, unless explicitly stated as originating from a non-human species, such as "mouse CD3," "monkey CD3," etc., the term "CD3" refers to human CD3.

[0239] The term “CD19” or “differentiation cluster 19” refers to the differentiation cluster 19 protein, an antigenic determinant detectable on leukemia progenitor cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and SwissProt. For example, the amino acid sequence of human CD19 can be found as UniProt / SwissProt accession number P15391, and the nucleotide sequence encoding human CD19 can be found at accession number NM_001178098. CD19 is expressed in most B-lineage cancers, including, for example, acute lymphoblastic leukemia, chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), and non-Hodgkin's lymphoma. It is also an early marker of B-cell progenitor cells. See, for example, Nicholson et al., 1997, Mol. Immun. [Molecular Immunology] 34(16-17):1157-1165. The term encompasses both "full-length" unprocessed human CD19 and any form of human CD19 produced by cell processing, provided that the antibody reported in this disclosure binds to it.

[0240] The term "CD20" is also known as B lymphocyte antigen CD20, B lymphocyte surface antigen B1, Leu-16, Bp35, BM5, and LF5; it is a human protein characterized in UniProt database entry P11836. CD20 is a hydrophobic transmembrane protein with a molecular weight of approximately 35 kDa, expressed on pre-B lymphocytes and mature B lymphocytes (Valentine, MA et al., J. Biol. Chem. 264 (1989) 11282-11287; Tedder, TF et al., Proc. Natl. Acad. Sci. USA 85 (1988) 208-212; Stamenkovic, I. et al., J. Exp. Med. 167 (1988) 1975-1980; Einfeld, DA et al., EMBO J. 7 (1988) 711-717; Tedder, TF et al., J. Immunol. 142 (1989) 2560-2568). The corresponding human gene is a transmembrane 4-domain, subfamily A member 1, also known as MS4A1. This gene encodes a member of the transmembrane 4A gene family. Members of this neonatal protein family are characterized by shared structural features and similar intron / exon splicing boundaries, and exhibit distinct expression patterns in hematopoietic cells and non-lymphoid tissues. This gene encodes a B lymphocyte surface molecule that functions during B cell development and differentiation into plasma cells. Family members are located at 11q12 within a cluster of family members. Alternative splicing of this gene produces two transcript variants encoding the same protein. Unless otherwise specified, the term “CD20” as used in this disclosure refers to any naturally occurring CD20 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term includes “full-length” unprocessed CD20 as well as any form of CD20 produced through cellular processing. The term also covers naturally occurring variants of CD20, such as spliced ​​variants or allelic variants. In one embodiment, CD20 is human CD20.

[0241] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (e.g., ortholeucine) or modified peptide backbones but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to naturally occurring amino acids.

[0242] The term "amino acid mutation" includes amino acid substitution (also known as amino acid replacement), deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be performed to achieve the final construct, provided that the final construct possesses the desired properties, such as reduced or absent binding to Fc receptors. Amino acid sequence deletions and insertions include deletions and insertions at the amino and / or carboxyl ends of the polypeptide chain. A specific amino acid mutation can be an amino acid substitution. In some embodiments, an amino acid mutation is a non-conservative amino acid substitution, i.e., replacing one amino acid with another amino acid that has a different structure and / or chemical properties. Amino acid substitution includes substitution by non-naturally occurring amino acids or by derivatives of 20 naturally occurring amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. Methods other than genetic engineering that alter amino acid side chain groups, such as chemical modification, are also expected to be available. Various names may be used in this disclosure to refer to the same amino acid mutation. In this disclosure, the amino acid residue at a specific site may be represented by the format "position + amino acid residue". For example, 102S indicates that the amino acid residue at position 102 is S. C102S indicates that the amino acid residue at position 102 has mutated from C to S. When the residue at a specific site is defined in the claim using the format "position + amino acid residue", the original residue at that site does not limit the scope of protection.

[0243] The term "antigen-binding molecule" is used in the broadest sense to encompass molecules that specifically bind antigens, including but not limited to antibodies, other peptides with antigen-binding activity, and antibody fusion proteins formed by the fusion of the two, as well as any molecule containing the aforementioned antibodies, peptides, or antibody fusion proteins, provided they exhibit the desired antigen-binding activity. The antigen-binding molecules disclosed herein comprise a variable region (VH) and a variable region (VL), which together constitute an antigen-binding domain. Exemplarily, the antigen-binding molecules in this disclosure are trispecific antigen-binding molecules (e.g., trispecific antibodies).

[0244] The term “antibody” is used in the broadest sense and covers a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity.

[0245] The term "natural antibody" refers to naturally occurring immunoglobulin molecules. For example, natural IgG antibodies are heterotetraglycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH, also known as the variable heavy domain or heavy chain variable region), followed by a heavy chain constant region. The natural IgG heavy chain constant region typically contains three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL, also known as the variable light domain or light chain variable domain), followed by a constant light domain (light chain constant region, CL).

[0246] The terms "full-length antibody," "intact antibody," and "complete antibody" are used interchangeably in this disclosure, referring to antibodies with a structure substantially similar to that of natural antibodies or with a heavy chain containing an Fc region as defined in this disclosure. The light chain of a natural intact antibody includes a variable region (VL) and a constant region (CL), with VL located at the amino terminus of the light chain. The constant region includes the κ and λ chains. The heavy chain includes a variable region (VH) and constant regions (CH1, CH2, and CH3), with VH located at the amino terminus of the heavy chain and the constant region located at the carboxyl terminus, where CH3 is closest to the carboxyl terminus of the polypeptide. The heavy chain can belong to any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0247] The term "multispecific antibody" refers to an antibody (including the antibody or its antigen-binding fragment) that can specifically bind to multiple different antigens or at least two different antigenic epitopes of the same antigen. In this disclosure, “anti-CD19, CD20, and CD3 antibodies are multispecific antibodies, optionally trispecific antibodies, comprising three distinct antigen-binding domains, one of which binds to antigen CD19, one of which binds to antigen CD20, and one of which binds to CD3. The term ‘multispecific antibody’ is used in the broadest sense in this disclosure and specifically covers antibodies having multi-epitope specificity. Multispecific antibodies include, but are not limited to, antibodies comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH and VL together constitute an antigen-binding domain (where VH / VL have multi-epitope specificity), antibodies having two or more VH and VL domains (each VH / VL unit binds to a different epitope), antibodies having two or more single variable domains (each single variable domain binds to a different epitope), full-length antibodies, and antibodies comprising one or more antibody fragments, as well as antibodies comprising antibody fragments covalently or non-covalently linked. Multispecific antibodies can be bispecific antibodies, trispecific antibodies, biantibodies, or similar molecules (see, for example, PNAS USA). (See 90(14), 6444-8(1993) for a description of biantibodies). In this disclosure, “trispecific antibody” should be understood as an antibody having three distinct antigen-binding domains defined by different antibody sequences. This can be understood as binding to different targets, but also includes binding to different epitopes within a single target.

[0248] The term "operably linked" refers to a functional relationship between two or more peptide or polypeptide domains or nucleic acid (e.g., DNA) segments. In this disclosure, the term "operably linked" means linking two or more amino acid segments to produce a functional polypeptide. For example, in the context of antigen-binding molecules of this disclosure, individual antigen-binding domains can be linked by peptide linkers. In the context of nucleic acids encoding fusion proteins, such as polypeptide chains of antigen-binding molecules of this disclosure, "operably linked" means linking two nucleic acids such that the amino acid sequences encoded by the two nucleic acids remain within the frame.

[0249] The term "association" in this disclosure refers to a functional relationship between two or more chains. Specifically, the term "association" means that two or more polypeptides associate with each other, for example, through non-covalent association via molecular interactions or through covalent association via one or more disulfide bridges or chemical crosslinks, thereby generating a functional antigen-binding molecule (e.g., a trispecific antibody), wherein a first antigen-binding domain, a second antigen-binding domain, and a third antigen-binding domain can bind their respective targets. Examples of association that may exist in the antigen-binding molecules of this disclosure include (but are not limited to) association between Fc regions in the Fc domain, association between the VH and VL regions in the Fab or Fv domain, and association between CH1 and CL in the Fab domain.

[0250] In the context of antigen-binding molecules (e.g., trispecific antibodies), the term "trivalent" refers to an antigen-binding molecule that has three antigen-binding domains.

[0251] The term "variable region" or "variable domain" in an antibody refers to the domain in the antibody heavy or light chain involved in antibody binding to the antigen. In this disclosure, the antibody heavy chain variable region (VH) and light chain variable region (VL) each contain four conserved frame regions (FRs) and three complementarity-determining regions (CDRs). The term "complementarity-determining region" or "CDR" refers to the region within the variable domain that primarily facilitates antigen binding; "frame" or "FR" refers to the variable domain residues other than the CDR residues. The VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; the VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus (also known as the N-terminus) to the carboxyl terminus (also known as the C-terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0252] The amino acid sequence boundaries of CDRs can be determined using various well-known schemes, such as the "Kabat" numbering rule, the "Chothia" numbering rule, the "ABM" numbering rule, the "contact" numbering rule, and the ImMunoGenTics (IMGT) numbering rule. The correspondence between various numbering systems is well known to those skilled in the art and is exemplified as shown in Table 1 below.

[0253] Table 1. Relationship between CDR numbering systems

[0254] Unless otherwise stated, the variable regions and CDRs in this disclosure embodiment are governed by the "Kabat" numbering rule. Although the Kabat numbering rule is used in specific implementations to define amino acid residues, corresponding technical solutions using other numbering systems are considered equivalent.

[0255] The term “antigen-binding fragment” encompasses full-length antibodies, Fab, modified Fab, Fab', Fab'-SH, modified Fab', F(ab')2, Fv, dsFv, Fab-Fv, Fab-dsFv, Fd, single-domain antibodies (sdAb, e.g., VH or VL or VHH), single-chain Fab (scFab), single-chain antibodies (e.g., scFv, sc(Fv)2), biantibodies, linear antibodies, bivalent or trivalent or quadrivalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope-binding fragments of any of the above (see, for example, Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for generating and preparing these antigen-binding fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0256] The term "antibody fragment" refers to a molecule that is distinct from the intact antibody but contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, dsFv, Fab, Fab′, Fab′-SH, F(ab′)2, Fd, single-domain antibodies (sdAb, such as VH, VL, or VHH), single-chain Fab (scFab), biantibodies, linear antibodies, single-chain antibodies (such as scFv, sc(Fv)2); and multispecific antibodies formed from antibody fragments.

[0257] The term "half-antibody" refers to a molecule that contains at least one antigen-binding domain and can associate with another molecule containing an antigen-binding domain via, for example, a disulfide bridge or molecular interaction (e.g., the club-and-mortar interaction between Fc heterodimers). A half-antibody may consist of one or more polypeptide chains (e.g., the two polypeptide chains of Fab). In some embodiments, the half-antibody further comprises an Fc region.

[0258] Examples of haptens are molecules comprising a heavy chain and a light chain of an antibody (e.g., an IgG antibody). In some embodiments, the hapten is a molecule comprising a first polypeptide and a second polypeptide, the first polypeptide comprising a VL domain and a CH1 domain, and the second polypeptide comprising (in order from N-terminus to C-terminus) a VH domain, a CL domain, a hinge domain, a CH2 domain, and a CH3 domain. In other embodiments, the hapten is a molecule comprising a first polypeptide and a second polypeptide, the first polypeptide comprising a VL domain and a CL domain, and the second polypeptide comprising (in order from N-terminus to C-terminus) a VH domain, a CH1 domain, a ScFv domain, a hinge domain, a CH2 domain, and a CH3 domain. Another example of a hapten is a molecule comprising a first polypeptide and a second polypeptide, the first polypeptide comprising a VL domain and a CL domain, and the second polypeptide comprising (in order from N-terminus to C-terminus) a VH domain, a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain, wherein the VL and VH domains form an antigen-binding domain. In some embodiments, the hapten may comprise more than one antigen-binding domain; for example, the hapten may comprise a molecule comprising a first polypeptide and a second polypeptide, the first polypeptide comprising (in order from N-terminus to C-terminus) a VH domain, a CH1 domain, an scFv domain, a CH2 domain, and a CH3 domain, and the second polypeptide comprising a VL domain and a CL domain.

[0259] Therefore, an antigen-binding molecule may contain at least one, more typically one, two, or even more than two half antibodies, and a half antibody may contain one or more antigen-binding domains.

[0260] In some embodiments, the first hapten will associate with the second hapten, for example, through heterodimerization. In other embodiments, the first hapten will be covalently linked to the second hapten, for example, via disulfide bridging or chemical cross-linking. In one specific embodiment, the first hapten will associate with the second hapten through covalent attachment and non-covalent interactions, such as disulfide bridging and mortise-and-tenon interactions.

[0261] The term "half-antibody" is intended for descriptive purposes only and does not indicate a specific configuration or method of manufacture. The descriptions of half-antibodies as "first" half-antibody, "second" half-antibody, "left" half-antibody, "right" half-antibody, etc., are merely for convenience and descriptive purposes.

[0262] The term "C-terminus," such as carboxyl-terminus, C-terminus, C-tail, C-terminus, or COOH-terminus, refers to the end of an amino acid chain (protein or polypeptide) terminated by a free carboxyl group (-COOH). When a protein is translated from messenger RNA, it is produced from the N-terminus to the C-terminus. The convention for writing peptide sequences is to place the C-terminus on the right and write the sequence from N-terminus to C-terminus. In some embodiments, the C-terminus of a polypeptide includes the last amino acid residue of the polypeptide, which contributes its amino group to form a peptide bond with the carboxyl group of its adjacent amino acid residue.

[0263] The term "N-terminus," etc. (e.g., amino-terminus, NH2-terminus, N-terminus, N-terminus, or amine-terminus), refers to the start of a protein or polypeptide, specifically the free amino group (-NH2) located at the end of the polypeptide. Normally, the amino group bonds to another carboxyl group in the protein to make it chained, but since only one of the two regions at the end of a protein is chained, the free amino group refers to the N-terminus. As mentioned above, by convention, peptide sequences in LTR language are written from N-terminus to C-terminus, from left to right. This associates the translation direction with the text direction (because when a protein is translated from messenger RNA, it is produced from N-terminus to C-terminus—an amino acid is added to the carbonyl terminus). In some embodiments, the N-terminus of the polypeptide contains the first amino acid of the polypeptide, which contributes its carboxyl group to form a peptide bond with the amino group of its adjacent amino acid residue.

[0264] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of an antibody heavy chain, including both native and engineered Fc regions. In some embodiments, the Fc region comprises two identical or different subunits. Suitable Fc regions for the antibodies described in this disclosure include the Fc regions of human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. In some embodiments, the boundaries of the Fc region may also vary, for example, by deleting the C-terminal lysine (residue 447 according to the EU numbering system) or by deleting both the C-terminal glycine and lysine (residues 446 and 447 according to the EU numbering system). Unless otherwise stated, the Fc region is numbered according to the EU numbering system, also known as the EU index.

[0265] The Fc region can be appropriately obtained by partially digesting IgG monoclonal antibodies with proteolytic enzymes such as pepsin, followed by eluting the components adsorbed on the protein A or protein G column. As the proteolytic enzyme, any enzyme capable of restrictively digesting full-length antibodies to produce Fab and F(ab')2 by appropriately setting the enzyme reaction conditions such as pH is acceptable; there is no particular limitation, and examples include pepsin and papain.

[0266] In this disclosure, the term "Fc region" or "Fc domain" refers to an antibody region that contains at least a CH2 domain and a CH3 domain. In this disclosure, the term "CH2 region" or "CH2 domain" is intended to refer to the CH2 region of an immunoglobulin. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to the EU numbering system (according to the IMGT website). However, the CH2 region can also be any other antibody isotype as described in this disclosure.

[0267] In this disclosure, the terms “CH3 region,” “CH3 domain,” or “CH3 structural domain” are intended to refer to the CH3 region of an immunoglobulin. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the EU numbering system (according to the IMGT website). However, the CH3 region can also be any other antibody isotype as described in this disclosure.

[0268] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a specific source or species, while the remaining portion of the heavy and / or light chain is derived from another different source or species.

[0269] The term "humanized" antibody refers to an antibody that retains the reactivity of a non-human antibody while exhibiting lower immunogenicity in humans. For example, this can be achieved by retaining the non-human CDR region and replacing the rest of the antibody with its human counterpart (i.e., the frame region portion of the constant region and the variable region).

[0270] The terms "human antibody," "fully human antibody," and "completely human antibody" are used interchangeably, referring to antibodies whose variable and constant regions are human sequences. This term encompasses antibodies derived from human genes but with sequence alterations, such as reduced potential immunogenicity, increased affinity, or the elimination of cysteine ​​residues or glycosylation sites that might cause undesirable folding. This term also covers antibodies recombined in non-human cells (which may confer glycosylations not characteristic of human cells). The term also includes antibodies generated in transgenic mice containing some or all human immunoglobulin heavy and light chain loci. The meaning of "human antibody" explicitly excludes humanized antibodies containing non-human antigen-binding residues.

[0271] The term "affinity" refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise specified, as used in this disclosure, binding "affinity" refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its ligand Y can typically be represented by the dissociation constant (KD). Affinity can be measured using conventional methods known in the art, including those described in this disclosure.

[0272] As used in this disclosure, the term "kassoc" or "ka" refers to the association rate of a specific antibody-antigen interaction, and the term "kdis" or "kd" refers to the dissociation rate of a specific antibody-antigen interaction. The term "KD" refers to the dissociation constant, which is derived from the ratio of kd to ka (i.e., kd / ka) and expressed as a molar concentration (M). The KD value of an antibody can be determined using methods known in the art. For example, it can be determined using a biosensing system such as a system for measuring surface plasmon resonance (e.g., Biacore), or by measuring affinity in solution using solution equilibrium titration (SET).

[0273] The term “surface plasmon resonance” refers to the optical phenomenon of analyzing real-time interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIAcore™ system (Biacore LifeSciences division of GE Healthcare, Piscataway, NJ).

[0274] The term "effector function" refers to biological activities attributable to the antibody's Fc region (either the native Fc region or the Fc region with amino acid sequence mutations) and that vary across antibody isotypes. Examples of antibody effector functions include, but are not limited to: C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0275] The term "monoclonal antibody" refers to a group of antibodies that are substantially homogeneous, meaning that the antibody molecules contained in this group have the same amino acid sequence, except for the possible small number of naturally occurring mutations. In contrast, polyclonal antibody formulations typically contain multiple different antibodies with varying amino acid sequences in their variable domains, and they generally target different epitopes specifically. "Monoclonal" indicates the characteristic of antibodies obtained from a substantially homogeneous group of antibodies and should not be interpreted as requiring the production of the antibody through any particular method.

[0276] The term "antigen" refers to a molecule or molecular part that can be bound by antigen-binding proteins, including, for example, antibodies. An antigen may have one or more epitopes that can interact with different antigen-binding proteins, such as antibodies.

[0277] The term "epitope" refers to a region on an antigen that is capable of specifically binding to an antibody or its antigen-binding fragment. Epitopes can be formed from a continuous string of amino acids (linear epitopes) or contain discontinuous amino acids (conformal epitopes), for example, due to the folding of the antigen (i.e., the tertiary folding of an antigen as a protein). The difference between conformational and linear epitopes is that in the presence of a denaturing solvent, the antibody loses binding to the conformational epitope. An epitope contains at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind the same epitope) can be performed using methods routine in the art, such as, but not limited to, alanine scanning, peptide blotting, peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see Prot. Sci. 9 (2000) 487-496), and cross-blocking.

[0278] The terms "specific binding," "specific binding," or "binding" refer to the ability of an antibody to bind to a specific antigen or epitope with a higher affinity than other antigens or epitopes. Typically, antibodies bind at an affinity of approximately 1 × 10⁻⁶. -7 M or smaller (e.g., about 1×10⁻⁶) -8 M, 1×10 -9 M, 1×10 -10 M, 1×10 -11 The equilibrium dissociation constant (KD) of an antibody (M or less) binds to an antigen or an epitope within the antigen. In some embodiments, the KD of the antibody binding to an antigen is 10% or less (e.g., 1%) of the KD of the antibody binding to a nonspecific antigen (e.g., BSA, casein). KD can be measured using known methods, such as by... Surface plasmon resonance assays are used to measure this. However, antibodies that specifically bind to antigens or epitopes within antigens may be cross-reactive to other related antigens, for example, to corresponding antigens from other species (homologous) (such as humans or monkeys, such as the cynomolgus (cyno), chimpanzee (chimp), or common marmoset (marmoset)).

[0279] The term "non-binding" means that the antibody cannot bind to an antigen or its epitope in the manner described above for specific binding. For example, when the antibody binds at approximately 1 × 10⁻⁶... -6 M or a larger equilibrium dissociation constant (KD) binds to the antigen or its epitope.

[0280] The terms "antigen-binding molecule that specifically binds to CD19, CD20, and CD3," "anti-CD19, CD20, and CD3 trispecific antibody," and "CD19xCD20xCD3 trispecific antibody" refer to antigen-binding molecules or antibodies capable of binding to CD19 or its epitopes, CD20 or its epitopes, and CD3 or its epitopes with sufficient affinity. For example, in the Biacore assay, the affinity (KD) for binding to human CD19 is below 7 nM.

[0281] In some embodiments, the term "first antigen-binding domain specifically binding to CD19" refers to an antigen-binding domain capable of binding to CD19 with a certain affinity, such that molecules containing this antigen-binding domain can be used as diagnostic and / or therapeutic agents targeting CD19. For example, the first antigen-binding domain specifically binding to CD19 has the following equilibrium dissociation constant (KD) for binding to human CD19: < about 7 nM, which is measured by surface plasmon resonance assay. In some embodiments, the term "second antigen-binding domain specifically binding to CD20" refers to an antigen-binding domain capable of binding to CD20 with a certain affinity, such that molecules containing this antigen-binding domain can be used as diagnostic and / or therapeutic agents targeting CD20. For example, the second antigen-binding domain specifically binding to CD20 has the following EC50 value (FACS detection) for binding to human CD20: < about 4 nM, which is measured by FACS. In this disclosure, antigen-binding domains include "antibody," "antigen-binding fragment," and "antibody fragment" (e.g., Fab, replaced by Fab, scFv) as defined in this disclosure.

[0282] The term "linker" refers to a connecting unit that links two polypeptide fragments. In this disclosure, linkers appearing in the same structural formula may be the same or different. A linker may be a "peptide linker" containing one or more amino acids, typically about 1-30, 2-24, or 3-15 amino acids. Linkers used in this disclosure may be the same or different. When a "-" appears in a structural formula, it indicates that the units on either side are directly connected by a covalent bond.

[0283] The term "peptide linker" can be any suitable peptide chain, as long as the antigen-binding molecule can exhibit the desired antigen-binding activity. For example, a peptide linker can be a flexible peptide containing 1-50 or 1-20 amino acid residues. In some embodiments, each of the peptide linkers independently has an L1-(GGGGS)n-L2 structure, wherein L1 is a bond, A, G, GS, GGG, GGS, GGGS, or GGGG (SEQ ID NO: 66), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, L2 is a bond, G, GG, GGG, or GGGG (SEQ ID NO: 66), and the peptide linker is not a bond. In some embodiments, the length of the peptide linker is 2-20 amino acid residues. In some embodiments, the peptide linker is represented by the following formula: (GS) a (GGS) b (GGGS) c (GGGGS) d (GGGGG) e a, b, c, d, and e are independent integers greater than or equal to 0; or the peptide linker is selected from: (EAAAK)3 (SEQ ID NO: 68), (EAAAR)3 (SEQ ID NO: 69), (EGGGK)3 (SEQ ID NO: 70), (EGGGR)3 (SEQ ID NO: 71), (DAAAR)3 (SEQ ID NO: 72), (DAAAK)3 (SEQ ID NO: 73), (DGGGR)3 (SEQ ID NO: 74) or (DGGGK)3 (SEQ ID NO: 75), SS (SEQ ID NO: 39), GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 41), EPKSS (SEQ ID NO: 42), RTVAA (SEQ ID NO: 44), ASEPKSS (SEQ ID NO: 45); or the peptide linker is (GxS)y, where x is selected from integers 1-5, and y is selected from integers 1-6, including but not limited to GGGS (SEQ ID NO: 68). (SEQ ID NO: 76), (GGGGS)2 (SEQ ID NO: 40), (GGGGS)2 (SEQ ID NO: 43), (GGGGS)3 (SEQ ID NO: 77); or the peptide linker is GGGGG (SEQ ID NO: 78). In some embodiments, the amino acid sequence of the peptide linker is as shown in SEQ ID NO: 39, 40, 41, 42, 43, 44 or 45. In some embodiments, the amino acid sequence of the peptide linker is as shown in SEQ ID NO: 39, 40 or 41.

[0284] The terms “antibody-dependent cell cytotoxicity,” “antibody-dependent cell-mediated cytotoxicity,” or “ADCC” refer to mechanisms that induce cell death that rely on the interaction between antibody-coated target cells and lytic effector cells (such as natural killer (NK) cells, monocytes, macrophages, and neutrophils) via Fcγ receptors (FcγR) expressed on the effector cells. For example, NK cells express FcγRIIIa, while monocytes express FcγRI, FcγRII, and FcγRIIIa. The ADCC activity of the antibodies disclosed herein can be assessed in vitro using cells expressing the antigen as target cells and NK cells as effector cells. Cell lysis is detected based on the release of markers (such as radioactive substrates, fluorescent dyes, or native intracellular proteins) from lysed cells.

[0285] The term "antibody-dependent phagocytosis (ADCP)" refers to the mechanism by which antibody-coated target cells are eliminated through internalization by phagocytes (such as macrophages or dendritic cells).

[0286] The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism that induces cell death in which the Fc effector domain of a target-binding antibody binds to and activates the complement component C1q. C1q then activates the complement cascade, leading to target cell death. Activation of complement can also result in the deposition of complement components on the surface of target cells, which promote CDC by binding to complement receptors (e.g., CR3) on leukocytes.

[0287] The term "nucleic acid" is used interchangeably with the term "polynucleotide" in this disclosure and refers to deoxyribonucleotides or ribonucleotides and their polymers in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, or non-naturally occurring, have similar binding properties to a reference nucleic acid, and are metabolized in a manner similar to that of a reference nucleotide. Examples of such analogs include, but are not limited to, thiophosphates, aminophosphates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide-nucleic acids (PNAs). "Isolated" nucleic acid refers to a nucleic acid molecule that has been separated from its components in its natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that typically contain such molecules but are present outside the chromosome or at a chromosomal location different from their natural chromosomal location. Isolated nucleic acids encoding polypeptides or fusion proteins refer to one or more nucleic acid molecules encoding polypeptides or fusion proteins, including one or more such nucleic acid molecules in a single or separate vector, and one or more such nucleic acid molecules present at one or more locations in the host cell. Unless otherwise stated, a particular nucleic acid sequence also implicitly encompasses variants of its conserved modifications (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly stated sequences. Specifically, as detailed below, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues.

[0288] The terms “polypeptide” and “protein” are used interchangeably in this disclosure.

[0289] The term "sequence identity" refers to the degree (percentage) to which two sequences share the same amino acids / nucleic acids at equivalent positions; wherein, when performing optimal alignment of two sequences, gaps are introduced where necessary to obtain the maximum percentage of sequence identity, and no conserved substitutions are considered part of the sequence identity. To determine the percentage of sequence identity, alignment can be performed using techniques known in the art, such as publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0290] The term "vector" refers to a polynucleotide molecule capable of transporting another polynucleotide linked to it. One type of vector is a "plasmid," which is a circular double-stranded DNA loop in which an additional DNA segment can be attached. Another type of vector is a viral vector, such as an adeno-associated virus vector (AAV or AAV2), in which an additional DNA segment can be attached to the viral genome. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and attachable mammalian vectors). Other vectors (e.g., non-attached mammalian vectors) can integrate into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome. The term "expression vector" or "expression construct" refers to a vector capable of transforming host cells and containing a nucleic acid sequence that directs and / or controls (alongside the host cell) the expression of one or more heterologous coding regions operatively linked to it. Expression constructs can include, but are not limited to, sequences that affect or control transcription, translation, and, in the presence of introns, influence RNA splicing of coding regions operatively linked to them.

[0291] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their derived progeny, regardless of passage number. Progeny may not be identical to parental cells in their nucleic acid contents and may contain mutations. This disclosure includes mutant progeny with the same function or biological activity as those screened or selected in the initially transformed cells. Host cells include prokaryotic and eukaryotic host cells, wherein eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.Fungal cells include yeast and filamentous fungal cells, including, for example, *Pichia pastoris*, *Pichia finlandica*, *Pichia trehalophila*, *Pichia koclamae*, *Pichia membranaefaciens*, *Pichia minuta* (Ogataea minuta, *Pichia lindneri*), *Pichia xiaopuntiae*, *Pichia thermotolerans*, *Pichia salictaria*, *Pichia guercuum*, *Pichia pijperi*, *Pichia stipitis*, *Pichia methanolica*, *Pichia* genus, *Saccharomyces cerevisiae*, *Saccharomyces* genus, and *Hansenula*. The fungi include *C. polymorpha*, *Kluyveromyces lactis*, *Candida albicans*, *Aspergillus*, *Aspergillus nidulans*, *Aspergillus niger*, *Aspergillus oryzae*, *Trichoderma reesei*, *Chrysosporium lucknowense*, *Fusarium* sp., *Fusarium graminearum*, *Fusarium venenatum*, *Physcomitrella patens*, and *Neurospora crassa*.

[0292] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur.

[0293] The term "pharmaceutical composition" refers to a mixture containing one or more antigen-binding molecules that specifically bind CD19, CD20 and CD3 as described in this disclosure, along with other chemical components, such as physiological / pharmaceutical carriers and excipients.

[0294] The term "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical formulation that is distinct from the active ingredient and non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0295] The terms “subject” or “individual” include both humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise specified, the terms “patient” or “subject” are used interchangeably in this disclosure. In some embodiments, the individual or subject is a human being.

[0296] "Administration" or "giving," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to the contact between an exogenous drug, therapeutic agent, diagnostic agent, or composition and the animal, human, subject, cell, tissue, organ, or biological fluid.

[0297] The term "sample" refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within the subject's body. Exemplary samples include biological fluids such as blood, serum and serous fluid, plasma, lymph, urine, saliva, cystic fluid, tears, excretions, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids in the abdominal cavity and other body cavities, fluids collected by bronchoalveolar lavage fluid, synovial fluid, liquid solutions in contact with the subject or biological sources, such as cell and organ culture media (including cell or organ conditioned media), lavage fluids, tissue biopsy samples, fine-needle aspiration, surgically removed tissue, organ cultures, or cell cultures.

[0298] "Treatment" and "treatment" (and their grammatical variations) refer to clinical interventions that attempt to alter the natural processes of the individual being treated, and can be implemented for prevention or during a clinicopathological process. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing / decreasing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and resolving or improving prognosis. In some implementations, antibodies disclosed herein are used to delay disease onset or slow disease progression.

[0299] The terms "recurrence," "relapse," and "relapsed" refer to the recovery of cancer or disease after a clinical assessment of disease resolution. A diagnosis of distant metastasis or local recurrence can be considered a recurrence.

[0300] The terms "refractory" or "resistant" refer to cancers or diseases that do not respond to treatment.

[0301] An "effective dose" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate such symptoms and / or underlying causes, prevent the occurrence of symptoms and / or underlying causes, and / or improve or mitigate damage caused by or associated with a disease state (e.g., lung disease). In some embodiments, an effective dose is a therapeutically effective dose or a preventatively effective dose. A "therapeuticly effective dose" is an amount sufficient to treat a disease state or symptom, particularly a state or symptom associated with that disease state, or otherwise prevent, inhibit, delay, or reverse the progression of the disease state or any other undesirable symptom associated with that disease. A "preventatively effective dose" is an amount that, when administered to a subject, will have a predetermined preventative effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or related symptoms. A complete therapeutic or preventative effect may not occur after the administration of a single dose, but may occur after the administration of a series of doses. Therefore, a therapeutically or preventatively effective dose may be administered in a single or multiple-dose manner. "Therapeutic effective dose" and "preventive effective dose" can vary depending on a number of factors, such as an individual's disease state, age, sex, and weight, as well as the ability of the treatment or combination of treatments to elicit the desired response in the individual. Exemplary indicators of an effective treatment or combination of treatments include, for example, improved health status in the patient.

[0302] This disclosure discloses antigen-binding molecules that specifically bind to CD19, CD20, and CD3.

[0303] This disclosure provides antigen-binding molecules that specifically bind to CD19, CD20, and CD3. Compared with existing CD20xCD3 bispecific antibodies, CD19xCD3 bispecific antibodies, or CD19xCD20xCD3 triple antibodies, these molecules possess numerous advantageous properties. For example, they allow for more specific targeting of specific cell subpopulations, exhibit antigen-binding activity (including specific binding activity to human antigens and good cross-binding activity with monkey antigens), effectively bind to cell lines with different antigen expression levels (i.e., varying levels of human CD19 and human CD20 antigen expression on cell surfaces), demonstrate good killing ability against various tumor cells (CD19 and CD20 expression covers high, medium, and low levels), maintain good killing effect on tumor cells under different E / T ratios, and exhibit good therapeutic activity, safety, pharmacokinetic properties, and drug-likeness (e.g., solubility, viscosity, purity, and stability).

[0304] Exemplary antigen-binding molecules that specifically bind to CD19, CD20, and CD3

[0305] For example, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 disclosed herein comprises:

[0306] The first antigen-binding domain that specifically binds to CD19 is Fab.

[0307] A second antigen-binding domain that specifically binds to CD20, wherein the second antigen-binding domain that specifically binds to CD20 is a substituted Fab; and

[0308] The third antigen-binding domain that specifically binds to CD3 is scFv;

[0309] The first antigen-binding domain that specifically binds to CD19 comprises a heavy chain variable region (VH) and a light chain variable region (VL). The VH comprises HCDR1, HCDR2, and HCDR3, with the amino acid sequence of HCDR1 shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 shown in SEQ ID NO: 2, and the amino acid sequence of HCDR3 shown in SEQ ID NO: 3. The VL comprises LCDR1, LCDR2, and LCDR3, with the amino acid sequence of LCDR1 shown in SEQ ID NO: 4, the amino acid sequence of LCDR2 shown in SEQ ID NO: 5, and the amino acid sequence of LCDR3 shown in SEQ ID NO: 6.

[0310] The second antigen-binding domain that specifically binds to CD20 comprises a heavy chain variable region (VH) and a light chain variable region (VL). The VH comprises HCDR1, HCDR2, and HCDR3, the amino acid sequence of which is shown in SEQ ID NO: 7, the amino acid sequence of which is shown in SEQ ID NO: 8, and the amino acid sequence of which is shown in SEQ ID NO: 9. The VL comprises LCDR1, LCDR2, and LCDR3, the amino acid sequence of which is shown in SEQ ID NO: 10, the amino acid sequence of which is shown in SEQ ID NO: 11, and the amino acid sequence of which is shown in SEQ ID NO: 12.

[0311] The third antigen-binding domain that specifically binds to CD3 comprises a heavy chain variable region (VH) and a light chain variable region (VL). The VH comprises HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is shown in SEQ ID NO: 13, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 14, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 15. The VL comprises LCDR1, LCDR2, and LCDR3. The amino acid sequence of LCDR1 is shown in SEQ ID NO: 16, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 17, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 18.

[0312] For example, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 disclosed herein comprises:

[0313] The first antigen-binding domain that specifically binds to CD19 is Fab.

[0314] A second antigen-binding domain that specifically binds to CD20, wherein the second antigen-binding domain that specifically binds to CD20 is a substituted Fab; and

[0315] The third antigen-binding domain that specifically binds to CD3 is scFv;

[0316] The first antigen-binding domain that specifically binds to CD19 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the amino acid sequence of the VH is shown in SEQ ID NO: 19 and the amino acid sequence of the VL is shown in SEQ ID NO: 20.

[0317] The second antigen-binding domain that specifically binds to CD20 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the amino acid sequence of the VH is shown in SEQ ID NO: 23 and the amino acid sequence of the VL is shown in SEQ ID NO: 24;

[0318] The third antigen-binding domain that specifically binds to CD3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the amino acid sequence of the VH is shown in SEQ ID NO: 25, and the amino acid sequence of the VL is shown in SEQ ID NO: 26.

[0319] For example, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 disclosed herein consists of a first chain, a second chain, a third chain, and a fourth chain, wherein:

[0320] The amino acid sequence of the first chain is shown in SEQ ID NO: 46, the amino acid sequence of the second chain is shown in SEQ ID NO: 47, the amino acid sequence of the third chain is shown in SEQ ID NO: 48, and the amino acid sequence of the fourth chain is shown in SEQ ID NO: 49.

[0321] For example, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 disclosed herein consists of a first chain, a second chain, a third chain, and a fourth chain, wherein:

[0322] The amino acid sequence of the first chain is shown in SEQ ID NO: 46, the amino acid sequence of the second chain is formed by adding amino acid residue K to the C-terminus of the sequence shown in SEQ ID NO: 47, the amino acid sequence of the third chain is formed by adding amino acid residue K to the C-terminus of the sequence shown in SEQ ID NO: 48, and the amino acid sequence of the fourth chain is shown in SEQ ID NO: 49.

[0323] For example, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 disclosed herein consists of a first chain, a second chain, a third chain, and a fourth chain, wherein:

[0324] The amino acid sequence of the first chain is shown in SEQ ID NO: 50, the amino acid sequence of the second chain is shown in SEQ ID NO: 51, the amino acid sequence of the third chain is shown in SEQ ID NO: 52, and the amino acid sequence of the fourth chain is shown in SEQ ID NO: 53.

[0325] For example, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 disclosed herein consists of a first chain, a second chain, a third chain, and a fourth chain, wherein:

[0326] The amino acid sequence of the first chain is shown in SEQ ID NO: 50, the amino acid sequence of the second chain is formed by adding amino acid residue K to the C-terminus as shown in SEQ ID NO: 51, the amino acid sequence of the third chain is formed by adding amino acid residue K to the C-terminus as shown in SEQ ID NO: 52, and the amino acid sequence of the fourth chain is shown in SEQ ID NO: 53.

[0327] Structure of antigen-binding molecules

[0328] The trispecific antibodies disclosed herein are not limited to a specific molecular structure, as long as they possess the desired antigen-binding function. For example, the trispecific antibodies disclosed herein can be trivalent (1+1+1). The antigen-binding domain in the trispecific antibody can be any antibody or antibody fragment with antigen-binding activity, fused via a peptide linker.

[0329] For example, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 disclosed herein comprises:

[0330] The first antigen-binding domain that specifically binds to CD19 is Fab.

[0331] A second antigen-binding domain that specifically binds to CD20, wherein the second antigen-binding domain that specifically binds to CD20 is a substituted Fab; and

[0332] The third antigen-binding domain that specifically binds to CD3 is scFv.

[0333] For example, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 disclosed herein comprises:

[0334] The first half antibody comprises a replaced Fab and Fc1 that bind to CD20;

[0335] The second half antibody comprises a Fab that binds to CD19, an scFv that binds to CD3, and an Fc2, wherein the first half antibody and the second half antibody are associated with each other via Fc1 and Fc2.

[0336] Exemplary examples include antigen-binding molecules that specifically bind to CD19, CD20, and CD3, wherein the antigen-binding molecules that specifically bind to CD19, CD20, and CD3 are (trivalent) trispecific antibodies and comprise:

[0337] The first half antibody comprises a replaced Fab and Fc1 that bind to CD20;

[0338] The second half antibody comprises a Fab that binds to CD19, an scFv that binds to CD3, and an Fc2, wherein the first half antibody and the second half antibody are associated with each other via Fc1 and Fc2.

[0339] For example, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 disclosed herein is a (trivalent) trispecific antibody and comprises:

[0340] The first hapten, comprising, from the N-terminus to the C-terminus, substituted Fab and Fc1 that bind CD20;

[0341] The second half antibody includes, from the N-terminus to the C-terminus, a Fab that binds CD19, an scFv that binds CD3, and an Fc2, wherein the first half antibody and the second half antibody are associated with each other via Fc1 and Fc2.

[0342] Exemplary examples include antigen-binding molecules that specifically bind to CD19, CD20, and CD3, wherein:

[0343] The antigen-binding molecule that specifically binds to CD19, CD20, and CD3 is a (trivalent) trispecific antibody and comprises:

[0344] The first hapten includes, from the N-terminus to the C-terminus, a CD20-binding substituted Fab and an Fc1, wherein the C-terminus of the CD20-binding substituted Fab and the N-terminus of the Fc1 are operatively linked.

[0345] The second half antibody includes, from the N-terminus to the C-terminus, a Fab that binds CD19, a scFv that binds CD3, and an Fc2, wherein the N-terminus of the scFv that binds CD3 is operatively connected to the C-terminus of the Fab that binds CD19, and the C-terminus of the scFv that binds CD3 is operatively connected to the N-terminus of the Fc2.

[0346] Furthermore, the first and second halves of the antibody are associated with each other via Fc1 and Fc2.

[0347] In some embodiments, antigen-binding molecules that specifically bind to CD19, CD20, and CD3, as described in any of the preceding embodiments, wherein:

[0348] The antigen-binding molecule that specifically binds to CD19, CD20, and CD3 is a (trivalent) trispecific antibody and comprises:

[0349] The first hapten includes, from the N-terminus to the C-terminus, a CD20-binding substituted Fab and an Fc1, wherein the C-terminus of the CD20-binding substituted Fab and the N-terminus of the Fc1 are operatively linked.

[0350] The second half antibody contains, from the N-terminus to the C-terminus, a Fab that binds to CD19, an scFv that binds to CD3, and an Fc2.

[0351] The Fab comprises a light chain variable region (VL), a light chain constant region (CL), a heavy chain variable region (VH), and a heavy chain constant region 1 (CH1);

[0352] The N-terminus of the CD3-bonded scFv is operably connected to the C-terminus of the CH1 of the CD19-bonded Fab, and the C-terminus of the CD3-bonded scFv is operably connected to the N-terminus of the Fc2.

[0353] Furthermore, the first and second halves of the antibody are associated with each other through the Fc region.

[0354] For example, the antigen-binding molecule that specifically binds to CD19, CD20 and CD3 disclosed herein has the structure shown in Format B of Figure 1.

[0355] In some embodiments, antigen-binding molecules that specifically bind to CD19, CD20, and CD3, as described in any of the preceding embodiments, wherein:

[0356] The antigen-binding molecule that specifically binds to CD19, CD20, and CD3 is a (trivalent) trispecific antibody and comprises:

[0357] The first hapten includes, from the N-terminus to the C-terminus, a CD20-binding substituted Fab and an Fc1, wherein the C-terminus of the CD20-binding substituted Fab and the N-terminus of the Fc1 are operatively linked.

[0358] The second half antibody contains, from the N-terminus to the C-terminus, a Fab that binds to CD19, an scFv that binds to CD3, and an Fc2.

[0359] The Fab comprises a light chain variable region (VL), a light chain constant region (CL), a heavy chain variable region (VH), and a heavy chain constant region 1 (CH1);

[0360] The N-terminus of the scFv of CD3 is operatively connected to the C-terminus of the CL of the Fab of CD19.

[0361] The C-terminus of CH1 of the Fab combined with CD19 is operatively connected to the N-terminus of Fc2.

[0362] Furthermore, the first and second halves of the antibody are associated with each other via Fc1 and Fc2.

[0363] In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments, wherein the replaced Fab comprises a light chain variable region (VL), a light chain constant region (CL), a heavy chain variable region (VH), and a heavy chain constant region 1 (CH1), and comprises any of the following substitutions:

[0364] (i) VL and VH are interchangeable;

[0365] (ii) CL and CH1 are interchangeable; or

[0366] (iii) VL and CL, VH and CH1 are interchangeable.

[0367] In some implementations, the replaced Fab comprises CL and CH1 interchangeably.

[0368] In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments, wherein the replaced Fab of the second antigen-binding domain specifically binding to CD20 comprises CL and CH1 interchangeably, or the replaced Fab of the second antigen-binding domain specifically binding to CD20 comprises VL and VH interchangeably, or the replaced Fab of the second antigen-binding domain specifically binding to CD20 comprises both VL-CL and VH-CH1 interchangeably. In some embodiments, the replaced Fab of the second antigen-binding domain specifically binding to CD20 comprises CL and CH1 interchangeably.

[0369] In some embodiments, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 as described in any of the preceding embodiments, wherein the Fab of the first antigen-binding domain that specifically binds to CD19 comprises a light chain variable region (VL), a light chain constant region (CL), a heavy chain variable region (VH), and a heavy chain constant region 1 (CH1), and the amino acid at position 126 of CH1 is cysteine, and the amino acid at position 121 of CL is cysteine, numbered according to the EU index.

[0370] In some embodiments, as described in any of the preceding embodiments, the antigen-binding molecule specifically binds to CD19, CD20, and CD3, wherein the heavy chain constant region CH1 of the first antigen-binding domain specifically binding to CD19 is the heavy chain constant region CH1 of human IgG1, and the amino acid at position 126 is cysteine, and the light chain constant region CL is the light chain constant region CL of human kappa, and the amino acid at position 121 is cysteine, numbered according to the EU index. In some embodiments, as described in any of the preceding embodiments, the antigen-binding molecule specifically binds to CD19, CD20, and CD3, wherein the heavy chain constant region CH1 of the first antigen-binding domain specifically binding to CD19 is the heavy chain constant region CH1 of human IgG1, and the amino acid at position 126 is cysteine, the amino acid at position 220 is non-cysteine, and the light chain constant region CL is the light chain constant region CL of human kappa, and the amino acid at position 121 is cysteine, the amino acid at position 214 is non-cysteine, numbered according to the EU index.

[0371] For example, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 disclosed herein comprises:

[0372] A first chain having the structure shown in equation (a), a second chain having the structure shown in equation (b), a third chain having the structure shown in equation (c), and a fourth chain having the structure shown in equation (d), wherein the structures shown in equations (a), (b), (c), and (d) are arranged from the N end to the C end:

[0373] (a) [CD20-VL]-[Connector 1]-[CH1-1];

[0374] (b)[CD20-VH]-[CL1]-[Fc1];

[0375] (c)[CD19-VH]-[CH1-2]-[Connector 2]-[CD3-VH]-[Connector 3]-[CD3-VL]-[Connector 4]-[Fc2];

[0376] (d)[CD19-VL]-[CL2];

[0377] The linkers 1, 2, 3 and 4 are the same or different peptide linkers, or linkers 1, 2, 3 and / or 4 are absent;

[0378] Fc1 and Fc2 are interchangeable;

[0379] The first and second chains combine with each other, the third and fourth chains combine with each other, and the second and third chains combine with each other to form antigen-binding molecules that specifically bind to CD19, CD20 and CD3.

[0380] For example, the structure of the antigen-binding molecule that specifically binds to CD19, CD20 and CD3 disclosed herein is shown in Format A of Figure 1.

[0381] antibody structure

[0382] In some implementations, the antibodies provided in this disclosure are full-length antibodies.

[0383] In some implementations, the antibodies provided in this disclosure are antibody fragments.

[0384] In some embodiments, the antibody fragment is a Fab, Fab′, Fab′-SH, or F(ab′)2 fragment, particularly a Fab fragment. “Fab” is a monovalent fragment consisting of VL, VH, CL, and CH1 domains. A “Fab fragment” can be generated by cleavage of an antibody with papain. “Fab′” contains VL, CL, VH, and CH1, and also contains a region between the CH1 and CH2 domains, allowing interchain disulfide bonds to form between the two heavy chains of two Fab′ fragments to form an F(ab′)2 molecule. “Fab′-SH” is a Fab′ fragment in which the cysteine ​​residues in the constant region have free thiol groups. “F(ab′)2” is a divalent fragment comprising two Fab fragments linked by disulfide bonds in the hinge region.

[0385] In other embodiments, the antibody fragment is a biantibody, triantibody, or tetraantibody. A biantibody is an antibody fragment having two antigen-binding sites, containing linked VH and VL within the same polypeptide chain (VH-VL). By using a short linker that prevents the two domains on the same chain from pairing, these domains are forced to pair with complementary domains on another chain, thereby creating two antigen-binding sites, the two antigens of which may be the same or different.

[0386] In other embodiments, the antibody fragment is a single-chain Fab fragment. A “single-chain Fab fragment” or “scFab” is a polypeptide consisting of VH, CH1, VL, CL, and a linker, wherein the antibody domain and the linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL. In some embodiments, the linker is a polypeptide having at least 30 amino acids. In other embodiments, the linker is a polypeptide having between 32 and 50 amino acids. The single-chain Fab fragment is stabilized via a native disulfide bond between CL and CH1. Additionally, these single-chain Fab molecules can be further stabilized by inserting cysteine ​​residues (e.g., at position 44 in the heavy chain variable region and position 100 in the light chain variable region, according to Kabat numbering) to create interchain disulfide bonds.

[0387] In other embodiments, the antibody fragment is an Fv fragment composed of the VH and VL domains of a single arm of the antibody.

[0388] In other embodiments, the antibody fragment is a single-chain variable fragment (scFv). An “scFv” is a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light and heavy chain variable regions are sequentially linked by a short, flexible peptide linker, capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, in this disclosure, the scFv may have VL and VH variable regions in any order; for example, relative to the N-terminus and C-terminus of the polypeptide, the scFv may comprise a VL-linker-VH or may comprise a VH-linker-VL.

[0389] In other embodiments, the antibody fragment is dsFv, which is obtained by linking polypeptides in which one amino acid residue in each VH and VL is replaced by a cysteine ​​residue via disulfide bonds between cysteine ​​residues. The amino acid residues to be replaced by cysteine ​​residues can be selected based on prediction of the antibody's three-dimensional structure using known methods (Protein Engineering. 7:697 (1994)).

[0390] In other embodiments, the antibody fragment is a single-domain antibody (dAb). A single-domain antibody is an antibody fragment containing all or part of the heavy chain variable domain or all or part of the light chain variable domain of the antibody.

[0391] In some embodiments, the antibodies provided in this disclosure are chimeric antibodies. In some embodiments, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In some embodiments, the chimeric antibody is a "class-switched" antibody, wherein the class or subclass has been changed from the class or subclass of the parent antibody.

[0392] In some embodiments, the antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody contains one or more variable regions, wherein the CDR or a portion thereof is derived from the non-human antibody, and the FR or a portion thereof is derived from the human antibody. Optionally, the humanized antibody may also contain a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody may be replaced with corresponding residues from the non-human antibody (e.g., an antibody providing the CDR sequence).

[0393] Humanized antibodies and their generation methods are reviewed in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5,821,337,7,527,791,6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describes specificity-determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describes “resurfuacing”); Dall'Acqua et al., Methods 36:43-60 (2005) (describes “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer 83:252-260 (2000) (describes the “guided selection” method for FR shuffling).

[0394] Human frame regions that can be used for humanization include, but are not limited to: frame regions selected using a "best-fit" method (see, for example, Sims et al., J. Immunol. 151:2296 (1993)); frame regions of the common sequence of human antibodies derived from specific subgroups of light chain variable regions or heavy chain variable regions (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:2623 (1993)); mature human (somatic mutant) frame regions or human germline frame regions (see, for example, Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and the frame regions obtained by screening FR libraries (see, for example, Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

[0395] Variants of antigen-binding molecules that specifically bind to CD19, CD20, and CD3

[0396] In some embodiments, amino acid sequence variants of antigen-binding molecules that specifically bind CD19, CD20, and CD3, as provided in this disclosure, are included. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing suitable modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions, and / or insertions, and / or substitutions of residues within the amino acid sequence of antigen-binding molecules that specifically bind CD19, CD20, and CD3. Any combination of deletions, insertions, and substitutions can be performed to obtain the final construct, provided that the final construct possesses the desired characteristics, such as antigen-binding properties.

[0397] Replace, insert, and delete variants

[0398] In some embodiments, antibody variants with one or more amino acid substitutions are provided. Substitution mutagenesis sites of interest include CDR and FR. Conserved substitutions are shown in Table 2-1 under the heading “Preferred Substitutions.” More substantial variations are provided in Table 2-1 under the heading “Exemplary Substitutions” and are further described below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into the antibody of interest, and the product can be screened for desired activities, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0399] Table 2-1. Substitution of amino acids

[0400] Based on common side-chain characteristics, amino acids can be grouped as follows:

[0401] (1) Hydrophobic: Leucine, Met, Ala, Val, Leu, Ile;

[0402] (2) Neutral and hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0403] (3) Acidic: Asp, Glu;

[0404] (4) Alkaline: His, Lys, Arg;

[0405] (5) Residues that affect chain orientation: Gly, Pro;

[0406] (6) Aromatic: Trp, Tyr, Phe.

[0407] Non-conservative replacement would require replacing a member of one of these categories with a member of another category.

[0408] One class of substitution variants involves replacing one or more CDR residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further research will have alterations (e.g., improvements) to certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody, and / or will substantially retain certain biological properties of the parent antibody. An exemplary substitution variant is an affinity-matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques (such as those described in this disclosure). In short, one or more CDR residues are mutated, and the variant antibody is displayed on a phage and screened for specific biological activities (e.g., binding affinity). CDRs can be altered (e.g., substituted), for example, to improve antibody affinity. Such alterations can be made to CDR “hotspots,” residues encoded by codons that undergo mutations at a high frequency during somatic maturation, and / or residues that contact the antigen, while testing the binding affinity of the resulting variant VH or VL. In some implementations of affinity maturation, diversity is introduced into the selected variant gene for maturation using any of a variety of methods, such as error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis. A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves CDR-directed approaches, where several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scan mutagenesis or modeling. In particular, HCDR3 and LCDR3 are frequently targeted.

[0409] In some embodiments, substitution, insertion, or deletion can occur within one or more CDRs, provided that such changes do not materially reduce the antibody's ability to bind to the antigen. For example, conserved changes (e.g., conserved substitutions, as provided in this disclosure) can be made to the CDRs that do not materially reduce binding affinity. Such changes can, for example, be external to the antigen-contacting residues in the CDR. In some embodiments of the variant VH and VL sequences provided above, each CDR is either unchanged or contains no more than one, two, or three amino acid substitutions.

[0410] One method for identifying residues or regions in an antibody that can serve as mutagenic targets is called "alanine scan mutagenesis." In this method, a residue or target group of residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., Ala or polyalanine) to determine if the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Furthermore, the contact points between the antibody and antigen can be identified by studying the crystal structure of the antigen-antibody complex. These contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine if they contain the desired properties.

[0411] Amino acid sequence insertions include fusion of the amino and / or carboxyl ends of peptides ranging in length from 1 residue to 100 or more residues, and intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N- or C-terminus of the antibody with an enzyme or a peptide that extends the serum half-life of the antibody.

[0412] Replaced Fab

[0413] In other embodiments, the antigen-binding domain disclosed herein is a replaced Fab domain. The Fab domain can be generated by proteolytic cleavage of an immunoglobulin molecule, using an enzyme such as papain, or through recombinant expression. The Fab domain typically includes a CH1 domain attached to a VH domain, which pairs with a CL domain attached to a VL domain.

[0414] In wild-type immunoglobulins, the VH domain pairs with the VL domain to form the Fv region, and the CH1 domain pairs with the CL domain to further stabilize the binding module. A disulfide bond between the two constant domains further stabilizes the Fab domain.

[0415] For the antigen-binding molecules disclosed herein (e.g., trispecific antibodies), it is advantageous to use a Fab heterodimerization strategy (i.e., substituted Fab) to allow proper association of Fab domains belonging to the same antigen-binding domain and minimize aberrant pairing of Fab domains belonging to different antigen-binding domains. For example, substituted Fabs as shown in Table 2-2 below can be used:

[0416] Table 2-2. Replaced Fab

[0417] Therefore, in some embodiments, proper association between two peptides of Fab is facilitated by exchanging the VL and VH domains of Fab with each other or by exchanging the CH1 and CL domains with each other, for example, as described in WO 2009 / 080251. The antigen-binding molecule disclosed herein is trispecific, meaning it contains at least three antigen-binding domains capable of specifically binding to three different antigenic determinants. In a specific embodiment, the trispecific antigen-binding molecule is capable of binding to three different antigenic determinants simultaneously. According to this disclosure, the antigen-binding domain is a replaced Fab. In one embodiment, the replaced Fab is human. In another embodiment, the replaced Fab is humanized. In yet another embodiment, the Fab fragment contains human heavy and light chain constant regions.

[0418] According to this disclosure, at least one of the replaced Fab fragments is a "Crossfab" fragment, wherein variable and / or constant domains of the Fab heavy and light chains are exchanged. This type of modification prevents mismatches between the heavy and light chains from different Fab fragments, thereby improving the yield and purity of the trispecific antigen-binding molecule disclosed herein in the recombinant production. In other words, the problem of heavy and light chain mismatches in trispecific antibody production is overcome by exchanging variable and / or constant domains of the heavy and light chains within one or more Fab fragments of the trispecific antigen-binding molecule, ensuring that Fab fragments of different specificities do not have the same domain arrangement and therefore do not "exchange" light chains.

[0419] Possible substitutions include: (i) substitution of the variable domains (VH and VL) of the Fab heavy chain and the light chain; (ii) substitution of the constant domains (CH1 and CL) of the Fab heavy chain and the light chain; or (iii) substitution of the Fab heavy chain and the light chain (VH-CH1 and VL-CL).

[0420] To achieve the desired result—preventing mismatches between heavy and light chains of different specificities—different substitutions must be made in Fab fragments of different specificities. For example, in Fab fragments that specifically bind CD20, the constant regions of the heavy and light chains can be interchanged.

[0421] The Fab domain can also be modified by replacing the CH1 and CL domains with alternative domains that promote proper assembly. For example, Wu et al., 2015, MAbs 7:364-76, described replacing the CH1 domain with the constant domain of the αT cell receptor and the CL domain with the β domain of the T cell receptor, and further replacing these domains with additional charge-charge interactions between the VL and VH domains by introducing 38D modification into the VL domain and 39K modification into the VH domain.

[0422] In some embodiments, the Fab of the antigen-binding molecule in this disclosure is a replaced Fab, which includes a heavy chain variable region, a light chain variable region, a titin chain, and an obscurin chain. In the replaced Fab, the original CH1 and CL of the Fab are replaced by the titin chain and the obscurin chain. For example, the sequences of the titin chain and the obscurin chain are derived from WO2021139758A1 and WO2022237882A1.

[0423] scFv structural domain

[0424] In some other embodiments, the antigen-binding domain disclosed herein is an scFv domain.

[0425] A "single-chain Fv" or "scFv" antibody fragment contains the VH and VL domains of an antibody within a single polypeptide chain, enabling expression as a single-chain polypeptide while retaining the specificity of the complete antibody from which it is derived. Typically, the scFv polypeptide further includes a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for target binding.

[0426] Unless otherwise stated, as used in this disclosure, the scFv may have VL and VH variable regions in any order relative to the N-terminus and C-terminus of the polypeptide, and the scFv may contain VL-connector-VH or may contain VH-connector-VL.

[0427] To generate scFv-encoded nucleic acids, the VH and VL-encoded DNA fragments are operatively linked to another fragment encoding a linker, such that the VH and VL sequences can be expressed as contiguous single-stranded proteins (with their VL and VH regions linked by a flexible linker) (see, for example, Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990, Nature 348:552-554).

[0428] Renovation of Fc District

[0429] In one aspect, the Fc region of the antigen-binding molecule disclosed herein contains one or more amino acid substitutions that reduce its binding to an Fc receptor, such as an Fcγ receptor, and reduce or eliminate effector function. Natural IgG Fc regions, specifically IgG1 or IgG4 Fc regions, may cause the antigen-binding molecule disclosed herein to target cells expressing Fc receptors rather than cells expressing antigens. The modified Fc region of this disclosure exhibits reduced binding affinity to Fc receptors and / or reduced effector function. In some embodiments, the modified Fc region exhibits a 50%, 80%, 90%, or 95% or more reduction in binding affinity to Fc receptors compared to the natural Fc region. In some embodiments, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fcγ receptor, such as FcγRI, FcγRIIa, FcγRIIB, or FcγRIIIa. In some embodiments, the modified Fc region also exhibits reduced binding affinity for complement, such as C1q, compared to the natural Fc region. In some embodiments, the modified Fc region does not exhibit reduced binding affinity for neonatal Fc receptors (FcRn) compared to the natural Fc region. In some embodiments, the modified Fc region has reduced effector functions, which may include, but are not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent phagocytosis (ADCP), reduced cytokine secretion, reduced antigen uptake by immune complex-mediated antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling-induced apoptosis, reduced dendritic cell maturation, or reduced T cell initiation. For the IgG1 Fc region, amino acid residues at positions 234, 235, 238, 265, 269, 270, 297, 327, and 329 replace potentially degraded effector functions. In some embodiments, the Fc region is the human IgG1 Fc region, and amino acid residues at positions 234 and 235 are designated as A, numbered according to the EU index. In some embodiments, the Fc region is the human IgG1 Fc region, and amino acid residues at positions 234 and 235 are designated as A, while amino acid residue at position 265 is designated as S, numbered according to the EU index. For the IgG4 Fc region, amino acid residues at positions 228, etc., replace potentially degraded effector functions.

[0430] Antigen-binding molecules can also contain disulfide bond modifications, such as 354C in the first subunit and 349C in the second subunit. To increase the serum half-life of antigen-binding molecules, mutations in 252Y, 254T, and 256E can be introduced.

[0431] When an antigen-binding molecule contains different binding modules fused to the two subunits of the Fc region, undesirable homodimerization may occur. To improve yield and purity, it is advantageous to introduce modifications that promote heterodimerization into the Fc region of the antigen-binding molecule disclosed herein. In some embodiments, the Fc region of this disclosure comprises a modification according to the knock-in-hole (KIH) technique, which involves introducing a protrusion (knob) at the interface of the first subunit and a hole (hole) at the interface of the second subunit. This allows the protrusion to be positioned within the hole, promoting heterodimer formation and inhibiting homodimer production. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first subunit with a larger side chain (e.g., tyrosine or tryptophan). The hole is created at the interface of the second subunit by replacing a large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). The protrusion and hole structures are prepared by altering the nucleic acid encoding the polypeptide; optional amino acid substitutions are shown in Tables 2-3.

[0432] Table 2-3. KIH mutation combinations

[0433] Besides the mortar and pestle technique, other techniques for modifying the CH3 domain of heavy chains to achieve heterodimerization are also known in the art, such as WO1996027011A1, WO1998050431A2, EP1870459A1, WO2007110205A2, WO2009089004A1, WO2010129304A2, WO2011143545A1, WO2012058768A1, WO2013157954A1 and WO2013096291A2.

[0434] The C-terminus of the Fc region can be a complete C-terminus ending with the amino acid residue PGK; or it can be a truncated C-terminus, for example, in which one or two C-terminal amino acid residues have been removed. In a preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. Thus, in some embodiments, the composition of a complete antibody may include an antibody population with all K447 residues and / or G446+K447 residues removed. In some embodiments, the composition of a complete antibody may include an antibody population without the removal of K447 residues and / or G446+K447 residues. In some embodiments, the composition of a complete antibody has an antibody population consisting of a mixture of antibodies with and without K447 residues and / or G446+K447 residues.

[0435] Recombination method

[0436] Antigen-binding molecules that specifically bind to CD19, CD20, and CD3 can be generated using recombinant methods. For these methods, one or more nucleic acids (e.g., isolated nucleic acids) encoding antigen-binding molecules that specifically bind to CD19, CD20, and CD3 are provided.

[0437] In some embodiments, this disclosure provides isolated nucleic acids encoding antigen-binding molecules that specifically bind CD19, CD20, and CD3 as described above. Such nucleic acids can be derived from independent polypeptide chains encoding any of the aforementioned. In another aspect, this disclosure provides one or more vectors (e.g., expression vectors) containing such nucleic acids. In yet another aspect, this disclosure provides host cells containing such nucleic acids. In some embodiments, a method for preparing a polypeptide or fusion protein is provided, wherein the method includes culturing host cells containing nucleic acids encoding said polypeptide or fusion protein, as provided above, under conditions suitable for expression, and optionally recovering said antigen-binding molecules that specifically bind CD19, CD20, and CD3 from the host cells (or host cell culture medium).

[0438] To recombinantly generate antigen-binding molecules that specifically bind to CD19, CD20, and CD3, nucleic acids encoding the proteins are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. These nucleic acids can be readily isolated and sequenced using standard procedures, or generated through recombinant methods or obtained through chemical synthesis.

[0439] Suitable host cells for cloning or expressing vectors encoding antigen-binding molecules that specifically bind to CD19, CD20, and CD3 include the prokaryotic or eukaryotic cells described in this disclosure. For example, they can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. Following expression, the expression can be separated from the bacterial cell paste in a soluble fraction and can be further purified.

[0440] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding fusion proteins, including fungal and yeast strains. Suitable host cells for expressing fusion proteins can also be derived from multicellular organisms (invertebrates and vertebrates); examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of fall armyworm (Spodoptera frugiperda) cells; plant cell cultures can also be used as hosts, such as US5959177, US 6040498, US6420548, US 7125978, and US6417429; and vertebrate cells, such as mammalian cell lines adapted for growth in suspension, can also be used as hosts. Other examples of suitable mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (293 or 293T cells); young hamster kidney cells (BHK); mouse seltoli cells (TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. Other suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines such as Y0, NSO, and Sp2 / 0. For reviews of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0441] Measurement

[0442] The antigen-binding molecules that specifically bind to CD19, CD20, and CD3 provided in this disclosure can be identified, screened, or characterized by their physical / chemical properties and / or biological activities using a variety of assays known in the art. In one aspect, the activity of the antigen-binding molecules that specifically bind to CD19, CD20, and CD3 disclosed herein can be tested, for example, by known methods such as ELISA, Western blotting, etc.

[0443] Treatment methods and routes of administration

[0444] Any antigen-binding molecules that specifically bind to CD19, CD20, and CD3 provided in this disclosure may be used to treat diseases. In another aspect, the use of antigen-binding molecules that specifically bind to CD19, CD20, and CD3 provided in this disclosure in the manufacture or preparation of medicaments. In some embodiments, the disease is a disease or condition related to CD19 and / or CD20. In some embodiments, the disease is a tumor or autoimmune disease.

[0445] In another aspect, pharmaceutical compositions comprising the antigen-binding molecules that specifically bind CD19, CD20, and CD3 are provided, for example, for any of the pharmaceutical uses or treatment methods described above. In some embodiments, the pharmaceutical composition comprises any of the antigen-binding molecules that specifically bind CD19, CD20, and CD3 provided in this disclosure and a pharmaceutically acceptable carrier. In other embodiments, the pharmaceutical composition further comprises at least one additional therapeutic agent.

[0446] The antigen-binding molecules that specifically bind to CD19, CD20, and CD3 disclosed herein can be used alone or in combination with other agents for treatment. For example, the antibodies disclosed herein can be administered co-administered with at least one additional therapeutic agent.

[0447] The antigen-binding molecules (and any other therapeutic agents) that specifically bind to CD19, CD20, and CD3 as disclosed herein may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Administration may be carried out via any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. This disclosure considers a variety of dosing schedules, including, but not limited to, single or multiple administrations at multiple time points, bolus administration, and pulsatile infusion.

[0448] The antigen-binding molecules that specifically bind to CD19, CD20, and CD3 disclosed herein will be formulated, administered, and applied in accordance with good medical practice. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to a medical practitioner. The antigen-binding molecules that specifically bind to CD19, CD20, and CD3 may be formulated with or without one or more agents currently used for the prevention or treatment of the stated condition. The effective amount of such other agents depends on the amount present in the pharmaceutical composition, the type of condition or treatment, and other factors. These are generally used at the same dosage and route of administration as described herein, or at about 1 to 99% of the dosage described herein, or at other dosages, and in any route determined empirically / clinically as appropriate.

[0449] For the prevention or treatment of disease, the appropriate dosage of the antigen-binding molecules that specifically bind to CD19, CD20, and CD3 (when used alone or in combination with one or more other additional therapeutic agents) disclosed herein will depend on the type of disease to be treated, the type of therapeutic molecule, the severity and duration of the disease, whether it is administered for prophylactic or therapeutic purposes, prior treatment, the patient's clinical history and response to the therapeutic molecule, and the judgment of the attending physician. The therapeutic molecule is appropriately administered to the patient either as a single dose or after a series of treatments.

[0450] Products

[0451] In another aspect of this disclosure, an article of manufacture (such as a medicine box) is provided, comprising materials that can be used to treat, prevent, and / or diagnose the aforementioned conditions. The article of manufacture comprises a container and a label or package insert on or in conjunction with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed from various materials such as glass or plastic. The container contains a composition, alone or in combination with another composition, that is effective in treating, preventing, and / or diagnosing the condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper puncturable by a hypodermic needle). At least one active agent in the composition is an antigen-binding molecule of the present disclosure that specifically binds to CD19, CD20, and CD3. The label or package insert indicates that the use of the composition is for the treatment of the selected condition. Furthermore, the article of manufacture may comprise: (a) a first container containing a composition comprising an antigen-binding molecule of the present disclosure that specifically binds to CD19, CD20, and CD3; and (b) a second container containing a composition comprising an additional therapeutic agent. The article of manufacture in the embodiments disclosed herein may further include a packaging insert indicating that the composition can be used to treat a specific condition. Alternatively, or additionally, the article of manufacture may further include a second (or third) container containing a pharmaceutically acceptable buffer solution. From a commercial and user perspective, it may further include other materials as desired, including additional buffers, diluents, filters, needles, and syringes.

[0452] Examples and Test Cases

[0453] The present disclosure is further described below with reference to examples and test cases, but these examples and test cases are not intended to limit the scope of the disclosure. Experimental methods in the examples and test cases of this disclosure that do not specify specific conditions are generally performed under conventional conditions, such as those described in Cold Spring Harbor's Antibody Technology Manual or Molecular Cloning Manual; or under conditions recommended by the raw material or commercial manufacturer. Reagents whose specific source is not specified are commercially available, conventional reagents.

[0454] Example 1: Preparation of anti-CD19xCD20xCD3 trispecific antibody

[0455] The molecular cloning of the trispecific antibodies disclosed herein was constructed using PCR or gene synthesis methods known in the art. The antibody sequences were constructed into the pHt plasmid vector using Polyethylenimine Max (Mw 40,000*)-High Potency Linear PEI (Polysciences, 24765-1) and... I Reduced Serum Medium, no Phenol Red (Gibco, 11058-021) plasmid was transfected into Expi-293F cells to obtain antibody protein. The plasmid transfection ratio was chain 1: chain 2: chain 3: chain 4 = 2:1:3:3.

[0456] The CD19, CD20, and CD3 arms of the anti-CD19XCD20xCD3 trispecific antibody disclosed herein can be derived from any suitable antibody. For example, the VH and VL sequences of the CD19 arm of the anti-CD19XCD20xCD3 trispecific antibody disclosed herein are SEQ ID NO: 19 and SEQ ID NO: 20 (same as tancituzumab, CAS: 1422527-84-1), the VH and VL sequences of the CD20 arm are SEQ ID NO: 23 and SEQ ID NO: 24 (same as Epcoritamab, CAS: 2134641-34-0), and the VH and VL sequences of the CD3 arm are SEQ ID NO: 25 and SEQ ID NO: 26.

[0457] Table 3. CDRs of CD19xCD20xCD3 Note: In the table above, the CDR sequence is numbered according to the Kabat number.

[0458] The CD19 variable region sequence is as follows:

[0459] CD19-VH

[0460] CD19-VL

[0461] CD19-VH (containing Q39D and G44C mutations)

[0462] CD19-VL (containing Q38K and A100C mutations)

[0463] The CD20 variable region sequence is as follows:

[0464] CD20-VH

[0465] CD20-VL

[0466] The CD3 variable region sequence is as follows:

[0467] CD3-VH

[0468] CD3-VL

[0469] The disclosed anti-CD19xCD20xCD3 trispecific antibody is an asymmetric molecule, with the complete molecule containing four distinct chains, as detailed below:

[0470] Format A:

[0471] Chain 1: [CD20-VL]-[linker 1a]-[IgG1(CH1)1];

[0472] Chain 2: [CD20-VH]-[CL1a]-[IgG1Fc(Knob)];

[0473] Chain 3: [CD19-VH]-[IgG1(CH1)2]-[linker 2a]-[CD3-VH]-[linker 3a]-[CD3-VL]-[linker 4a]-[IgG1Fc(Hole)];

[0474] Chain 4: [CD19-VL]-[CL2a]; Chains 1, 2, 3, and 4 are all arranged from the N end to the C end;

[0475] in:

[0476] When IgG1Fc(Knob) is IgG1Fc(Knob)-1, IgG1Fc(Hole) is IgG1Fc(Hole)-1;

[0477] When IgG1Fc(Knob) is IgG1Fc(Knob)-2, IgG1Fc(Hole) is IgG1Fc(Hole)-2;

[0478] Format B:

[0479] Chain 1: [CD20-VL]-[linker 1a]-[IgG1(CH1)1];

[0480] Chain 2: [CD20-VH]-[CL1a]-[IgG1Fc(Knob)];

[0481] Chain 3: [CD19-VH]-[IgG1(CH1)3]-[IgG1Fc(Hole)];

[0482] Chain 4: [CD19-VL]-[CL2a]-[Connector 5a]-[CD3-VH]-[Connector 3a]-[CD3-VL];

[0483] The structures of chain 1, chain 2, chain 3, and chain 4 are all arranged from the N end to the C end;

[0484] in:

[0485] When IgG1Fc(Knob) is IgG1Fc(Knob)-2, IgG1Fc(Hole) is IgG1Fc(Hole)-2;

[0486] Format C:

[0487] Chain 1: [CD19-VL]-[Connector 6a]-[CD3-VH];

[0488] Chain 2: [CD3-VL]-[linker 6a]-[CD19-VH]-[linker 7a]-[IgG1Fc(Knob)];

[0489] Chain 3: [CD20-VH]-[IgG1(CH1)1]-[IgG1Fc(Hole)];

[0490] Chain 4: [CD20-VL]-[CL3a];

[0491] The structures of chain 1, chain 2, chain 3, and chain 4 are all arranged from the N end to the C end;

[0492] in:

[0493] When IgG1Fc(Knob) is IgG1Fc(Knob)-3, IgG1Fc(Hole) is IgG1Fc(Hole)-3;

[0494] The CD19-VH and CD19-VL are VH and VL that specifically bind to the antigen-binding domain of CD19; the CD20-VH and CD20-VL are VH and VL that specifically bind to the antigen-binding domain of CD20; and the CD3-VH and CD3-VL are VH and VL that specifically bind to the antigen-binding domain of CD3.

[0495] Figure 1 shows a schematic diagram of the anti-CD19XCD20xCD3 trispecific antibody format.

[0496] The relevant sequences are as follows:

[0497] >IgG1Fc(Knob)-1 (containing point mutations L234A, L235A, D265S, S354C, and T366W)

[0498] >IgG1Fc(Knob)-2 (containing point mutations L234A, L235A, S354C, and T366W)

[0499] >IgG1Fc(Knob-3

[0500] >IgG1Fc(Hole)-1 (containing point mutations L234A, L235A, D265S, Y349C, T366S, L368A, Y407V, H435R, Y436F)

[0501] >IgG1Fc(Hole)-2 (containing point mutations L234A, L235A, Y349C, T366S, L368A, Y407V, H435R, Y436F)

[0502] >IgG1Fc(Hole-3

[0503] >IgG1(CH1)1

[0504] >IgG1(CH1)2 (containing the point mutation F126C)

[0505] >IgG1(CH1)3 (containing point mutations F126C and C220S)

[0506] >CL1a (including point mutations R108A and T109S)

[0507] >CL2a (including point mutations S121C and C214S)

[0508] >CL3a

[0509] >Connector 1a

[0510] >Connector 2a

[0511] >Connector 3a

[0512] >Connector 4a

[0513] >Connector 5a

[0514] >Connector 6a

[0515] >Connector 7a

[0516] Construct anti-CD19xCD20xCD3 trispecific antibodies according to Table 4:

[0517] Table 4. Anti-CD19xCD20xCD3 Trispecific Antibodies Disclosed in This Study

[0518] For example, the specific amino acid sequence of the anti-CD19xCD20xCD3 trispecific antibody having the structure shown in Format A in this disclosure is as follows:

[0519] The sequence of TsAb-1 is shown below:

[0520] Chain 1

[0521] Chain 2

[0522] Chain 3

[0523] Chain 4

[0524] The sequence of TsAb-2 is shown below:

[0525] Chain 1

[0526] Chain 2

[0527] Chain 3

[0528] Chain 4

[0529] For example, the specific amino acid sequence of the anti-CD19xCD20xCD3 trispecific antibody having the structure shown in Format B in this disclosure is as follows:

[0530] The sequence of TsAb-3 is shown below:

[0531] Chain 1

[0532] Chain 2

[0533] Chain 3

[0534] Chain 4

[0535] For example, the specific amino acid sequence of the anti-CD19xCD20xCD3 trispecific antibody having the structure shown in Format C in this disclosure is as follows:

[0536] The sequence of TsAb-4 is shown below:

[0537] Chain 1

[0538] Chain 2

[0539] Chain 3

[0540] Chain 4

[0541] Note: In the above sequences, the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; the underlined parts are CDR sequences determined according to the Kabat numbering system; the non-underlined parts are FR sequences; the double-underlined and bolded parts are amino acid point mutations; the italicized parts are Cκ or constant regions, and the italicized and underlined parts are linker regions. CDR and variable region mutation sites are uniformly numbered using Kabat, and Fc region mutation sites are numbered using Eu.

[0542] The positive antibody used in this disclosure is a trispecific antibody B-Ab (targeting CD19 antigen, CD20 antigen, and CD3 receptor expressed on the surface of T cells), and the antibody sequence is derived from WO2020253393A1. The negative control antibody used in this disclosure is C25-hIgG1 (where the VH / VL sequence is derived from the C25 antibody of patent US6114143A).

[0543] The light and heavy chain amino acid sequences of B-Ab are as follows:

[0544] Chain 1

[0545] Chain 2

[0546] Chain 3

[0547] Chain 4

[0548] Negative control antibody C25-hIgG1:

[0549] C25-hIgG1 heavy chain

[0550] >C25-hIgG1 light chain

[0551] Note: Single underscore: variable area.

[0552] Test case

[0553] Test Example 1: Binding activity of anti-CD19xCD20xCD3 trispecific antibody with human CD19 antigen and human CD3 antigen.

[0554] Antibody samples were prepared into a 4 μg / mL solution using 1×HBS-EP buffer (pH 7.4) (Cytiva, BR-1006-69). Antibodies were affinity-captured using a Protein G biosensor chip (Cytiva, 29179315). Then, a certain concentration of human CD19 antigen (catalog number CD1-HM119, Kacrusbio) and human CD3D&E antigen (catalog number CDD-H52W1, Acrobiosystem) were passed through the chip surface. The reaction signals were monitored in real-time using a Biacore (Cytiva, Biacore T200) instrument to obtain binding and dissociation curves. After dissociation in each experimental cycle, the biosensor chip was washed and regenerated with 10 mM glycine-HCl (pH 1.5) (Cytiva, BR-1003-54). A 1:1 binding model was used for data fitting. The detection results are shown in Table 5.

[0555] Table 5. Affinity of CD19xCD20xCD3 antibodies to antigens as tested by Biacore

[0556] The results showed that the disclosed anti-CD19xCD20xCD3 trispecific antibody can bind well to human CD19 antigen and human CD3 antigen.

[0557] Test Example 2: Binding activity of anti-CD19xCD20xCD3 trispecific antibody with human CD20-K562 cell line

[0558] The binding activity of the anti-CD19xCD20xCD3 trispecific antibody to human CD20-K562 cells was tested using FACS. 1×10 6 / mL human CD20-K562 cells were blocked with 10% FBS, and then different concentrations of anti-CD19xCD20xCD3 trispecific antibody (C25-hIgG1 as a negative control) were added and incubated at room temperature for 60 min. The cells were then washed four times with 2% FBS, and then incubated with Protein A-Flu647 secondary antibody (Changzhou Tiandi Renhe Biotechnology, SLP03302) at room temperature for 15 min. After washing twice with 2% FBS, the fluorescence signal values ​​were read using flow cytometry.

[0559] Table 6. Binding activity of CD19xCD20xCD3 trispecific antibody to human CD20-K562 cells as determined by FACS

[0560] The results showed that the disclosed anti-CD19xCD20xCD3 antibody (TsAb-1) had good in vitro binding activity against human CD20 protein expressed in K562 cells.

[0561] Test Example 3: Detection of the killing effect of anti-CD19xCD20xCD3 trispecific antibodies of different formats on Raji tumor cells

[0562] Antibodies were diluted with complete medium (RPMI 1640 + 10% FBS) at an initial concentration of 40 nM, then diluted 20-fold for a total of 9 concentration points, and 100 μL / well was added to each well of a 96-well plate. Raji tumor cells were counted and resuspended in complete medium to a concentration of 2E5 / mL, then added to each well of a 96-well plate at 50 μL / well. Frozen PBMCs (Shanghai Xuanfeng Biotechnology Co., Ltd., XW0119014) were rapidly thawed at 37°C, resuspended in complete medium, centrifuged at 1500 rpm for 15 minutes, the supernatant was discarded, and the cells were resuspended in complete medium to a concentration of 1E6 / mL, then added to each well of a 96-well plate at 50 μL / well. Control wells (wells containing only Raji and PBMCs, and wells containing only Raji cells) were also prepared. The 96-well plates were incubated in medium for 48 hours. After incubation, the LDH detection kit (Promega, G1780) was used to perform the test according to the instructions, and the results were analyzed using Graphpad Prism 8.0 software to obtain Emax and EC. 50 .

[0563] Table 7. Killing effect of anti-CD19xCD20xCD3 trispecific antibodies of different formats on tumor cells

[0564] The results showed that TsAb-1, among the anti-CD19xCD20xCD3 trispecific antibodies disclosed herein, had better in vitro killing activity on Raji tumor cells compared to antibodies with other structures (TsAb-3, TsAb-4).

[0565] Test Example 4: In vitro killing effect of anti-CD19xCD20xCD3 trispecific antibody on tumor cells in combination with rituximab under occult conditions.

[0566] The killing ability of the disclosed anti-CD19xCD20xCD3 trispecific antibody against the JeKo-1 tumor cell line was tested by in vitro killing function experiments of total T cells; and the killing ability of the anti-CD19xCD20xCD3 trispecific antibody under CD20 target competition was characterized in the case of rituximab combined with occupancy.

[0567] Currently, the first-line treatment for DLBCL is rituximab + CHOP or rituximab + Pola-CHP, with rituximab being an indispensable component of the first-line therapy. In addition, there are two major categories of second-line therapies (rituximab + GemOx, rituximab + PolaBenda) that also include rituximab. When using anti-CD19xCD20xCD3 trispecific antibodies to simulate later-line treatment, this experimental model is needed to characterize the in vitro cytotoxic activity of the trispecific antibody in the presence of rituximab because rituximab already occupies space in the patient's body.

[0568] Collect the expanded JeKo-1 / LucG tumor cell line and adjust the target cell concentration to 1×10⁻⁶ cells using complete culture medium (RPMI 1640 + 10% FBS). 5 Human PBMCs were resuspended in complete culture medium after resuscitation, centrifuged at 400g for 5 min, and the supernatant was removed. They were then resuspended in complete culture medium, counted, and sorted using the EasySep STEM CELL sorting kit. TM The Human T Cell Isolation Kit sorts T cells, and the resulting T cell count is adjusted to 5 × 10⁻⁶ cells. 5The cell plating process for the rituximab competition assay is as follows: The T cell suspension and target cell suspension were mixed in equal volumes. 100 μL of the cell suspension was added to each well of a 96-well plate containing 1600 nM rituximab (50 μL / well). The plate was mixed thoroughly and incubated for 30 min. For the rituximab-free group, an equal volume of complete culture medium was added. The anti-CD19xCD20xCD3 trispecific antibody was diluted with complete culture medium at an initial concentration of 40 nM (4X final concentration), with 10-fold dilutions, 9 dose points, and a final dose point with an antibody concentration of 0. 50 μL was added to each well. The treated cells were incubated at 37°C in a 5% CO2 incubator for 72 hours. The viability of tumor cells was then detected using the following chemiluminescence method. First, the culture plate was removed, and 50 μL of ONE-Glo was added to the plate. TM The Luciferase Assay System was used. After incubation at room temperature for 5 minutes, luminescence was detected using Envision, and the cytotoxic effect of the antibody at different concentrations was calculated. Data processing was as follows: Graphpad Prism 8.0 software was used to plot dose-response curves based on the logarithmic concentration of the antibody and the luminescence signal value, yielding the cytotoxic EC50 mediated by the anti-CD19xCD20xCD3 trispecific antibody. 50 Wells containing only target cells and T cells without the addition of trispecific antibodies were set to 0% kill rate, and the kill rate of the trispecific antibodies against tumor cells was calculated. The results are shown in Table 8.

[0569] Table 8. In vitro killing activity of T cells mediated by anti-CD19xCD20xCD3 trispecific antibodies against JeKo-1 / LucG tumor cell lines.

[0570] Note: ECG with Shift = 400 nM rituximab present 50 EC without rituximab 50 This ratio indicates the degree of reduction in the cytotoxic activity of the trispecific antibody in the presence of rituximab: the larger the ratio, the greater the reduction in the cytotoxic activity of the trispecific antibody in the presence of rituximab; and vice versa.

[0571] The results showed that the disclosed anti-CD19xCD20xCD3 trispecific antibodies (TsAb-1, TsAb-2) exhibited superior in vitro tumor cell killing activity compared to the positive antibody B-Ab in competition with 400 nM rituximab.

[0572] Test Example 5: In vitro killing effect of anti-CD19xCD20xCD3 trispecific antibody on tumor cells in combination with tancituzumab under occlusive tumor conditions.

[0573] The killing ability of the disclosed anti-CD19xCD20xCD3 trispecific antibody against the JeKo-1 tumor cell line was tested by in vitro killing function experiments of T cells in human PBMCs; and the killing ability of the anti-CD19xCD20xCD3 trispecific antibody was characterized when the CD19 target was competed for by tancituzumab in combination with occupancy.

[0574] Currently, the anti-CD19 monoclonal antibody tancituzumab is undergoing phase III clinical trials in combination with lenalidomide and rituximab + CHOP, potentially becoming a new first-line treatment for DLBCL. Furthermore, the combination of tancituzumab and lenalidomide is currently the only non-chemotherapy second-line combination therapy for DLBCL. When using anti-CD19xCD20xCD3 trispecific antibodies to simulate later-line treatment, this experimental model is needed to characterize the in vitro killing activity of the trispecific antibodies in the presence of tancituzumab, since tancituzumab already occupies spaces in the patient's body.

[0575] Collect the expanded and cultured JeKo-1 / LucG tumor cell line, and adjust the target cell concentration to 1*10⁻⁶ cells using complete culture medium (RPMI 1640 + 10% FBS). 5 cells / mL. Human PBMCs were resuspended in complete culture medium after resuscitation, centrifuged at 400g for 5 min, and the supernatant was removed. They were then resuspended in complete culture medium, counted, and B cells were removed using a STEM CELL sorting kit. The cell count of the PBMCs after B cell removal was adjusted to 5*102. 5 The cell plating process for the tancituzumab competition assay is as follows: The PBMC suspension (with B cells removed) and target cell suspension were mixed in equal volumes. 100 μL of the cell suspension was added to each well of a 96-well plate containing 2750 nM tancituzumab (50 μL / well). The plate was mixed thoroughly and incubated for 30 min. For the tancituzumab-free group, an equal volume of complete culture medium was added. The anti-CD19xCD20xCD3 trispecific antibody was diluted with complete culture medium at an initial concentration of 40 nM (4X final concentration), with 10-fold dilutions, 9 dose points, and a final dose point with an antibody concentration of 0. 50 μL was added to each well. The treated cells were incubated at 37°C in a 5% CO2 incubator for 72 hours. The viability of tumor cells was then detected using the following chemiluminescence method. First, the culture plate was removed, and 50 μL of ONE-Glo was added to the plate. TMThe Luciferase Assay System was used. After incubation at room temperature for 5 minutes, luminescence was detected using Envision, and the cytotoxic effect of the antibody at different concentrations was calculated. Data processing was as follows: Graphpad Prism 8.0 software was used to plot dose-response curves based on the logarithmic concentration of the antibody and the luminescence signal value, yielding the cytotoxic EC50 mediated by the anti-CD19xCD20xCD3 trispecific antibody. 50 Wells containing only target cells and PBMCs without the trispecific antibody were set as 0% kill, and the kill rate of the trispecific antibody against tumor cells was calculated. The results are shown in Table 9.

[0576] Table 9. In vitro killing ability of T cells in PBMCs against JeKo-1 / LucG tumor cell line mediated by anti-CD19xCD20xCD3 trispecific antibodies.

[0577] Note: Shift = EC when tanxitosumab is present 50 EC without tancituzumab 50 This ratio indicates the degree of reduction in the cytotoxic activity of the trispecific antibody in the presence of tancituzumab: the larger the ratio, the greater the reduction in the cytotoxic activity of the trispecific antibody in the presence of tancituzumab; and vice versa.

[0578] The results showed that the disclosed anti-CD19xCD20xCD3 antibody (TsAb-1) exhibited superior in vitro tumor cell killing activity compared to the positive antibody B-Ab in competition with 687.5 nM tancituzumab.

[0579] Test Example 6: Inhibitory effect of anti-CD19xCD20xCD3 trispecific antibody on subcutaneous xenografts of human mantle cell lymphoma JeKo-1 tumor-bearing mice

[0580] JeKo-1 cells (ATCC) in logarithmic growth phase were subcutaneously injected into the right rib area of ​​NOG mice (Vitallix), with a cell count of 2.5 × 10⁻⁶ cells. 6 Each mouse was given 100 μL of PBMC (containing 50 μL of matrix gel). Five days later, the PBMCs were injected at 5 × 10⁻⁶ ppm. 6 Cells were injected intraperitoneally at a dose of 200 μL per mouse into tumor-bearing mice. One week later, the mice were divided into groups of seven. The day of grouping was defined as Day 0 of the experiment, and the mice began receiving intraperitoneal injections of high and medium doses of anti-CD19xCD20xCD3 trispecific antibody once a week. Tumor volume and animal weight were monitored and recorded twice a week.

[0581] Tumor volumes in each group of animals were expressed as mean ± standard deviation (Mean ± SEM) and plotted using Graphpad Prism 10 software. Two-way ANOVA was used for statistical analysis.

[0582] The formula for calculating tumor volume (V) is: V = 1 / 2 × a × b 2 Where a and b represent length and width, respectively.

[0583] Relative tumor proliferation rate: TGR (%) = [(T-T0) / (C-C0)] × 100%

[0584] Tumor inhibition rate: TGI (%) = 100% - TGR (%).

[0585] When the average tumor volume in the treatment group is smaller at the end of the experiment than it was at the beginning of the experiment, then:

[0586] Tumor inhibition rate: TGI (%) = 100% + (T0 - T) / T0 × 100%

[0587] Where T0 and T are the tumor volumes in the treatment group at the beginning and end of the experiment, respectively. C0 and C are the tumor volumes in the PBS solvent control group at the beginning and end of the experiment, respectively. The results are shown in Table 10.

[0588] Table 10. Efficacy of anti-CD19xCD20xCD3 trispecific antibodies in the JeKo-1 model

[0589] Note: qw means once a week, 2w means every two weeks, and ip means intraperitoneal injection.

[0590] The results showed that, compared with the PBS solvent control group (Vehicle group), the anti-CD19xCD20xCD3 trispecific antibody TsAb-1 and the positive antibody B-Ab significantly inhibited the growth of JeKo-1 subcutaneous tumors at medium and high doses, exhibiting a clear dose-dependent effect. On day 14, in the high-dose TsAb-1 and B-Ab groups, 5 out of 7 animals showed complete regression of subcutaneous tumors, with tumor inhibition rates reaching 189.3% and 115.5%, respectively. TsAb-1 demonstrated superior efficacy compared to the positive antibody B-Ab in this model.

[0591] Test Example 7: Inhibitory effect of anti-CD19xCD20xCD3 trispecific antibody on subcutaneous xenografts of human Burkitt's lymphoma Raji tumor-bearing mice.

[0592] Raji cells in logarithmic growth phase (ATCC): PBMC = (2 × 10⁻⁶) 6 ): (0.5×10 6The anti-CD19xCD20xCD3 trispecific antibody was mixed at a ratio of 4:1 and subcutaneously injected into the right rib area of ​​NOG mice (Vitallix), 200 μL per mouse (containing 100 μL of matrix gel), with 7 mice per group. The day of grouping was defined as Day 0 of the experiment, and intraperitoneal injections of high, medium, and low doses of the anti-CD19xCD20xCD3 trispecific antibody were started once a week. Tumor volume and animal weight were monitored and recorded twice a week.

[0593] Tumor volumes in each group of animals were expressed as mean ± standard deviation (Mean ± SEM) and plotted using Graphpad Prism 10 software. Two-way ANOVA was used for statistical analysis.

[0594] The formula for calculating tumor volume (V) is: V = 1 / 2 × a × b 2 Where a and b represent length and width, respectively.

[0595] Relative tumor proliferation rate: TGR (%) = [(T-T0) / (C-C0)] × 100%

[0596] Tumor inhibition rate: TGI (%) = 100% - TGR (%).

[0597] When the average tumor volume in the treatment group is smaller at the end of the experiment than it was at the beginning of the experiment, then:

[0598] Tumor inhibition rate: TGI (%) = 100% + (T0 - T) / T0 × 100%

[0599] Where T0 and T are the tumor volumes in the treatment group at the beginning and end of the experiment, respectively. C0 and C are the tumor volumes in the PBS solvent control group at the beginning and end of the experiment, respectively. The results are shown in Table 11.

[0600] Table 11. Efficacy of anti-CD19xCD20xCD3 trispecific antibodies in the Raji model Note: qw means once a week, 2w means every two weeks, and ip means intraperitoneal injection.

[0601] The results showed that, compared with the PBS solvent control group (Vehicle group), both the anti-CD19xCD20xCD3 trispecific antibody TsAb-1 and the positive antibody B-Ab significantly inhibited the growth of Raji subcutaneous tumors at medium and high doses. TsAb-1 showed a dose-dependent and extremely strong tumor-suppressing effect in this model, and was superior to the positive antibody B-Ab.

[0602] Test Example 8: Efficacy evaluation of anti-CD19xCD20xCD3 trispecific antibody in a BRGSF mouse model of SLE induced by PBMCs in SLE patients

[0603] Using BRGSF mice from Shanghai Kinuowei Biotechnology Co., Ltd. as test animals, a lupus phenotype model was induced in vivo using PBMCs from SLE patients (purchased from Miaoshun (Shanghai) Biotechnology Co., Ltd.) to evaluate the efficacy of the anti-CD19xCD20xCD3 trispecific antibody. Female BRGSF mice were intraperitoneally injected with SLE PBMCs for 14 days and then randomly divided into three groups: a control group receiving 0.005 mpk TsAb-1, a control group receiving 0.1 mpk TsAb-1, and a PBS solvent, and a blank control group not injected with PBMCs. Intraperitoneal injections were administered once weekly for a total of 5 weeks, starting the day after grouping.

[0604] Animal body weight was measured weekly. Blood samples were collected from mice anesthetized with isoflurane on days 35 and 49 to detect human IgG and anti-dsDNA IgG levels. At the experimental endpoint, left and right kidneys were collected and flash-frozen in liquid nitrogen at -80°C and fixed in neutral formalin, respectively, for further analysis of hIgG deposition in kidney tissue and renal pathology.

[0605] Human IgG was detected using Neobioscience's Human IgG (Total) ELISA Kit.

[0606] The Anti-dsDNA IgG assay was performed using the Human Anti-dsDNA IgG High Sensitivity ELISA Kit from Alpha Diagnostic International.

[0607] Excel 2019 statistical software was used: the mean was calculated using AVERAGE; the SD value was calculated using STDEV; and the SEM value was calculated using STDEV / SQRT. GraphPad Prism 10 was used for chart processing and statistical analysis; p-values ​​for differences between groups were calculated using analysis of variance.

[0608] ELISA analysis of human antibodies in mouse plasma at weeks 3 and 5 post-administration revealed significantly elevated levels of human IgG and anti-dsDNA IgG in the peripheral blood of mice injected with PBMCs, which was significantly reversed by TsAb-1. Compared to the PBS-mediated control group, the efficacy of TsAb-1 exhibited both dose- and time-dependent effects. At the experimental endpoint, the results of human IgG deposition in kidney tissue were completely consistent with the ELISA results.

[0609] Approximately 5 weeks after PBMC injection, graft-versus-host disease (GVHD) caused a trend of weight loss in the animals, so the experimental observation was terminated at week 7. Kidney pathological analysis revealed kidney damage in the model animals, with a trend of reduced kidney damage observed in the 0.1 mpk TsAb-1 administration group.

[0610] Therefore, in a BRGSF mouse model reconstructed in the PBMCs of SLE patients, TsAb-1 was able to dose-dependently clear human antibodies from the peripheral blood of mice. Multiple administrations of 0.1 mpk TsAb-1 in the presence of GVHD interference showed a trend toward alleviating kidney injury.

[0611] Although the invention has been described in detail with the aid of accompanying drawings and examples for clarity of understanding, these descriptions and examples should not be construed as limiting the scope of this disclosure. All patent and scientific literature disclosures cited in this disclosure are clearly and fully incorporated by reference.

Claims

1. An antigen-binding molecule that specifically binds to CD19, CD20, and CD3, comprising: The first antigen-binding domain that specifically binds to CD19 is Fab or a substituted Fab. A second antigen-binding domain that specifically binds to CD20, wherein the second antigen-binding domain that specifically binds to CD20 is Fab or a substituted Fab; and The third antigen-binding domain that specifically binds to CD3 is scFv.

2. The antigen-binding molecule that specifically binds to CD19, CD20, and CD3 according to claim 1, wherein: The first antigen-binding domain that specifically binds to CD19 comprises a heavy chain variable region (VH) and a light chain variable region (VL). The VH comprises HCDR1, HCDR2, and HCDR3. HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, and HCDR3 comprises the amino acid sequence of SEQ ID NO:

3. The VL comprises LCDR1, LCDR2, and LCDR3. LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO:

6. Preferably, The first antigen-binding domain that specifically binds to CD19 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises SEQ ID NO: 19, 21, or an amino acid sequence having at least 80% sequence identity with it, and the VL comprises SEQ ID NO: 20, 22, or an amino acid sequence having at least 80% sequence identity with it. More preferably, The first antigen-binding domain that specifically binds to CD19 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 19 and the VL comprises the amino acid sequence of SEQ ID NO:

20.

3. The antigen-binding molecule that specifically binds to CD19, CD20, and CD3 according to claim 1 or 2, wherein: The second antigen-binding domain that specifically binds to CD20 comprises a heavy chain variable region (VH) and a light chain variable region (VL). The VH comprises HCDR1, HCDR2, and HCDR3. HCDR1 comprises the amino acid sequence of SEQ ID NO: 7, HCDR2 comprises the amino acid sequence of SEQ ID NO: 8, and HCDR3 comprises the amino acid sequence of SEQ ID NO:

9. The VL comprises LCDR1, LCDR2, and LCDR3. LCDR1 comprises the amino acid sequence of SEQ ID NO: 10, LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence of SEQ ID NO:

12. Preferably, The second antigen-binding domain that specifically binds to CD20 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises SEQ ID NO: 23, or an amino acid sequence having at least 80% sequence identity with it, and the VL comprises SEQ ID NO: 24, or an amino acid sequence having at least 80% sequence identity with it. More preferably, The second antigen-binding domain that specifically binds to CD20 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 23 and the VL comprises the amino acid sequence of SEQ ID NO:

24.

4. The antigen-binding molecule that specifically binds to CD19, CD20, and CD3 according to any one of claims 1 to 3, wherein: The third antigen-binding domain that specifically binds to CD3 comprises a heavy chain variable region (VH) and a light chain variable region (VL). The VH comprises HCDR1, HCDR2, and HCDR3. HCDR1 comprises the amino acid sequence of SEQ ID NO: 13, HCDR2 comprises the amino acid sequence of SEQ ID NO: 14, and HCDR3 comprises the amino acid sequence of SEQ ID NO:

15. The VL comprises LCDR1, LCDR2, and LCDR3. LCDR1 comprises the amino acid sequence of SEQ ID NO: 16, LCDR2 comprises the amino acid sequence of SEQ ID NO: 17, and LCDR3 comprises the amino acid sequence of SEQ ID NO:

18. Preferably, The third antigen-binding domain that specifically binds to CD3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises SEQ ID NO: 25, or an amino acid sequence having at least 80% sequence identity with it, and the VL comprises SEQ ID NO: 26, or an amino acid sequence having at least 80% sequence identity with it. More preferably, The third antigen-binding domain that specifically binds to CD3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 25 and the VL comprises the amino acid sequence of SEQ ID NO:

26.

5. The antigen-binding molecule that specifically binds to CD19, CD20, and CD3 according to any one of claims 1 to 4, wherein: The first antigen-binding domain that specifically binds to CD19 comprises a heavy chain variable region (VH) and a light chain variable region (VL). The VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; the VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6; and The second antigen-binding domain that specifically binds to CD20 comprises a heavy chain variable region (VH) and a light chain variable region (VL). The VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 7, HCDR2 comprises the amino acid sequence of SEQ ID NO: 8, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 9; the VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 10, LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 12; and The third antigen-binding domain that specifically binds to CD3 comprises a heavy chain variable region (VH) and a light chain variable region (VL). The VH comprises HCDR1, HCDR2, and HCDR3. HCDR1 comprises the amino acid sequence of SEQ ID NO: 13, HCDR2 comprises the amino acid sequence of SEQ ID NO: 14, and HCDR3 comprises the amino acid sequence of SEQ ID NO:

15. The VL comprises LCDR1, LCDR2, and LCDR3. LCDR1 comprises the amino acid sequence of SEQ ID NO: 16, LCDR2 comprises the amino acid sequence of SEQ ID NO: 17, and LCDR3 comprises the amino acid sequence of SEQ ID NO:

18. Preferably, The first antigen-binding domain that specifically binds to CD19 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises SEQ ID NO: 19, 21, or an amino acid sequence having at least 80% sequence identity with it, and the VL comprises SEQ ID NO: 20, 22, or an amino acid sequence having at least 80% sequence identity with it; and The second antigen-binding domain that specifically binds to CD20 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises SEQ ID NO: 23, or an amino acid sequence having at least 80% sequence identity therewith, and the VL comprises SEQ ID NO: 24, or an amino acid sequence having at least 80% sequence identity therewith; and The third antigen-binding domain that specifically binds to CD3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises SEQ ID NO: 25, or an amino acid sequence having at least 80% sequence identity with it, and the VL comprises SEQ ID NO: 26, or an amino acid sequence having at least 80% sequence identity with it. More preferably, The first antigen-binding domain that specifically binds to CD19 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 19, and the VL comprises the amino acid sequence of SEQ ID NO: 20; and The second antigen-binding domain that specifically binds to CD20 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 23, and the VL comprises the amino acid sequence of SEQ ID NO: 24; and The third antigen-binding domain that specifically binds to CD3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 25 and the VL comprises the amino acid sequence of SEQ ID NO:

26.

6. The antigen-binding molecule that specifically binds to CD19, CD20, and CD3 according to any one of claims 1 to 5, wherein the replaced Fab comprises a light chain variable region (VL), a light chain constant region (CL), a heavy chain variable region (VH), and a heavy chain constant region 1 (CH1), and comprises any one of the following substitutions: (i) VL and VH are interchangeable; (ii) CL and CH1 are interchangeable; or (iii) VL and CL, VH and CH1 are interchangeable; Preferably, The replaced Fab comprises CL and CH1 interchangeably.

7. The antigen-binding molecule that specifically binds to CD19, CD20 and CD3 according to any one of claims 1 to 6, wherein the Fab comprises a light chain variable region (VL), a light chain constant region (CL), a heavy chain variable region (VH) and a heavy chain constant region 1 (CH1), and the amino acid at position 126 of CH1 is cysteine, and the amino acid at position 121 of CL is cysteine, numbered according to the EU index; Preferably, the Fab of the first antigen-binding domain that specifically binds to CD19 comprises a light chain variable region (VL), a light chain constant region (CL), a heavy chain variable region (VH), and a heavy chain constant region 1 (CH1), wherein the amino acid at position 126 of CH1 is cysteine, and the amino acid at position 121 of CL is cysteine, and the numbering is based on the EU index.

8. The antigen-binding molecule that specifically binds to CD19, CD20 and CD3 according to claim 6, wherein CH1 is CH1 derived from human IgG1, IgG2, IgG3 or IgG4, and CL is CL derived from human kappa or lambda; Preferably, CH1 is CH1 derived from human IgG1, and CL is CL derived from human kappa; More preferably, CH1 contains the amino acid sequence of SEQ ID NO: 33, 34 or 35, and CL contains the amino acid sequence of SEQ ID NO: 36, 37 or 38; Most preferably, CH1 and CL are selected from one or two of the following groups: CH1 contains the amino acid sequence of SEQ ID NO: 33, and CL contains the amino acid sequence of SEQ ID NO: 36; or CH1 contains the amino acid sequence of SEQ ID NO: 34, and CL contains the amino acid sequence of SEQ ID NO: 37; or CH1 contains the amino acid sequence of SEQ ID NO: 35, and CL contains the amino acid sequence of SEQ ID NO: 37; or CH1 contains the amino acid sequence of SEQ ID NO: 33, and CL contains the amino acid sequence of SEQ ID NO:

38.

9. The antigen-binding molecule that specifically binds to CD19, CD20 and CD3 according to any one of claims 1 to 8, further comprising an Fc region, wherein the Fc region is preferably an Fc region derived from IgG, and more preferably an Fc region derived from IgG1; More preferably, the Fc region contains one or more amino acid substitutions that can reduce the binding of the Fc region to the Fcγ receptor; Most preferably, the Fc region is the human IgG1 Fc region, and the amino acids at positions 234 and 235 are A, and the amino acid at position 265 is S; or The Fc region is the human IgG1 Fc region, and the amino acids at positions 234 and 235 are A, numbered according to the EU index.

10. The antigen-binding molecule that specifically binds to CD19, CD20 and CD3 according to any one of claims 1 to 9, comprising an Fc region, wherein the Fc region comprises a first subunit Fc1 and a second subunit Fc2 capable of associating with each other; Preferably, Fc1 includes a protruding structure according to the pestle and mortar technique and Fc2 includes a hole structure according to the pestle and mortar technique; More preferably, Fc1 has C at position 354 and W at position 366; and Fc2 has C at position 349, S at position 366, A at position 368, and V at position 407, with the numbering based on the EU index. More preferably, Fc1 contains the amino acid sequence of SEQ ID NO: 27, 28 or 29; and Fc2 contains the amino acid sequence of SEQ ID NO: 30, 31 or 32. More preferably, Fc1 and Fc2 are selected from any of the following: Fc1 contains the amino acid sequence of SEQ ID NO: 27 and Fc2 contains the amino acid sequence of SEQ ID NO: 30; or Fc1 contains the amino acid sequence of SEQ ID NO: 28 and Fc2 contains the amino acid sequence of SEQ ID NO: 31; or Fc1 contains the amino acid sequence SEQ ID NO: 29 and Fc2 contains the amino acid sequence SEQ ID NO: 32; or Fc1 contains the amino acid sequence of SEQ ID NO: 27 and Fc2 contains the amino acid sequence of SEQ ID NO: 31; or Fc1 contains the amino acid sequence of SEQ ID NO: 28 and Fc2 contains the amino acid sequence of SEQ ID NO:

30.

11. The antigen-binding molecule that specifically binds to CD19, CD20 and CD3 according to any one of claims 1 to 10, wherein the antigen-binding molecule that specifically binds to CD19, CD20 and CD3 is an antibody, preferably a multispecific antibody, and more preferably a trispecific antibody.

12. The antigen-binding molecule that specifically binds to CD19, CD20, and CD3 according to claim 11, wherein: The antigen-binding molecule that specifically binds to CD19, CD20, and CD3 is a trispecific antibody and comprises: The first hapten, comprising substituted Fab and Fc1 binding to CD20; and The second half-antibody comprises Fab that binds to CD19, scFv that binds to CD3, and Fc2. And wherein the first and second halves of the antibody are associated with each other via Fc1 and Fc2; or The antigen-binding molecule that specifically binds to CD19, CD20, and CD3 is a trispecific antibody and comprises: The first hapten, comprising Fab and Fc1 binding to CD20; and The second half antibody comprises a Fab that binds to CD19, an scFv that binds to CD3, and an Fc2, wherein the first half antibody and the second half antibody are associated with each other via Fc1 and Fc2. Preferably, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 is a trispecific antibody and comprises: The first hapten, comprising, from the N-terminus to the C-terminus, substituted Fab and Fc1 that bind CD20; The second half-antibody, from the N-terminus to the C-terminus, includes a Fab that binds to CD19, an scFv that binds to CD3, and an Fc2. And wherein the first and second halves of the antibody are associated with each other via Fc1 and Fc2; or The antigen-binding molecule that specifically binds to CD19, CD20, and CD3 is a trispecific antibody and comprises: The first half antibody contains Fab and Fc1 binding to CD20 from the N-terminus to the C-terminus. The second half antibody includes, from the N-terminus to the C-terminus, a Fab that binds CD19, an scFv that binds CD3, and an Fc2, wherein the first half antibody and the second half antibody are associated with each other through Fc1 and Fc2. More preferably, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 is a trispecific antibody and comprises: The first hapten includes, from the N-terminus to the C-terminus, a CD20-binding replaced Fab and an Fc1, wherein the C-terminus of the CD20-binding replaced Fab is operatively connected to the N-terminus of the Fc1. The second half antibody comprises, from the N-terminus to the C-terminus, a Fab that binds CD19, a scFv that binds CD3, and an Fc2; wherein the N-terminus of the scFv that binds CD3 is operatively connected to the C-terminus of the Fab that binds CD19, and the C-terminus of the scFv that binds CD3 is operatively connected to the N-terminus of the Fc2. Furthermore, the first and second halves of the antibody are associated with each other via Fc1 and Fc2; Most preferably, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 comprises: A first chain having the structure shown in equation (a), a second chain having the structure shown in equation (b), a third chain having the structure shown in equation (c), and a fourth chain having the structure shown in equation (d), wherein the structures shown in equations (a), (b), (c), and (d) are arranged from the N end to the C end: (a) [CD20-VL]-[Connector 1]-[CH1-1]; (b)[CD20-VH]-[CL1]-[Fc1]; (c)[CD19-VH]-[CH1-2]-[Connector 2]-[CD3-VH]-[Connector 3]-[CD3-VL]-[Connector 4]-[Fc2]; (d)[CD19-VL]-[CL2]; The linker 1, linker 2, linker 3 and linker 4 may be the same or different peptide linkers, or the linker 1, linker 2, linker 3 and / or linker 4 may not exist; Fc1 and Fc2 are interchangeable; The first and second chains combine with each other, the third and fourth chains combine with each other, and the second and third chains combine with each other to form antigen-binding molecules that specifically bind to CD19, CD20 and CD3.

13. The antigen-binding molecule that specifically binds to CD19, CD20, and CD3 according to any one of claims 1 to 12, comprising: i) a first chain containing an amino acid sequence of SEQ ID NO: 46, or having at least 80% sequence identity with it; a second chain containing an amino acid sequence of SEQ ID NO: 47, or having at least 80% sequence identity with it; a third chain containing an amino acid sequence of SEQ ID NO: 48, or having at least 80% sequence identity with it; and a fourth chain containing an amino acid sequence of SEQ ID NO: 49, or having at least 80% sequence identity with it; or ii) A first chain containing an amino acid sequence of SEQ ID NO: 50, or having at least 80% sequence identity with it; a second chain containing an amino acid sequence of SEQ ID NO: 51, or having at least 80% sequence identity with it; a third chain containing an amino acid sequence of SEQ ID NO: 52, or having at least 80% sequence identity with it; and a fourth chain containing an amino acid sequence of SEQ ID NO: 53, or having at least 80% sequence identity with it; or iii) A first chain containing SEQ ID NO: 54, or an amino acid sequence having at least 80% sequence identity with it; a second chain containing SEQ ID NO: 55, or an amino acid sequence having at least 80% sequence identity with it; a third chain containing SEQ ID NO: 56, or an amino acid sequence having at least 80% sequence identity with it; and a fourth chain containing SEQ ID NO: 57, or an amino acid sequence having at least 80% sequence identity with it. Preferably, the antigen-binding molecule that specifically binds to CD19, CD20, and CD3 comprises: i) A first strand containing the amino acid sequence of SEQ ID NO: 46, a second strand containing the amino acid sequence of SEQ ID NO: 47, a third strand containing the amino acid sequence of SEQ ID NO: 48, and a fourth strand containing the amino acid sequence of SEQ ID NO:

49. ii) A first strand containing the amino acid sequence of SEQ ID NO: 50, a second strand containing the amino acid sequence of SEQ ID NO: 51, a third strand containing the amino acid sequence of SEQ ID NO: 52, and a fourth strand containing the amino acid sequence of SEQ ID NO: 53; More preferably, the antigen-binding molecule that specifically binds to CD19, CD20 and CD3 comprises: a first chain containing the amino acid sequence of SEQ ID NO: 46, a second chain containing the amino acid sequence of SEQ ID NO: 47, a third chain containing the amino acid sequence of SEQ ID NO: 48 and a fourth chain containing the amino acid sequence of SEQ ID NO:

49.

14. A pharmaceutical composition comprising an antigen-binding molecule that specifically binds to CD19, CD20 and CD3 according to any one of claims 1 to 13, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients.

15. A nucleic acid encoding an antigen-binding molecule that specifically binds to CD19, CD20 and CD3 as described in any one of claims 1 to 13.

16. A host cell comprising the nucleic acid as described in claim 15.

17. Use of the antigen-binding molecule that specifically binds to CD19, CD20 and CD3 as described in any one of claims 1 to 13, or the pharmaceutical composition of claim 14, in the preparation of a medicament for treating a disease or condition; Preferably, the disease or symptom is a tumor or an autoimmune disease; More preferably, the tumor is a hematologic malignancy; the autoimmune disease is systemic lupus erythematosus or lupus nephritis; More preferably, the tumor is lymphoma or leukemia; More preferably, the tumor is non-Hodgkin's lymphoma or acute lymphoblastic leukemia; Most preferably, the tumor is a B-cell non-Hodgkin lymphoma.

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