Preparation and application of targeted BAFF-R and CD3 bispecific antibody

Abstract

The invention relates to the technical field of biology, in particular to an anti-BAFF-R and anti-CD3 bispecific antibody and application thereof. The bispecific antibody disclosed by the invention can be specifically combined with a cell surface B cell activating factor receptor (BAFF-R) and an immune cell surface antigen molecular cluster 3 (CD3); wherein the anti-BAFF-R antibody is a full human source single domain antibody. The invention also provides a preparation method of the bispecific antibody with different combination configurations of 1 + 1, 2 + 1 and 2 + 2 and an in-vitro and in-vivo biological activity evaluation method. The bispecific antibody disclosed by the invention has relatively high affinity and can be used for treating BAFFR expressed tumor diseases and autoimmune diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a construction and preparation method of a bispecific antibody targeting BAFF-R and CD3, and application of the antibody in diseases. Background Art

[0002] Bispecific antibodies are artificial antibodies composed of two different antibody fragments that can specifically recognize and bind to two different antigens or two different antigenic epitopes. As early as 1960, the concept of bispecific antibodies was proposed by Nisonoff of the Roswell Park Memorial Institute in New York. Due to the limitations of production conditions, the development of bispecific antibodies was extremely slow. In 1975, Kohler and Milstein proposed the hybridoma technology of myeloma cell and spleen cell fusion, and the subsequent development of antibody humanization technology, which accelerated the development of monoclonal antibodies; until the 1990s, the emergence of knobs-into-holes technology significantly reduced antibody mismatching, and large-scale production of bispecific antibodies became possible, and the development of bispecific antibodies gradually accelerated.

[0003] There are three main modes for bispecific antibodies to select targets: one is to bind to tumor cell antigens and CD3, CD28 or 4-1BB on T cells respectively, and use T cells to kill tumor cells; the second is to bind to two targets on cells, which has a double blocking effect or a bridging effect; the third is to bind to two domains of the same target, which has a more effective blocking effect. CD3 is an important component of the T cell receptor complex and plays an important role in T cell activation, proliferation, and immune response. Bispecific antibodies targeting tumor cells and T cells have always been a research hotspot for tumor treatment. In 2007, the first bispecific antibody, catumaxomab, was approved by the EMA for marketing. It targets EpCAM and CD3 and is used to treat malignant ascites. In 2014, Amgen's bispecific antibody blinatumomab targeting CD19 and CD3 was approved by the US FDA for marketing for the treatment of relapsed or refractory B-cell acute lymphoblastic leukemia. In November 2017, the US FDA approved Roche's emicizumab for the treatment of hemophilia A. The antibody binds to coagulation factors IXa and X to simulate the coagulation process. 2022 is the year of the explosion of bispecific antibodies. From 2022 to June 2023, a total of 6 bispecific antibodies were approved for marketing worldwide, of which 5 targeted CD3 and tumor targets. In January 2022, Immunocore's bispecific antibody Tebentafusp targeting gp100 / CD3 was approved by the U.S. FDA for the treatment of adult patients with HLA-A*02:01-positive unresectable or metastatic uveal melanoma; in June 2022, Roche's bispecific antibody Mosunetuzumab targeting CD20 / CD3 was conditionally approved by the EU for adult patients with relapsed or refractory (R / R) follicular lymphoma (FL) who had received at least two previous systemic therapies, and was approved for marketing by the U.S. FDA in December 2022; in August 2022, Johnson & Johnson's bispecific antibody Teclistamab targeting BCMA / CD3 was conditionally approved by the EU for the treatment of relapsed and refractory multiple myeloma (R / R In March 2023, Roche's CD20 / CD3-targeting bispecific antibody Glofitamab was approved by Canada for the treatment of relapsed or refractory (R / R) diffuse large B-cell lymphoma (DLBCL); in May 2023, AbbVie / Genmab's CD20 / CD3-targeting bispecific antibody Epcoritamab was approved by the US FDA for the treatment of relapsed or refractory (R / R) diffuse large B-cell lymphoma (DLBCL).These five bispecific antibodies are different from blinatumomab approved by the FDA in 2014. They are all IgG bispecific antibodies. The complete Fc end ensures the half-life of the antibody, solving the problem of short half-life of biTe bispecific antibodies and poor patient compliance caused by frequent administration. Currently, bispecific antibodies targeting CD3 and tumor cell surface antigens have been expanded to solid tumors in addition to the research on blood tumors, including CD19, CD20, BCMA, GPRC5D, etc., with targets including Her2, EGFR, EpCAM, claudin18.2, PSMA, etc.

[0004] BAFF-R is one of the three known receptors of BAFF (B cell activating factor). It is a type III transmembrane protein receptor expressed on excessive T2-type B lymphocytes and marginal zone and follicular mature B2 cell populations. It belongs to the TNF receptor superfamily. BAFF-R is composed of 184 amino acids, of which the extracellular region located at the N-terminus contains 78 amino acids. BAFF-R has high specificity and is mainly expressed in B cells. It only binds to BAFF and does not bind to other members of the TNF family. BAFF-R mRNA is highly expressed in secondary lymphoid organs (spleen and lymph nodes), lowly expressed in peripheral blood leukocytes and thymus, but not expressed in bone marrow and fetal liver. BAFF-R protein is only expressed in mature normal B lymphocytes and B lymphoma cells. In addition, it has been reported that about 5% of spleen CD4+T cells (80% of CD4 + CD25 + BAFF-R is also expressed on T-reg.

[0005] BAFF-R is the main mediating receptor for BAFF to regulate B cell differentiation. The BAFF / BAFF-R signaling pathway plays an important role in the proliferation of B lymphoma cells. Upregulated expression of BAFF-R is associated with the disease progression of B cell lymphoma and pre-B-ALL. Mice with BAFF-R mutations or knockouts show obvious symptoms of B cell reduction. In addition, the BAFF / BAFF-R signaling pathway also plays an important role in autoimmune diseases.

[0006] Monoclonal antibodies targeting BAFF-R have been used in multiple clinical tests for autoimmune diseases, such as Novartis's biologic Ianalumab (VAY736). A randomized, parallel, double-blind, placebo-controlled Phase 2b dose-exploratory study conducted in 56 centers in 19 countries showed that Ianalumab treatment for 24 weeks can significantly improve the ESSDAI score of patients with moderate to severe Sjögren's syndrome, and it is well tolerated and safe. In May 2022, Novartis has launched a Phase 3 clinical trial of ianalumab for the treatment of lupus nephritis in China; ianalumab has also launched a study for the treatment of relapsed or refractory chronic lymphocytic leukemia (R / RCLL) (NCT02137889). Genentech has also conducted research on anti-BR3 antibodies for the treatment of autoimmune diseases. There are currently two reports on CAR-T targeting BAFF-R. Turazzi et al. first tried BAFF-R CAR-T for the treatment of B-ALL and obtained good in vitro functional data. Hong Qin et al. from City of Hope also reported that BAFF-R CAR-T can effectively eliminate CD19 knockout tumor cells in animal models. Studies have reported that the BAFF-R signaling pathway is the main bypass signal for the inhibition of the B cell TCR signaling pathway. For patients who are resistant to TCR pathway inhibitors (including antibodies targeting CD19 and Car-T, TBK inhibitors), BAFF-R pathway inhibitors are also an option.

[0007] Therefore, BAFF-R is an effective therapeutic target for a variety of B lymphocyte tumors, autoimmune diseases, and other diseases characterized by BAFF-R expression. The development of immunotherapy products targeting BAFF-R is of great significance and application value. Summary of the invention

[0008] The object of the present invention is to provide a method for constructing and preparing a bispecific antibody targeting BAFF-R and CD3, a method for evaluating biological activity, and a method for evaluating safety.

[0009] In a first aspect of the present invention, a single domain antibody against B cell activating factor receptor (BAFF-R) is provided, wherein the VHH chain of the single domain antibody comprises the following complementary determining regions CDR: CDR1 shown in SEQ ID NO.3, CDR2 shown in SEQ ID NO.4, and CDR3 shown in SEQ ID NO.5;

[0010] Any of the above amino acid sequences also includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and is able to retain BAFF-R binding affinity.

[0011] In another preferred embodiment, the VHH chain of the single-domain antibody further includes a framework region (FR).

[0012] In another preferred embodiment, the framework region FR is of human, mouse, rabbit or camel origin.

[0013] In another preferred embodiment, the framework region FR includes a human FR region, a mouse FR region or a camel FR region.

[0014] In another preferred embodiment, the VHH chain of the single-domain antibody targeting BAFF-R has an amino acid sequence as shown in SEQ ID NO.: 1 or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the above sequence.

[0015] In another preferred embodiment, the single-domain antibody targeting BAFF-R is a fully human antibody.

[0016] The second aspect of the present invention provides an antibody targeting BAFF-R, wherein the antibody comprises one or more VHH chains of the single-domain antibody targeting BAFF-R as described in the first aspect of the present invention.

[0017] In another preferred embodiment, the antibody is a monomer, a bivalent antibody, and / or a multivalent antibody.

[0018] In another preferred embodiment, the antibody is a fully human antibody, an animal antibody, a humanized antibody, a chimeric antibody or a chimeric antigen receptor antibody (CAR).

[0019] In another preferred embodiment, the CDR region of the humanized antibody comprises 1, 2, or 3 amino acid changes.

[0020] In another preferred embodiment, the animal is a non-human mammal, preferably a mouse, sheep, rabbit or camel.

[0021] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0022] In another preferred embodiment, the antibody is a monoclonal antibody.

[0023] In another preferred embodiment, the antibody is a fully human monoclonal antibody.

[0024] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids does not exceed 40% of the total number of amino acids in the initial amino acid sequence, preferably 20%, more preferably 10%.

[0025] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids is 1-7, preferably 1-3, and more preferably 1.

[0026] In another preferred embodiment, the at least one amino acid sequence that has been added, deleted, modified and / or substituted has an amino acid sequence with a homology of at least 80%.

[0027] The third aspect of the present invention provides a multispecific antibody, wherein the multispecific antibody comprises: the single domain antibody targeting BAFF-R according to the first aspect of the present invention or the antibody targeting BAFF-R according to the second aspect of the present invention.

[0028] In another preferred embodiment, the multispecific antibody further comprises a second antigen binding region targeting a target selected from the group consisting of CD3, CD28, 4-1BB, BCMA, CD73, GPC3, HER2, PMSA, OX40, GLP-1, Trop2, FGL1, LFA-3, 2B4, 5T4, α-4 integrin, α-V integrin, α4β7 integrin, α4β7 integrin, α-SMA, AGR2, Apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9, carbonic anhydrase 9, CD2, CD6, CD9, CDlla, CD19, CD20, CD22, CD24, CD25, CD27, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD69, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD107a, CD117, CD123, CD125, C D132(IL-2Rg), CD133, CD137, CD138, CD160, CD166, CD172A, CD248, CEACAM5(CEA), CEACAM6(NCA- 90), CLAUDIN-3, CLAUDIN-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FRα), FSP-1, GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIlb / IIIa receptor, Gpl30, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HLA-DR, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL -12R, IL-12Rβl, IL13, IL13R, IL13Ra2, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R(wsxl), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, IL1 receptor accessory protein (IL1RAP), insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, KLRG1, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, Nicastrin, Notch receptor, Notch 1, Notch 2, Notch 3, Notch 4. NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PSCA, PSMA, PS GR, RAAG12, RAGE, SLC44A4, Siglecl5, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, TLR4, TL R6, TLR7, TLR8, TLR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, WISP-3, or a combination thereof.

[0029] In another preferred embodiment, the second antigen binding region is a fully human antibody, an animal antibody, a humanized antibody or a chimeric antibody.

[0030] In another preferred embodiment, the multispecific antibody comprises one or more second antigen binding regions.

[0031] In another preferred embodiment, the multispecific antibody contains or does not contain the Fc segment of the antibody.

[0032] In another preferred embodiment, the Fc segment of the antibody is of IgG1 subtype or IgG4 subtype.

[0033] In another preferred embodiment, the Fc segment of the antibody comprises L234A / L235A mutation or S228P / F234A / L235A mutation.

[0034] In another preferred embodiment, the Fc segment of the antibody comprises a knobs-into-holes structure; preferably, the knobs side is a S354C / T366W combined mutation, and the holes side is a Y349C / T366S / L368A / Y407V combined mutation.

[0035] In another preferred embodiment, the antigen binding region is an antibody or an antibody fragment, and the antibody fragment includes: (i) Fab fragment; (ii) F(ab')2 fragment; (iii) Fd fragment; (iv) Fv fragment; (v) single-chain Fv (scFv) molecule; (vi) dAb fragment.

[0036] In a fourth aspect of the present invention, a bispecific antibody is provided, wherein the bispecific antibody comprises:

[0037] Targeting the first antigen binding region of BAFF-R; and

[0038] Targeting the second antigen binding region of CD3; wherein,

[0039] The first antigen binding region targeting BAFF-R comprises the following complementary determining regions CDR: CDR1 shown in SEQ ID NO.3, CDR2 shown in SEQ ID NO.4 and CDR3 shown in SEQ ID NO.5.

[0040] In another preferred embodiment, the second antigen binding region targeting CD3 comprises a heavy chain variable region and a light chain variable region targeting CD3, wherein:

[0041] The CD3-targeting heavy chain variable region comprises the following complementary determining regions CDR: CDR1 shown in SEQ ID NO: 44, CDR2 shown in SEQ ID NO: 45, and CDR3 shown in SEQ ID NO: 46;

[0042] The CD3-targeting light chain variable region comprises the following complementary determining regions CDR: CDR1 shown in SEQ ID NO: 47, CDR2 with an amino acid sequence of GTN, and CDR3 shown in SEQ ID NO: 48;

[0043] Any of the above amino acid sequences also includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and is able to retain CD3 binding affinity.

[0044] In another preferred embodiment, the amino acid sequence of the heavy chain variable region targeting CD3 is shown as SEQ ID NO.10, and the amino acid sequence of the light chain variable region targeting CD3 is shown as SEQ ID NO.12; or

[0045] The amino acid sequence of the heavy chain variable region targeting CD3 is shown in SEQ ID NO.30, and the amino acid sequence of the light chain variable region targeting CD3 is shown in SEQ ID NO.32.

[0046] In another preferred embodiment, the nucleotide sequence encoding the heavy chain variable region targeting CD3 is shown as SEQ ID NO.11, and the nucleotide sequence encoding the light chain variable region targeting CD3 is shown as SEQ ID NO.13; or

[0047] The nucleotide sequence encoding the heavy chain variable region targeting CD3 is shown in SEQ ID NO.31, and the nucleotide sequence encoding the light chain variable region targeting CD3 is shown in SEQ ID NO.33.

[0048] In another preferred embodiment, the second antigen binding region targeting CD3 is a fully human antibody, an animal antibody, a humanized antibody or a chimeric antibody.

[0049] In another preferred embodiment, the second antigen binding region is an antibody or an antibody fragment, and the antibody fragment includes: (i) Fab fragment; (ii) F(ab')2 fragment; (iii) Fd fragment; (iv) Fv fragment; (v) single-chain Fv (scFv) molecule; (vi) dAb fragment.

[0050] In another preferred example, the amino acid sequence of the Fab fragment targeting CD3 is shown as SEQ ID NO.14; the nucleotide sequence encoding the Fab fragment targeting CD3 is shown as SEQ ID NO.15.

[0051] In another preferred embodiment, the bispecific antibody is a symmetric or asymmetric dimer.

[0052] In another preferred example, the first antigen binding region is connected to the N segment or C segment of the second antigen binding region.

[0053] In another preferred example, the first antigen binding region is connected to the N segment or C segment of the Fab fragment encoding targeting CD3.

[0054] In another preferred embodiment, the first antigen binding region is connected to the N segment or C segment of the heavy chain or light chain of the Fab fragment encoding targeting CD3, preferably, connected to the N segment of the heavy chain of the Fab fragment encoding targeting CD3.

[0055] In another preferred embodiment, the bispecific antibody further comprises an antibody Fc segment.

[0056] In another preferred embodiment, the first antigen binding region is connected to the N segment or C segment of the Fc segment, preferably, connected to the C segment of the Fc segment.

[0057] In another preferred embodiment, the bispecific antibody has the following structure:

[0058] (a) Two heavy chains, the first heavy chain comprises, from N-terminus to C-terminus, anti-BAFF-R VH, human IgG Fc region; the second heavy chain comprises, from N-terminus to C-terminus, anti-CD3 VH, antibody heavy chain constant region (CH1), human IgG Fc region;

[0059] (b) a light chain, comprising, from N-terminus to C-terminus, an anti-CD3 VL, an antibody light chain constant region (CL),

[0060] Wherein, a disulfide bond exists between the antibody heavy chain constant region (CH1) and the antibody light chain constant region (CL).

[0061] In another preferred embodiment, the anti-CD3 VL is paired with the anti-CD3 VH, and the antibody heavy chain constant region (CH1) is paired with the antibody light chain constant region (CL).

[0062] In another preferred embodiment, the anti-BAFF-R VH and the first antigen binding region targeting BAFF-R have the same meaning.

[0063] In another preferred embodiment, the antibody heavy chain constant region (CH1) is connected to the human IgG Fc region via a hinge region.

[0064] In another preferred example, there is a disulfide bond between the two human IgG Fc regions contained in the bispecific antibody.

[0065] In another preferred embodiment, the two human IgG Fc regions contained in the bispecific antibody together constitute the Fc segment of the antibody. Preferably, the Fc segment of the antibody is of IgG4 subtype, and more preferably, comprises S228P / F234A / L235A mutations.

[0066] In another preferred embodiment, the Fc segment of the antibody comprises a knobs-into-holes structure; preferably, the knobs side is a S354C / T366W combined mutation, and the holes side is a Y349C / T366S / L368A / Y407V combined mutation.

[0067] In another preferred embodiment, the bispecific antibody has the following structure:

[0068] (a) two heavy chains, wherein the first heavy chain comprises, from N-terminus to C-terminus, anti-BAFF-R VH, anti-CD3 VH, antibody heavy chain constant region (CH1), and human IgG Fc region; the second heavy chain comprises, from N-terminus to C-terminus, anti-BAFF-R VH and human IgG Fc region;

[0069] (b) a light chain, comprising, from N-terminus to C-terminus, an anti-CD3 VL, an antibody light chain constant region (CL),

[0070] Wherein, a disulfide bond exists between the antibody heavy chain constant region (CH1) and the antibody light chain constant region (CL).

[0071] In another preferred embodiment, the anti-CD3 VL is paired with the anti-CD3 VH, and the antibody heavy chain constant region (CH1) is paired with the antibody light chain constant region (CL).

[0072] In another preferred embodiment, the bispecific antibody has the following structure:

[0073] (a) two identical heavy chains, wherein the heavy chains comprise, from N-terminus to C-terminus, anti-BAFF-R VH, anti-CD3 VH, antibody heavy chain constant region (CH1), and human IgG Fc region;

[0074] (b) two identical light chains, wherein the light chains comprise, from N-terminus to C-terminus, anti-CD3 VL, antibody light chain constant region (CL),

[0075] Wherein, a disulfide bond exists between the antibody heavy chain constant region (CH1) and the antibody light chain constant region (CL).

[0076] In another preferred embodiment, the anti-CD3 VL is paired with the anti-CD3 VH, and the antibody heavy chain constant region (CH1) is paired with the antibody light chain constant region (CL).

[0077] In another preferred embodiment, the bispecific antibody, wherein:

[0078] The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 38 or 16, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the above sequence,

[0079] The light chain comprises an amino acid sequence as shown in SEQ ID NO: 36, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the above sequence.

[0080] In another preferred embodiment, the bispecific antibody, wherein:

[0081] The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 40 or 16, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the above sequence,

[0082] The light chain comprises an amino acid sequence as shown in SEQ ID NO: 36, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the above sequence.

[0083] In another preferred embodiment, the bispecific antibody, wherein:

[0084] The heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 42, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the above sequence,

[0085] The light chain comprises an amino acid sequence as shown in SEQ ID NO: 36, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the above sequence.

[0086] In a fifth aspect, the present invention provides a recombinant protein, wherein the recombinant protein has:

[0087] (i) the single domain antibody targeting BAFF-R as described in the first aspect of the present invention, the antibody targeting BAFF-R as described in the second aspect of the present invention, the multispecific antibody as described in the third aspect of the present invention, or the bispecific antibody as described in the fourth aspect of the present invention; and

[0088] (ii) optional polypeptide molecules or fragments having therapeutic functions; and / or

[0089] (iii) Optional functional domains that enhance the physicochemical properties or druggability of the protein.

[0090] The sixth aspect of the present invention provides an immunoconjugate, wherein the immunoconjugate comprises:

[0091] (a) an antibody portion, wherein the antibody portion is a single domain antibody targeting BAFF-R as described in the first aspect of the present invention, an antibody targeting BAFF-R as described in the second aspect of the present invention, a multispecific antibody as described in the third aspect of the present invention, or a bispecific antibody as described in the fourth aspect of the present invention; and

[0092] (b) a conjugated moiety conjugated to the antibody portion, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, an enzyme, or a combination thereof.

[0093] In another preferred embodiment, the immunoconjugate is a single domain antibody drug conjugate.

[0094] In another preferred embodiment, the antibody portion and the coupling portion are coupled via a chemical bond or a linker.

[0095] In another preferred embodiment, the coupling moiety is a chemical marker and a biological marker.

[0096] In another preferred embodiment, the chemical label is an isotope, an immunotoxin and / or a chemical drug.

[0097] In another preferred embodiment, the biological marker is biotin, avidin or an enzyme marker.

[0098] In another preferred embodiment, the coupling moiety is a drug or a toxin.

[0099] In another preferred embodiment, the drug is a cytotoxic drug.

[0100] In another preferred embodiment, the cytotoxic drug is selected from the following group: anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or a combination thereof.

[0101] Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.

[0102] In another preferred embodiment, the toxin is selected from the following group:

[0103] auristatins (e.g., auristatin E, auristatin F, MMAE and MMAF), chlortetracycline, maytansinoids, ricin, ricin A-chain, combretastatin, duocarmycin, dolastatin, adriamycin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotonin, calicheamicin, saponin, officinalis) inhibitors, glucocorticoids, or a combination thereof.

[0104] In another preferred embodiment, the coupling moiety is a detectable label.

[0105] In another preferred embodiment, the detectable marker comprises a radionuclide, and the radionuclide comprises:

[0106] (i) a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and / or

[0107] (ii) therapeutic isotopes, wherein the therapeutic isotopes are selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133 Yb-169, Yb-177, or a combination thereof.

[0108] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold single-domain particles / single-domain rods, viral particles, liposomes, single-domain magnetic particles, prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (for example, cisplatin) or any form of single-domain particles, etc.

[0109] In another preferred embodiment, the immunoconjugate contains: a multivalent (eg, bivalent) single domain antibody targeting BAFF-R as described in the first aspect of the present invention or an antibody targeting BAFF-R as described in the second aspect of the present invention.

[0110] In another preferred embodiment, the multivalency means that the amino acid sequence of the immunoconjugate contains multiple repeats of the single domain antibody targeting BAFF-R as described in the first aspect of the present invention or the antibody targeting BAFF-R as described in the second aspect of the present invention.

[0111] In another preferred embodiment, the detection is in vivo detection or in vitro detection.

[0112] In another preferred embodiment, the immunoconjugate is used for diagnosing and / or treating tumors expressing BAFF-R protein.

[0113] In another preferred embodiment, the immunoconjugate has the following molecular formula:

[0114]

[0115] in:

[0116] nAbs are single-domain antibodies targeting BAFF-R, antibodies targeting BAFF-R, or multispecific antibodies;

[0117] LU is a linker (also called a connector);

[0118] D is for medicine;

[0119] Furthermore, the subscript p is a value selected from 1-10.

[0120] In another preferred embodiment, LU is selected from maleimidocaproyl (MC), maleimide (MAL), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate) (SMCC) linkers connected to the antibody portion, and contains one or more linkers of valine-citrulline (VC), valine-alanine (VA), glycine-glycine-phenylalanine-glycine (GGFG), alanine-alanine-alanine (AAA), p-aminobenzyloxycarbonyl (PAB), and polyethylene glycol (PEG).

[0121] In another preferred embodiment, the antibody portion is covalently bound to the linker by reacting with a portion selected from the following group: maleimidocaproyl (MC), maleimide (MAL), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate) (SMCC), etc.

[0122] In another preferred embodiment, D is selected from the following group of compounds having anti-tumor activity:

[0123] (i) Tubulin inhibitors, such as maytansine derivatives (DM1, DM4), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF);

[0124] (ii) Acting on DNA toxins, such as duocarmycin and pyrrolobenzodiazepine (PBD);

[0125] (iii) Topoisomerase inhibitors, such as camptothecin, SN38, exitecan, and Dxd.

[0126] The seventh aspect of the present invention provides a pharmaceutical composition, comprising:

[0127] (i) the single domain antibody targeting BAFF-R as described in the first aspect of the present invention, the antibody targeting BAFF-R as described in the second aspect of the present invention, the multispecific antibody as described in the third aspect of the present invention, the bispecific antibody as described in the fourth aspect of the present invention, the recombinant protein as described in the fifth aspect of the present invention, or the immunoconjugate as described in the sixth aspect of the present invention;

[0128] (ii) a pharmaceutically acceptable carrier.

[0129] In another preferred embodiment, the pharmaceutical composition includes a single drug, a compound drug, or a synergistic drug.

[0130] In another preferred embodiment, the pharmaceutical composition further comprises other biologically active substances, such as drugs for treating tumors.

[0131] In another preferred embodiment, the administration method of the pharmaceutical composition is selected from the following group: subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, oral administration, or oral and nasal spraying and aerosol inhalation.

[0132] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: liquid, solid, or gel.

[0133] In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

[0134] In another preferred embodiment, the pharmaceutical composition is an injection.

[0135] The eighth aspect of the present invention provides a use of an active ingredient, wherein the active ingredient is selected from the following group: a single domain antibody targeting BAFF-R as described in the first aspect of the present invention, an antibody targeting BAFF-R as described in the second aspect of the present invention, a multispecific antibody as described in the third aspect of the present invention, a bispecific antibody as described in the fourth aspect of the present invention, a recombinant protein as described in the fifth aspect of the present invention, or an immunoconjugate as described in the sixth aspect of the present invention, or a combination thereof, wherein the active ingredient is used for (a) preparing a detection reagent, a detection plate or a kit; and / or (b) preparing a drug for preventing and / or treating a BAFF-R related disease.

[0136] In another preferred embodiment, the BAFF-R related diseases include tumor occurrence, growth and / or metastasis, tumor resistance related diseases, autoimmune diseases, inflammation, and metabolism related diseases.

[0137] In another preferred embodiment, the BAFF-R related diseases include BAFF-R highly expressed tumors.

[0138] In another preferred embodiment, the tumor includes leukemia, lymphoma, myeloma, breast cancer (such as triple-negative breast cancer), lung cancer (such as non-small cell lung cancer), pancreatic cancer, malignant glioma, gastric cancer, liver cancer, esophageal cancer, kidney cancer, colorectal cancer, bladder cancer, prostate cancer, endometrial cancer, ovarian cancer, cervical cancer, and angiosarcoma.

[0139] The ninth aspect of the present invention provides a polynucleotide encoding a polypeptide selected from the group consisting of:

[0140] (1) the single domain antibody targeting BAFF-R as described in the first aspect of the present invention, the antibody targeting BAFF-R as described in the second aspect of the present invention, the multispecific antibody as described in the third aspect of the present invention, or the bispecific antibody as described in the fourth aspect of the present invention; or

[0141] (2) The recombinant protein as described in the fifth aspect of the present invention.

[0142] In another preferred embodiment, the polynucleotide includes RNA, DNA or cDNA.

[0143] The tenth aspect of the present invention provides a vector, wherein the vector contains the polynucleotide as described in the ninth aspect of the present invention.

[0144] In another preferred embodiment, the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.

[0145] The eleventh aspect of the present invention provides a host cell, wherein the host cell contains the vector described in the tenth aspect of the present invention or the polynucleotide described in the ninth aspect of the present invention is integrated into its genome.

[0146] In a twelfth aspect of the present invention, there is provided a method for in vitro detection (including diagnostic or non-diagnostic) of BAFF-R in a sample, the method comprising the steps of:

[0147] (1) contacting the sample in vitro with the single domain antibody targeting BAFF-R according to the first aspect of the present invention, the antibody targeting BAFF-R according to the second aspect of the present invention, or the immunoconjugate according to the sixth aspect of the present invention;

[0148] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of BAFF-R in the sample.

[0149] In another preferred embodiment, the detection includes diagnostic or non-diagnostic.

[0150] The thirteenth aspect of the present invention provides a method for preparing a recombinant polypeptide, the method comprising:

[0151] (a) culturing the host cell according to the eleventh aspect of the present invention under conditions suitable for expression; and

[0152] (b) isolating a recombinant polypeptide from the culture, wherein the recombinant polypeptide is a single domain antibody targeting BAFF-R as described in the first aspect of the present invention, an antibody targeting BAFF-R as described in the second aspect of the present invention, a multispecific antibody as described in the third aspect of the present invention, or a recombinant protein as described in the fifth aspect of the present invention.

[0153] In the fourteenth aspect of the present invention, a method for treating BAFF-R related diseases is provided, the method comprising: administering to a subject in need thereof a single domain antibody targeting BAFF-R as described in the first aspect of the present invention, a multispecific antibody comprising the antibody, a recombinant protein, an immunoconjugate, or a pharmaceutical composition thereof, or a combination thereof.

[0154] In another preferred embodiment, the method further comprises: administering other drugs or treatment methods to a subject in need for combined treatment.

[0155] In another preferred embodiment, the other drugs or treatment methods include: anti-tumor immunotherapy drugs, tumor targeted drugs, tumor chemotherapy drugs, and tumor radiotherapy.

[0156] In another preferred embodiment, the anti-tumor immunotherapy drugs include PD-1 and PD-L1 monoclonal antibodies.

[0157] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0158] Figure 1 :α-BAFF-R×α-CD3 bispecific antibody 1+1 structural model diagram; Figure 1 A: α-BAFF-RscFab×α-CD3 scFab bispecific antibody 1+1 structural model; Figure 1 B: α-BAFF-R VH×α-CD3 scFab bispecific antibody 1+1 structural model

[0159] Figure 2 :α-BAFF-R×α-CD3 bispecific antibody mediates activation of TDCC reporter cells by K562-BAFF-R

[0160] Figure 3:α-BAFF-R×α-CD3 bispecific antibody mediates K562 activation of TDCC reporter cells

[0161] Figure 4 :α-BAFF-R×α-CD3 bispecific antibody mediates T cell killing of Nalm 6 cells

[0162] Figure 5 :α-BAFF-R×α-CD3 bispecific antibody mediates T cell killing of K562 cells

[0163] Figure 6 :Comparative analysis of amino acids in the extracellular domains of BAFF-R from humans, monkeys, and mice

[0164] Figure 7 :Schematic diagram of different structural configurations of α-BAFF-R VH×α-CD3 Fab bispecific antibody

[0165] Figure 7 A: α-BAFF-R VH×α-CD3 Fab bispecific antibody 1+1 structural model

[0166] Figure 7 B: α-BAFF-R VH×α-CD3 Fab bispecific antibody 2+1 structural model

[0167] Figure 7 C: α-BAFF-R VH×α-CD3 Fab bispecific antibody 2+2 structural model

[0168] Figure 8 :α-BAFF-R VH×α-CD3 Fab bispecific antibody mediates K562-BAFF-R activation of TDCC reporter cells

[0169] Fig. 9 :α-BAFF-R VH×α-CD3 Fab bispecific antibody mediates activation of TDCC reporter cells by Nalm6

[0170] Fig.10 :α-BAFF-R VH×α-CD3 Fab bispecific antibody mediates Mec-1 activation of TDCC reporter cells

[0171] Fig.11 :α-BAFF-R VH×α-CD3 Fab bispecific antibody mediates Jeko-1 activation of TDCC reporter cells

[0172] Fig.12 :α-BAFF-R VH×α-CD3 Fab bispecific antibody mediates K562 activation of TDCC reporter cells

[0173] Fig.13 :α-BAFF-R VH×α-CD3 Fab bispecific antibody mediates PBMC cells (Donor 301C) to kill Jeko-1 cells for 24 hours

[0174] Fig.14 :α-BAFF-R VH×α-CD3 Fab bispecific antibody mediates PBMC cells (Donor 301C) to kill Jeko-1 cells for 48 hours

[0175] Fig.15 :α-BAFF-R VH×α-CD3 Fab bispecific antibody mediates PBMC cells (Donor 605C) to kill Jeko-1 cells for 24 hours

[0176] Fig.16 :α-BAFF-R VH×α-CD3 Fab bispecific antibody mediates PBMC cells (Donor 605C) to kill Jeko-1 cells for 48 hours

[0177] Fig.17 :α-BAFF-R VH×α-CD3 Fab bispecific antibody promotes IFN-γ secretion in PBMC cells (Donor 303C)

[0178] Fig.17 A: Bispecific antibody, Jeko-1 and PBMC cells (Donor 303C) were co-incubated for 24 hours, and PBMC cytokine IFN-γ secretion was analyzed

[0179] Fig.17 B: Bispecific antibody, Jeko-1 and PBMC cells (Donor 303C) were co-incubated for 48 hours, and PBMC cytokine IFN-γ secretion was analyzed

[0180] Fig.17 C: Bispecific antibody and PBMC cells (Donor 303C) were co-incubated for 24 hours, and PBMC cytokine IFN-γ secretion analysis

[0181] Fig.17 D: Bispecific antibody and PBMC cells (Donor 303C) were co-incubated for 48 hours, and PBMC cytokine IFN-γ secretion analysis

[0182] Fig.18 :α-BAFF-R VH×α-CD3 Fab bispecific antibody promotes IFN-γ secretion in PBMC cells (Donor 605C)

[0183] Fig.18 A: Bispecific antibody, Jeko-1 and PBMC cells (Donor 605C) were co-incubated for 24 hours, and PBMC cytokine IFN-γ secretion was analyzed

[0184] Fig.18 B: Bispecific antibody, Jeko-1 and PBMC cells (Donor 605C) were co-incubated for 48 hours, and PBMC cytokine IFN-γ secretion analysis

[0185] Fig.18 C: Bispecific antibody and PBMC cells (Donor 605C) were co-incubated for 24 hours, and PBMC cytokine IFN-γ secretion analysis

[0186] Fig.18 D: Bispecific antibody and PBMC cells (Donor 605C) were co-incubated for 48 hours, and PBMC cytokine IFN-γ secretion analysis

[0187] Fig.19 :α-BAFF-R VH×α-CD3 Fab bispecific antibody promotes IL-2 secretion in PBMC cells (Donor 303C)

[0188] Fig.19 A: Bispecific antibody, Jeko-1 and PBMC cells (Donor 303C) were co-incubated for 24 hours, and PBMC cytokine IL-2 secretion was analyzed

[0189] Fig.19 B: Bispecific antibody, Jeko-1 and PBMC cells (Donor 303C) were co-incubated for 48 hours, and PBMC cytokine IL-2 secretion analysis

[0190] Fig.18 C: Bispecific antibody and PBMC cells (Donor 303C) were co-incubated for 24 hours, and PBMC cytokine IL-2 secretion analysis

[0191] Fig.19 D: Bispecific antibody and PBMC cells (Donor 303C) were co-incubated for 48 hours, and PBMC cytokine IL-2 secretion analysis

[0192] Fig. 20 :α-BAFF-R VH×α-CD3 Fab bispecific antibody promotes IL-2 secretion in PBMC cells (Donor 605C)

[0193] Fig. 20A: Bispecific antibody, Jeko-1 and PBMC cells (Donor 605C) were co-incubated for 24 hours, and PBMC cytokine IL-2 secretion was analyzed

[0194] Fig. 20 B: Bispecific antibody, Jeko-1 and PBMC cells (Donor 605C) were co-incubated for 48 hours, and PBMC cytokine IL-2 secretion analysis

[0195] Fig. 20 C: Bispecific antibody and PBMC cells (Donor 605C) were co-incubated for 24 hours, and PBMC cytokine IL-2 secretion analysis

[0196] Fig. 20 D: Bispecific antibody and PBMC cells (Donor 605C) were co-incubated for 48 hours, and PBMC cytokine IL-2 secretion analysis

[0197] Fig.21 :α-BAFF-R VH×α-CD3 Fab bispecific antibody promotes TNF-α secretion in PBMC cells (Donor 303C)

[0198] Fig.21 A: Bispecific antibody, Jeko-1 and PBMC cells (Donor 303C) were co-incubated for 24 hours, and PBMC cytokine TNF-α secretion was analyzed

[0199] Fig.21 B: Bispecific antibody, Jeko-1 and PBMC cells (Donor 303C) were co-incubated for 48 hours, and PBMC cytokine TNF-α secretion was analyzed

[0200] Fig.21 C: Bispecific antibody and PBMC cells (Donor 303C) were co-incubated for 24 hours, and PBMC cytokine TNF-α secretion analysis

[0201] Fig.21 D: Bispecific antibody and PBMC cells (Donor 303C) were co-incubated for 48 hours, and PBMC cytokine TNF-α secretion was analyzed

[0202] Fig. 22 :α-BAFF-R VH×α-CD3 Fab bispecific antibody promotes TNF-α secretion in PBMC cells (Donor 605C)

[0203] Fig. 22 A: Bispecific antibody, Jeko-1 and PBMC cells (Donor 605C) were co-incubated for 24 hours, and PBMC cytokine TNF-α secretion was analyzed

[0204] Fig. 22 B: Bispecific antibody, Jeko-1 and PBMC cells (Donor 605C) were co-incubated for 48 hours, and PBMC cytokine TNF-α secretion was analyzed

[0205] Fig. 22 C: Bispecific antibody and PBMC cells (Donor 605C) were co-incubated for 24 hours, and PBMC cytokine TNF-α secretion analysis

[0206] Fig. 22 D: Bispecific antibody and PBMC cells (Donor 605C) were co-incubated for 48 hours, and PBMC cytokine TNF-α secretion analysis

[0207] Fig.23 :α-BAFF-R VH×α-CD3 Fab bispecific antibody promotes IL-6 secretion in PBMC cells (Donor 303C)

[0208] Fig.23 A: Bispecific antibody, Jeko-1 and PBMC cells (Donor 303C) were co-incubated for 24 hours, and PBMC cytokine IL-6 secretion was analyzed

[0209] Fig.23 B: Bispecific antibody, Jeko-1 and PBMC cells (Donor 303C) were co-incubated for 48 hours, and PBMC cytokine IL-6 secretion analysis

[0210] Fig.23 C: Bispecific antibody and PBMC cells (Donor 303C) were co-incubated for 24 hours, and PBMC cytokine IL-6 secretion analysis

[0211] Fig.23 D: Bispecific antibody and PBMC cells (Donor 303C) were co-incubated for 48 hours, and PBMC cytokine IL-6 secretion analysis

[0212] Fig.24 :α-BAFF-R VH×α-CD3 Fab bispecific antibody promotes IL-6 secretion in PBMC cells (Donor 605C)

[0213] Fig.24 A: Bispecific antibody, Jeko-1 and PBMC cells (Donor 605C) were co-incubated for 24 hours, and PBMC cytokine IL-6 secretion was analyzed

[0214] Fig.24B: Bispecific antibody, Jeko-1 and PBMC cells (Donor 605C) were co-incubated for 48 hours, and PBMC cytokine IL-6 secretion analysis

[0215] Fig.24 C: Bispecific antibody and PBMC cells (Donor 605C) were co-incubated for 24 hours, and PBMC cytokine IL-6 secretion analysis

[0216] Fig.24 D: Bispecific antibody and PBMC cells (Donor 605C) were co-incubated for 48 hours, and PBMC cytokine IL-6 secretion analysis

[0217] Fig.25 : After a single dose, the concentration of bispecific antibody 0205-2 in SD rat serum changes with time

[0218] Fig.26 : After a single dose, the concentration of bispecific antibody 0205-3 in SD rat serum changes with time

[0219] Fig. 27 : After the first administration, the subcutaneous tumor volume (mm3) of mice in the bispecific antibody 0205-2 group and the PBS control group changed with the observation time

[0220] Fig.28 : After the first administration, the subcutaneous tumor volume (mm3) of mice in the PBS group changed with the observation time

[0221] Fig.29 : After the first administration, the subcutaneous tumor volume (mm3) of mice in the bispecific antibody 0205-2 administration 1 mpk group changed with the observation time

[0222] Fig.30 : After the first administration, the subcutaneous tumor volume (mm3) of mice in the bispecific antibody 0205-2 10 mpk group changed with the observation time

[0223] Fig.31 : After the first administration, the weight (g) of mice in each group changes with the observation time DETAILED DESCRIPTION

[0224] After extensive and in-depth research, the inventors have discovered for the first time a fully human single-domain antibody targeting BAFF-R and a bispecific antibody thereof. Anti-BAFF-R and anti-CD3 bispecific antibodies of different configurations (e.g., 1+1, 2+1 and 2+2 different combination configurations) can specifically bind to cell surface B cell activating factor receptor (BAFF-R) and immune cell surface antigen molecular cluster 3 (CD3), have high affinity, and can be used to treat tumor diseases and autoimmune diseases expressed by BAFFR. The present invention was completed on this basis.

[0225] the term

[0226] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0227] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0228] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0229] As used herein, the terms "Nanoantibodies of the present invention", "anti-BAFF-R Nanoantibodies of the present invention", and "BAFF-R single domain antibodies of the present invention" are used interchangeably, all referring to single domain antibodies that specifically recognize and bind to BAFF-R (including human BAFF-R). Particularly preferred is a single domain antibody whose amino acid sequence of the VHH chain is as shown in SEQ ID NO.: 1, i.e., clone No. 5 in the example.

[0230] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0231] As used herein, the terms "single domain antibody (VHH)" and "nanobody" have the same meaning, referring to the variable region of the heavy chain of a monoclonal antibody, and constructing a single domain antibody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete functions. Usually, an antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.

[0232] As used herein, the term "variable" means that some parts of the variable region in an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three fragments called complementary determining regions (CDRs) or hypervariable regions in the variable regions of light and heavy chains. The more conservative part of the variable region is called the framework region (FR). The variable regions of natural heavy and light chains each contain four FR regions, which are roughly in a β-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, 647-669 pages (1991)). The constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

[0233] As used herein, "Chothia", "Kabat", "IMGT", "AbM" refer to the determination of complementary binding determining regions CDR under the above-mentioned different assignment systems. The assignment systems include, for example, Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International Immuno GeneTics database (IMGT), and Chothia definition based on loop structure position.

[0234] As known to those skilled in the art, immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules combined with the antibodies or fragments of the present invention to form conjugates. The present invention also includes cell surface markers or antigens combined with the anti-BAFF-R protein antibodies or fragments thereof.

[0235] As used herein, the term "heavy chain variable region" is synonymous with "V H ” can be used interchangeably.

[0236] As used herein, the term "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0237] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity determining regions CDR1, CDR2, and CDR3.

[0238] In a preferred embodiment of the present invention, the heavy chain of the antibody comprises the above-mentioned heavy chain variable region and heavy chain constant region.

[0239] In the present invention, the terms "antibodies of the present invention", "proteins of the present invention", or "polypeptides of the present invention" are used interchangeably, and all refer to polypeptides that specifically bind to BAFF-R protein, such as proteins or polypeptides having a heavy chain variable region. They may or may not contain an initial methionine.

[0240] The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, as long as the variable region is identical to or at least 90% homologous to the heavy chain variable region of the antibodies of the present invention, preferably at least 95% homologous.

[0241] Generally, the antigen binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called the variable region (CDR). This segment is divided into four framework regions (FR). The amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction. These CDRs form a ring structure, and the β-folds formed by the FRs in between are close to each other in spatial structure. The CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR region.

[0242] The variable regions of the heavy chains of the antibodies of the present invention are of particular interest because they are at least partially involved in binding to antigen. Therefore, the present invention includes molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, and most preferably more than 98%) homology with the CDRs identified herein.

[0243] The antibody of the present invention refers to a polypeptide having BAFF-R protein binding activity and including the above CDR region. The term also includes variant forms of polypeptides having the same function as the antibody of the present invention and including the above CDR region. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions, insertions and / or substitutions, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present invention.

[0244] Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0245] The present invention also provides other polypeptides, such as fusion proteins comprising single domain antibodies or fragments thereof. In addition to almost full-length polypeptides, the present invention also includes fragments of single domain antibodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0246] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids having similar or similar properties compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table A.

[0247] Table A

[0248] Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0249] The present invention also provides a polynucleotide molecule encoding the above-mentioned antibody or its fragment or its fusion protein. The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0250] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only a mature polypeptide; a coding sequence of a mature polypeptide and various additional coding sequences; a coding sequence of a mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0251] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may include additional coding and / or non-coding sequences.

[0252] The present invention also relates to polynucleotides that hybridize with the above sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of denaturing agents during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0253] The full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Usually, a fragment with a very long sequence can be obtained by synthesizing multiple small fragments first and then connecting them. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.

[0254] Bispecific Antibodies

[0255] In the present invention, a multispecific antibody containing the BAFF-R single domain antibody of the present invention is also included, wherein the multispecific antibody further includes a second antigen binding region targeting a target selected from the group consisting of: CD3, CD28, 4-1BB, BCMA, CD73, GPC3, HER2, PMSA, OX40, GLP-1, Trop2, FGL1, LFA-3, 2B4, 5T4, α-4 integrin, α-V integrin, α4β7 integrin, α4β7 integrin, α-SMA, AGR2, Apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9, carbonic anhydrase 9, CD2, CD6, CD9, CDlla, CD19, CD20, CD22, CD24, CD25, CD27, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD69, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD107a, CD117, CD123, CD125, C D132(IL-2Rg), CD133, CD137, CD138, CD160, CD166, CD172A, CD248, CEACAM5(CEA), CEACAM6(NCA- 90), CLAUDIN-3, CLAUDIN-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FRα), FSP-1, GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIlb / IIIa receptor, Gpl30, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HLA-DR, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL -12R, IL-12Rβl, IL13, IL13R, IL13Ra2, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R(wsxl), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, IL1 receptor accessory protein (IL1RAP), insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, KLRG1, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, Nicastrin, Notch receptor, Notch 1, Notch 2, Notch 3, Notch 4. NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PSCA, PSMA, PS GR, RAAG12, RAGE, SLC44A4, Siglecl5, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, TLR4, TL R6, TLR7, TLR8, TLR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, WISP-3, or a combination thereof.

[0256] Preferably, the multispecific antibody comprises one or more second antigen binding regions, or further comprises a third antigen binding region.

[0257] A preferred bispecific antibody is an anti-BAFF-R and anti-CD3 bispecific antibody. The bispecific antibody of the present invention can specifically bind to the cell surface B cell activating factor receptor (BAFF-R) and immune cell surface antigen molecular cluster 3 (CD3); wherein the anti-BAFF-R antibody is a fully human single domain antibody. The bispecific antibody of the present invention is a symmetrical or asymmetrical dimer, such as 1+1, 2+1 and 2+2 different combination configurations of bispecific antibodies.

[0258] Preferably, the bispecific antibody has the following structure:

[0259] (a) two heavy chains, wherein the first heavy chain comprises, from N-terminus to C-terminus, anti-BAFF-R VH and human IgG Fc region; the second heavy chain comprises, from N-terminus to C-terminus, anti-CD3 VH, antibody heavy chain constant region (CH1), and human IgG Fc region;

[0260] (b) a light chain, comprising, from N-terminus to C-terminus, an anti-CD3 VL, an antibody light chain constant region (CL),

[0261] The anti-BAFF-R VH is the heavy chain variable region of a single domain antibody targeting CD3,

[0262] The anti-CD3 VL is paired with the anti-CD3 VH, and the antibody heavy chain constant region (CH1) is paired with the antibody light chain constant region (CL). In one embodiment of the present application, the bispecific antibody (VH-Fab-Fc 1+1) comprises: a first heavy chain having an amino acid sequence such as SEQ ID NO.16, a second heavy chain having an amino acid sequence such as SEQ ID NO.38, and a light chain having an amino acid sequence such as SEQ ID NO.36.

[0263] In another preferred embodiment, the bispecific antibody has the following structure:

[0264] (a) two heavy chains, wherein the first heavy chain comprises, from N-terminus to C-terminus, anti-BAFF-R VH, anti-CD3 VH, antibody heavy chain constant region (CH1), and human IgG Fc region; the second heavy chain comprises, from N-terminus to C-terminus, anti-BAFF-R VH and human IgG Fc region;

[0265] (b) a light chain, comprising, from N-terminus to C-terminus, an anti-CD3 VL, an antibody light chain constant region (CL),

[0266] The anti-BAFF-R VH is the heavy chain variable region of a single domain antibody targeting CD3,

[0267] The anti-CD3 VL is paired with the anti-CD3 VH, and the antibody heavy chain constant region (CH1) is paired with the antibody light chain constant region (CL). In one embodiment of the present application, the bispecific antibody (VH2-Fab-Fc 2+1) comprises: a first heavy chain having an amino acid sequence such as SEQ ID NO.16, a second heavy chain having an amino acid sequence such as SEQ ID NO.40, and a light chain having an amino acid sequence such as SEQ ID NO.36.

[0268] In another preferred embodiment, the bispecific antibody has the following structure:

[0269] (a) two heavy chains, wherein the heavy chains comprise, from N-terminus to C-terminus, anti-BAFF-R VH, anti-CD3 VH, antibody heavy chain constant region (CH1), and human IgG Fc region;

[0270] (b) two light chains, wherein the light chains comprise, from N-terminus to C-terminus, anti-CD3 VL, antibody light chain constant region (CL),

[0271] The anti-BAFF-R VH is the heavy chain variable region of a single domain antibody targeting CD3,

[0272] The anti-CD3 VL is paired with the anti-CD3 VH, and the antibody heavy chain constant region (CH1) is paired with the antibody light chain constant region (CL). In one embodiment of the present application, the bispecific antibody (VH2-Fab2-Fc 2+2) comprises: a first heavy chain having an amino acid sequence such as SEQ ID NO.16, a second heavy chain having an amino acid sequence such as SEQ ID NO.42, and a light chain having an amino acid sequence such as SEQ ID NO.36.

[0273] Antibody preparation

[0274] The sequence of the DNA molecule of the antibody of the present invention or its fragment can be obtained by conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences of the light chain and the heavy chain can be fused together to form a single-chain antibody.

[0275] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0276] In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0277] At present, the DNA sequence encoding the antibody (or its fragment, or its derivative) of the present invention can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0278] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0279] The host cell can be a prokaryotic cell, such as a bacterial cell, a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include (but are not limited to): CHO-S, HEK-293 cells.

[0280] Typically, the transformed host cells are cultured under conditions suitable for the expression of the antibodies of the present invention, and then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc., conventional separation and purification means well known to those skilled in the art to obtain the antibodies of the present invention.

[0281] The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of the monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibodies can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0282] The antibodies of the present invention can be expressed in cells, on cell membranes, or secreted outside cells. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting out method), centrifugation, osmotic sterilization, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0283] Immunoconjugates

[0284] The present invention also provides an immunoconjugate (ADC) based on the antibody of the present invention, preferably a single domain antibody-drug conjugate (NDC).

[0285] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, wherein the antibody is coupled to the effector molecule, and preferably chemically coupled. The effector molecule is preferably a drug with therapeutic activity. In addition, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug or a radionuclide.

[0286] The antibody of the present invention and the effector molecule can be coupled via a coupling agent. Examples of the coupling agent can be any one or more of a non-selective coupling agent, a coupling agent utilizing a carboxyl group, a peptide chain, and a coupling agent utilizing a disulfide bond. The non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde. The coupling agent utilizing a carboxyl group can be any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (the coupling site is an acylhydrazone).

[0287] Certain residues on antibodies (such as Cys or Lys, etc.) are used to connect to a variety of functional groups, including imaging agents (such as chromophores and fluorescent groups), diagnostic agents (such as MRI contrast agents and radioisotopes), stabilizers (such as ethylene glycol polymers) and therapeutic agents. Antibodies can be coupled to functional agents to form antibody-functional agent conjugates. Functional agents (such as drugs, detection agents, stabilizers) are coupled (covalently linked) to antibodies. Functional agents can be directly or indirectly connected to antibodies through linkers.

[0288] Single domain antibodies can be coupled to drugs to form antibody drug conjugates (NDCs). Typically, NDCs contain a linker between the drug and the antibody. The linker can be a degradable or non-degradable linker. Degradable linkers are typically easily degraded in the intracellular environment, such as degradation of the linker at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (such as lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronidase-containing linkers. Peptide linkers can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (such as linkers that are hydrolyzed when the pH is less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (such as disulfide linkers). Non-degradable linkers typically release drugs under conditions where the antibody is hydrolyzed by proteases.

[0289] Prior to attachment to the antibody, the linker has an active reactive group capable of reacting with certain amino acid residues, and attachment is achieved through the active reactive group. Thiol-specific active reactive groups are preferred and include, for example, maleimide compounds, halogenated amides (e.g., iodinated, brominated or chlorinated); halogenated esters (e.g., iodinated, brominated or chlorinated); halogenated methyl ketones (e.g., iodinated, brominated or chlorinated), benzyl halides (e.g., iodinated, brominated or chlorinated); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, maleimide attached to the antibody via thiosuccinimide.

[0290] The drug can be any cytotoxic, cytostatic or immunosuppressive drug. In an embodiment, a linker connects the antibody and the drug, and the drug has a functional group that can form a bond with the linker. For example, the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group that can form a bond with the linker. In the case where the drug is directly connected to the linker, the drug has a reactive group before being connected to the antibody.

[0291] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folate antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines), and vinca alkaloids.

[0292] In the present invention, drug-linkers can be used to form NDC in one simple step. In other embodiments, bifunctional linker compounds can be used to form NDC in a two-step or multi-step process. For example, a cysteine ​​residue is reacted with a reactive moiety of a linker in a first step, and in a subsequent step, a functional group on the linker is reacted with a drug to form an NDC.

[0293] Typically, the functional groups on the linker are selected to react specifically with the appropriate reactive groups on the drug moiety. As a non-limiting example, an azide-based moiety can be used to react specifically with a reactive alkynyl group on the drug moiety. The drug is covalently attached to the linker by a 1,3-dipolar cycloaddition between the azide and the alkynyl group. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other attachment strategies, such as those described in Bioconjugation Technology, 2nd Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that when a complementary pair of reactive functional groups is selected for selective reaction of the drug moiety and the linker, each member of the complementary pair can be used for both the linker and the drug.

[0294] The present invention also provides a method for preparing NDC, which may further include: combining an antibody and a drug-linker compound under conditions sufficient to form an antibody conjugate (NDC).

[0295] In certain embodiments, the methods of the invention include: combining the antibody with the bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the invention further include: combining the antibody-linker conjugate with the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody via the linker.

[0296] In some embodiments, the structure of the immunoconjugate, preferably a single domain antibody drug conjugate NDC, is shown in the following molecular formula:

[0297]

[0298] in:

[0299] nAb is a single domain antibody targeting BAFF-R, an antibody targeting BAFF-R, or a multispecific antibody as described above.

[0300] LU is the linker / connector;

[0301] D is for medicine;

[0302] And the subscript p is a value selected from 1 to 10.

[0303] application

[0304] The present invention also provides uses of the antibodies of the present invention, such as for preparing diagnostic preparations, or preparing drugs for preventing and / or treating BAFF-R-related diseases. The BAFF-R-related diseases include tumor occurrence, growth and / or metastasis, tumor resistance-related diseases, autoimmune diseases, inflammation, metabolism-related diseases, etc.

[0305] The uses of the antibodies or ADCs of the present invention include (but are not limited to):

[0306] (i) Diagnosis, prevention and / or treatment of tumor occurrence, growth and / or metastasis, especially tumors with high expression of BAFF-R. The tumors include (but are not limited to): leukemia, lymphoma, myeloma, breast cancer (such as triple-negative breast cancer), lung cancer (such as non-small cell lung cancer), pancreatic cancer, malignant glioma, gastric cancer, liver cancer, esophageal cancer, kidney cancer, colorectal cancer, bladder cancer, prostate cancer, endometrial cancer, ovarian cancer, cervical cancer, angiosarcoma, etc.; especially triple-negative breast cancer, non-small cell lung cancer, pancreatic cancer, malignant glioma, more preferably triple-negative breast cancer and / or non-small cell lung cancer.

[0307] (ii) Diagnosis, prevention and / or treatment of autoimmune diseases, including but not limited to systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, type I diabetes, psoriasis, multiple sclerosis, myasthenia gravis, Sjögren's syndrome, immune thrombocytopenia, and immune hemolysis.

[0308] (iii) diagnosing, preventing and / or treating inflammation, including (but not limited to) rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, gout, Reiter's syndrome, psoriatic arthropathy, infectious arthritis, tuberculous arthritis, viral arthritis, fungal arthritis, glomerulonephritis, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, acute lung injury, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis.

[0309] (iv) diagnosing, preventing and / or treating metabolic-related diseases, including (but not limited to) diabetes, diet-induced obesity and adipose inflammation.

[0310] Pharmaceutical composition

[0311] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein or its NDC or corresponding CAR-T cell, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium. The prepared pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0312] The antibody of the present invention can also be expressed in cells by nucleotide sequences for cell therapy, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.

[0313] The pharmaceutical composition of the present invention can be directly used to bind to BAFF-R protein molecules, and thus can be used to prevent and treat diseases such as tumors. In addition, other therapeutic agents can also be used simultaneously.

[0314] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned monoclonal antibody (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 5 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used with other therapeutic agents.

[0315] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms / kg body weight, and in most cases does not exceed about 50 milligrams / kg body weight, preferably the dosage is about 10 micrograms / kg body weight to about 20 milligrams / kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

[0316] For NDC, since the single domain antibody-drug conjugate provided by the present invention can target a specific cell population and bind to a specific protein (antigen) on the cell surface, the drug is released into the cell in an active form through endocytosis of the conjugate or drug penetration, the single domain antibody-drug conjugate of the present invention can be used to treat target diseases, and the antibody-drug conjugate mentioned above can be administered to a subject (e.g., a human) in a therapeutically effective amount through a suitable route. The subject in need of treatment may be a patient who is at risk or suspected of having a disease related to the activity or expression of a specific antigen. Such patients can be identified by routine physical examination.

[0317] When treated with the single domain antibody-drug conjugate of the present invention, it can be delivered by conventional methods in the art. For example, it can be introduced into cells by using liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres. Alternatively, the nucleic acid or vector can be delivered locally by direct injection or by using an infusion pump.

[0318] The main advantages of the present invention include

[0319] 1. The BAFF-R targeting single domain antibody of the present invention obtained by screening a fully human phage antibody library has low immunogenicity and high safety.

[0320] 2. The BAFF-R targeting single domain antibody of the present invention has species cross-activity and can bind to human, monkey and mouse BAFF-R proteins.

[0321] 3. The bispecific antibody targeting BAFF-R single domain antibody and CD3 of the present invention can specifically and effectively mediate PBMC killing of BAFF-R positive target cells and has good in vivo biological activity and safety.

[0322] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify detailed conditions are usually based on conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0323] Example 1 Screening of specific antibodies targeting BAFF-R protein

[0324] Using a fully human phage antibody library (including a fully human single-chain antibody library and a fully human single-domain antibody library), an affinity panning method was used to enrich antibodies that specifically bind to BAFF-R, and an enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FCM) were used to screen antibody clones that specifically bind to BAFF-R antigen and BAFF-R positive cells. The brief method is as follows: biotinylated BAFF-R antigen (BAFFR-His-biotin, BAF-HM40RB, Kactus) was bound to SA-coupled magnetic beads (22307-10, Beaver Biotechnology), magnetic beads coupled with Streptavidin were used as negative antigens to remove non-specifically bound phage clones in the phage library, and magnetic beads bound to BAFF-R antigen were used as positive antigens to enrich for binding phage clones, and this enrichment process was repeated 3-4 rounds. As shown in Table 1, after four rounds of enrichment, the recovery rates of the four fully human phage antibody libraries all reached 10-3, which can be used for monoclonal screening and identification.

[0325] Table 1. Recovery rate and enrichment multiple of phage antibody library after each round of panning

[0326]

[0327] The selected phage monoclones were determined by ELISA using BAFFR-His-biotin as positive antigen and Streptavidin (21125, Pierce) as negative antigen to identify specific binding phage clones that only bind to BAFFR-His-biotin but not to Streptavidin; at the same time, the BAFF-R overexpressing cell line K562-BAFF-R was used as positive cells and K562 as negative cells, and FCM was used to identify specific binding phage clones that bind to K562-BAFF-R but not to K562. Based on the results of ELISA and FCM, the phage clones with dual specific binding were identified as candidate positive clones. After sequencing, the amino acid sequence of the candidate positive clones was determined.

[0328] Example 2 Analysis of Cytotoxic Activity of Candidate Clones

[0329] In order to identify clones with better T-cell dependent cellular cytotoxicity (TDCC) activity, the candidate clones were combined with the CD3 antibody OKT3 to form a 1+1 bispecific antibody, that is, in one bispecific antibody molecule, both the BAFF-R antibody and the CD3 antibody are monovalent. Figure 1As shown: the molecular structure of OKT3 is scFab, and the molecular structure of BAFF-R candidate clones is VH or scFab (single domain antibody is constructed as VH, and single chain antibody is constructed as scFab); to facilitate antibody purification, the antibody Fc is retained, and to eliminate ADCC, ADCP and CDC effects, Fc uses IgG4 subtype combined with S228P / F234A / L235A mutations; to reduce the risk of Fc mismatch, the knobs-into-holes structure is introduced, the CD3 antibody side is selected for knobs design, and the BAFF-R antibody side is selected for holes design. After codon optimization, the antibody amino acid sequence was constructed into the pcDNA3.4 plasmid, namely OKT3 scFab-Fc PAA Ks, α-BAFF-R VH-Fc PAA Hs, and α-BAFF-R scFab-FcPAA Hs. The expression plasmids expressing OKT3 scFab antibody and BAFF-R VH or scFab antibody were co-transfected into CHOS cells, and the expression supernatant was collected after 7 days of suspension culture. The supernatant was centrifuged at 10,000 g for 30 minutes to remove debris, and then filtered through a 0.22 μm filter membrane for antibody Protein A affinity chromatography purification. The purified antibody was concentrated and exchanged into PBS for antibody TDCC activity analysis.

[0330] The activity analysis of α-BAFF-R×α-CD3 bispecific antibody was carried out in two steps.

[0331] First, based on the TDCC reporter gene cell line (Jurkat-NFAT-luc2, Nanjing IASOBiotechnology Co., Ltd.), the activation activity of α-BAFF-R×α-CD3 bispecific antibody on the CD3 signaling pathway was evaluated.

[0332] T cells were used as effector cells to evaluate the killing activity of α-BAFF-R×α-CD3 bispecific antibodies against BAFF-R positive target cells. Based on the activation activity and killing activity, the α-BAFF-R×α-CD3 bispecific antibodies were ranked to determine the α-BAFF-R clone with better activity. The brief experimental method of the CD3 signaling pathway activation test based on the TDCC reporter gene cell line is as follows: K562 (K562-BAFF-R) stably overexpressing BAFF-R is used as the positive target cell, K562 is used as the negative control cell, and Jurkat-NFAT-luc2 is used as the effector cell; 2E4 positive target cells or control cells are added to each well of a 96-well plate, and an equal amount of effector cells are added to each well. The bispecific antibody is diluted 5 times and added to the 96-well plate. The final dilution concentration gradient of the antibody is 100nM, 20nM, 4nM, 0.8nM, 0.16nM, 0.032nM, 0.0064nM, 0.00nM. After incubation at 37°C for 14-16 hours, the luciferase substrate (ONE-Glo TM EX Luciferase Assay System, E8120, Promega) was used to detect the luminescence intensity and calculate the EC50 curve of the luminescence intensity changing with the antibody concentration.

[0333] like Figure 2 The bispecific antibodies composed of clone 02, clone 04, clone 05, clone 06, and clone 07 can all mediate the activation of CD3 signaling pathway by K562-BAFF-R target cells, among which clone 02, clone 05, and clone 06 have stronger activation activity; Figure 3 As shown, when BAFF-R is not expressed on the surface of target cells, these five bispecific antibodies cannot cause activation of the CD3 signaling pathway.

[0334] The brief experimental method of the T cell-based target cell killing test is as follows: Nalm6 (Nalm6-luc) that stably overexpresses luciferase is used as a positive target cell, and K562 (K562-luc) that stably overexpresses luciferase is used as a negative control cell; T cells are used as effector cells. T cells are positively sorted from PBMCs by CD3 magnetic beads, CD3 / CD28 activation magnetic beads are added, and cultured in T cell culture medium for 9-11 days. 2E4 positive target cells or control cells are added to each well of a 96-well plate, and 4 times the amount of effector cells are added to each well to make the effector-target ratio 4:1. After 5-fold gradient dilution of the bispecific antibody, it is added to the 96-well plate. The final dilution concentration gradient of the antibody is 10nM, 2nM, 0.4nM, 0.08nM, 0.016nM, 0.0032nM, 0.00064nM, 0.00nM. After incubation at 37°C for 14-16 hours, the luciferase substrate (ONE-Glo TM EX Luciferase Assay System, E8120, Promega) was used to detect the luminescence intensity, calculate the target cell killing efficiency, and calculate the EC50 curve of the killing efficiency changing with the concentration.

[0335] like Figure 4 The bispecific antibodies formed by the combination of clone 02, clone 04, clone 05, clone 06, and clone 07 can mediate the killing of target cells Nalm6 by PBMC, among which clone 05 has the strongest activity, while clone 07 has a weaker activity, and clone 02 has no activity. Figure 5 As shown, when BAFF-R is not expressed on the surface of target cells, these 5 bispecific antibodies cannot cause killing of target cells.

[0336] Example 3 Study on Antibody Cross-species Reactivity

[0337] According to the BAFF-R protein information provided by Uniprot, the similarity of the amino acid in the extracellular region of human BAFF-R with that of cynomolgus monkey and mouse is 96.16% and 48.57%, respectively, and the similarity of the amino acid in the extracellular region of cynomolgus monkey BAFF-R with that of mouse is 47.14% (Table 2, Figure 6 ).

[0338] Table 2. Comparison of amino acid similarities among human, monkey and mouse BAFF-R extracellular domains

[0339]

[0340] To determine the cross-binding activity of BAFF-R antibody between monkey and mouse, the enzyme-linked immunosorbent assay (ELISA) was used to detect the strength of antibody binding to mouse and monkey BAFF-R antigens. Monoclonal antibody was constructed from clone 5, and the antibody Fc was S228P / L235E. After the antibody was prepared by CHOS expression, it was used for the study of species cross-activity. Different species BAFF-R antigens (BAFFR-human-bio-his, BAF-HM40RB, KACTUS; BAFFR-cyno-His, BAF-CM10R, KACTUS; BAFFR-mouse-rFc, 50353-M31H, Sino) were diluted to 2μg / mL with PBS, and 100μL / well was coated on a 96-well ELISA plate at 4℃ overnight. 3% BSA was used for blocking at 37℃ for 2 hours. The antibody was diluted 5-fold, 500nM, 100nM, 20nM, 4nM, 0.8nM, 0.16nM, 0.032nM, 0.0064nM, 0.00nM, 100μL / well, incubated at 37℃ for 2 hours. HRP-labeled anti-human IgG antibody (HRP Donkey anti-human IgG, 410902, Biolegend) was diluted 1:2000, 100μL / well, incubated at 37℃ for 1 hour. After washing three times with 0.01% PBST, 100μL / well substrate TMB was added, reacted in the dark for 3 minutes, 50μL / well 2M H2SO4 was used to terminate the reaction, and A450 was detected using an enzyme reader.

[0341] Calculate the maximum binding value and binding EC according to the change of A450 with concentration gradient 50 As shown in Table 3, clone 5 binds to the EC of human, monkey, and mouse BAFF-R. 50 They were 0.965nM, 1.906nM and 3.402nM respectively, indicating that clone 5 had both monkey and mouse cross-activity, with the monkey cross-activity being stronger and the mouse cross-activity being slightly weaker.

[0342] Table 3. Cross-binding data of antibody No. 5 and human, monkey and mouse BAFF-R

[0343]

[0344] Example 4 Preferred cloned bispecific antibody structure design and preparation

[0345] The preferred BAFF-R single domain antibody clone was combined with CD3 antibody SP34 to construct a bispecific antibody. The three configurations are as follows Figure 7 Configuration 1 is a 1+1 structure, that is, in a bispecific antibody molecule, both the BAFF-R antibody and the CD3 antibody are monovalent ( Figure 7A); Configuration 2 is a 2+1 structure, that is, in a bispecific antibody molecule, the BAFF-R antibody is bivalent, while the CD3 antibody is monovalent ( Figure 7 B); Configuration 3 is a 2+2 structure, that is, in a bispecific antibody molecule, both the BAFF-R antibody and the CD3 antibody are bivalent ( Figure 7 C) Among the three configurations, BAFF-R antibodies are all single domain antibodies, while CD3 antibody SP34 is a Fab structure.

[0346] Similarly, in order to enhance the half-life of the antibody and facilitate antibody purification, the Fc part of the antibody was retained. At the same time, in order to eliminate the ADCC, ADCP and CDC effects, the Fc used the IgG4 subtype combined with S228P / F234A / L235A mutations. In the asymmetric structure, in order to reduce the risk of Fc mispairing, a knobs-into-holes structure was introduced, with knobs design selected on the CD3 antibody side and holes design selected on the BAFF-R antibody side. At the same time, a pair of disulfide bonds were introduced into the Fc structure, and the knobs side was a combined mutation of S354C / T366W, and the holes side was a combined mutation of Y349C / T366S / L368A / Y407V. The antibody amino acid sequences were codon-optimized and then constructed into pcDNA3.4 plasmids, and the plasmids were named SP34-H PAA Ks, 05VH-SP34-H PAA Ks, 05VH-SP34-H PAA, SP34-L and 05VH-hFc PAA Hs. The plasmid combination for expressing 1+1 bispecific antibody was SP34-H PAA Ks, SP34-L and 05VH-hFc PAA Hs; the plasmid combination for expressing 2+1 bispecific antibody was 05VH-SP34-H PAA Ks, SP34-L and 05VH-hFc PAA Hs; the plasmid combination for expressing 2+2 bispecific antibody was 05VH-SP34-H PAA and SP34-L; the bispecific antibodies of 1+1, 2+1, and 2+2 configurations were named VH-Fab-Fc1+1, VH2-Fab-Fc 2+1, and VH2-Fab2-Fc 2+2, and the antibody numbers were 0205-1, 0205-2, and 0205-3, respectively (Table 4).

[0347] According to the plasmid combination in Table 4, the plasmids were co-transfected into CHOS cells, and the expression supernatant was collected after 7 days of suspension culture. The supernatant was centrifuged at 10,000g for 30 minutes to remove debris, and then filtered through a 0.22μm filter membrane for antibody Protein A affinity chromatography purification. The purified antibody was concentrated and the buffer was replaced with a cationic chromatography MES buffer. The protein A purified antibody was deeply purified using a cationic chromatography column to remove mismatched antibodies. The deeply purified antibody was used for binding kinetics, in vitro and in vivo biological activity analysis, etc.

[0348] Table 4. Combination of bispecific antibody expression plasmids

[0349]

[0350]

[0351] Example 5 Study on the binding kinetics of antibodies and antigens

[0352] To study the binding kinetics of α-BAFF-R×α-CD3 bispecific antibody with human BAFF-R protein and human CD3 protein, the bio-layer interferometry (BLI) technology was used to analyze the binding dissociation constant and affinity of the bispecific antibody with BAFF-R protein and CD3 protein based on the ForteBio Octet molecular interaction analysis system.

[0353] His-tagged human BAFF-R antigen (BAR-H52H3, Acro) was diluted with binding buffer (1×PBS containing 0.02% Tween and 0.1% BSA, PH=7.4) to 5 μg / mL and bound to the biosensor HIS1K (18-0038, ForteBio) with a thickness of 0.25 nm; the sensor after binding with the antigen was equilibrated in the sample diluent (1×PBS containing 0.02% Tween and 0.1% BSA, PH=7.4) for 180 seconds, placed in the antibody gradient dilution solution (200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM) for binding for 60 seconds, and then placed in the sample diluent for dissociation for 60-180 seconds; finally, the sensor was regenerated step by step using 10mM glycine and binding buffer (1×PBS containing 0.02% Tween and 0.1% BSA, PH=7.4). The data were analyzed using the data analysis system provided by the Octet molecular interaction analysis system to obtain the binding, dissociation constants and affinity data. As shown in Table 5, the affinity of the 05 monoclonal antibody to human BAFF-R is 2.63nM, and the affinity of the bispecific antibodies 0205-1, 0205-2, and 0205-3 to human BAFF-R is 1.78nM, 1.12nM, and 1.27nM, respectively.

[0354] Table 5. Kinetic parameters of α-BAFF-R monoclonal antibody and α-BAFF-R×α-CD3 bispecific antibody binding to BAFF-R

[0355]

[0356] His-tagged human CD3E&D (CDD-H52W1, Acro) and human CD3E&G (CDG-H52W6, Acro) were diluted to 5 μg / mL with binding buffer (1×PBS containing 0.02% Tween and 0.1% BSA, PH=7.4) and the binding thickness was 0.4 nm via the biosensor HIS1K (18-0038, ForteBio); the sensor after binding with the antigen was equilibrated in the sample diluent (1×PBS containing 0.02% Tween and 0.1% BSA, PH=7.4) for 180 sec, placed in a gradient dilution solution of antibodies (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM,) for 60 sec, and then placed in the sample diluent for dissociation for 60-180 sec; finally, 10 mM glycine and binding buffer (containing 0.02% Tween and 0.1% The sensor was regenerated with 1×PBS containing BSA, pH=7.4. The data were analyzed using the data analysis system of the Octet molecular interaction analysis system to obtain the binding, dissociation constants and affinity data. As shown in Table 5, the affinities of monoclonal antibody SP34 with human CD3E&D and humanCD3E&G were 6.38nM and 7.87nM, respectively; the affinities of bispecific antibody 0205-1 with CD3E&D and human CD3E&G were 10.7nM and 6.68nM, respectively; the affinities of bispecific antibody 0205-2 with CD3E&D and human CD3E&G were 10.3nM and 11.1nM, respectively; the affinities of bispecific antibody 0205-3 with CD3E&D and human CD3E&G were 10.6nM and 9.03nM, respectively. Although the N-terminus of the heavy chain of the anti-CD3 antibodies in 0205-2 and 0205-3 is linked to a single domain antibody, their binding to the CD3E&D or CD3E&G heterodimer complex is not affected or is minimally affected by the N-terminal single domain antibody.

[0357] Table 6. Kinetic parameters of α-CD3 monoclonal antibody and α-BAFF-R×α-CD3 bispecific antibody binding to CD3E&D and CD3E&G

[0358]

[0359] Example 6 Study on the activation activity of bispecific antibodies on CD3 signaling pathway

[0360] The first step for the α-BAFF-R×α-CD3 bispecific antibody to exert its biological function is bispecific antibody-mediated T cell activation, and T cells are activated in the presence of BAFF-R positive target cells, but not in the absence of BAFF-R positive cells. Based on the TDCC reporter gene cell line (Jurkat-NFAT-luc2), the activation activity and specificity of the α-BAFF-R×α-CD3 bispecific antibody on the CD3 signaling pathway were evaluated. Human chronic myeloid leukemia cell line K562 (K562-BAFF-R) stably overexpressing BAFF-R, human acute lymphoblastic leukemia cell line Nalm6, human chronic lymphocytic leukemia cell line MEC-1, and human mantle cell lymphoma cell line Jeko-1 were used as positive target cells, K562 was used as negative control cells, and Jurkat-NFAT-luc2 was used as effector cells. 2E4 positive target cells or negative control cells were added to each well of a 96-well plate, and an equal amount of effector cells were added to each well. The bispecific antibody was diluted 5-fold and added to the 96-well plate. The final dilution concentration gradient of the antibody was 100nM, 20nM, 4nM, 0.8nM, 0.16nM, 0.032nM, 0.0064nM, and 0.00nM. After incubation at 37°C for 14-16 hours, luciferase substrate (ONE-Glo TM EX Luciferase Assay System, E8120, Promega) was used to detect the luminescence intensity and calculate the EC50 curve of the luminescence intensity changing with the antibody concentration.

[0361] like Figure 8-11 As shown in Tables 7-10, the target cells are K562-BAFF-R with high BAFF-R expression. 0205-1, 0205-2 and 0205-3 can mediate the activation of TDCC reporter gene cells, and EC 50 When the target cells were natural tumor cell lines expressing BAFF-R, such as Nalm6, Mec-1, and Jeko-1, the three bispecific antibodies mediated TDCC reporter gene cell activation activities that were significantly different, with 0205-3 having the best activity, and EC 50 Between 0.02nM and 0.065; 0205-2 is second, EC 50 Between 0.06nM and 0.5; 0205-1 had the worst activity.

[0362] like Fig.12As shown in Table 11, the levels of TDCC reporter gene activation mediated by the three bispecific antibodies in BAFF-R negative cells were all low, and only at the highest concentrations of 0205-1 and 0205-3, the TDCC reporter gene cells were slightly activated. Because 0205-3 is a bivalent CD3 antibody, this result suggests that 0205-3 may have certain safety risks and needs to be evaluated more in subsequent experiments.

[0363] Table 7. Parameters of α-BAFF-R VH×α-CD3 Fab bispecific antibody-mediated activation of K562-BAFF-R on TDCC reporter cells

[0364]

[0365] Table 8. Parameters of α-BAFF-R VH×α-CD3 Fab bispecific antibody-mediated activation of TDCC reporter cells by Nalm6

[0366]

[0367] Table 9. Parameters of Mec-1 activation of TDCC reporter cells mediated by α-BAFF-R VH×α-CD3 Fab bispecific antibody

[0368]

[0369] Table 10. Parameters of α-BAFF-R VH×α-CD3 Fab bispecific antibody-mediated activation of Jeko-1 on TDCC reporter cells

[0370]

[0371] Table 11. Parameters of α-BAFF-R VH×α-CD3 Fab bispecific antibody-mediated activation of K562 on TDCC reporter cells

[0372]

[0373] Example 7 Study on the killing activity of PBMC on target cells mediated by bispecific antibodies

[0374] To simulate the activity of bispecific antibodies in the body to mediate immune cell killing of BAFF-R positive target cells, PBMCs were used as effector cells, Jeko-1 stably overexpressing luciferase (Jeko-1-luc) was used as positive target cells, and K562 stably overexpressing luciferase (K562-luc) was used as negative control cells. 2E4 positive target cells or control cells were added to each well of a 96-well plate, and 10 times the amount of effector cells were added to each well to make the effector-target ratio 10:1. After 5-fold gradient dilution of the antibody, it was added to the 96-well plate. The final dilution concentration gradient of the antibody was 1nM, 0.4nM, 0.04nM, 0.008nM, 0.0016nM, 0.00032nM, and 0.0nM. After incubation at 37°C for 24 hours and 48 hours, the luciferase substrate (ONE-Glo TM EX Luciferase Assay System, E8120, Promega), detect the luminescence intensity, calculate the target cell killing efficiency, and calculate the EC50 curve of the killing efficiency changing with the concentration. In this experiment, Regeneron's bispecific antibody REGN1979 analog targeting CD20 and CD3 was used as a positive reference.

[0375] like Figure 13-16 As shown in Tables 12 and 13, the activity reaction rankings of the three bispecific antibodies in the PBMCs of the two donors were consistent. No killing activity of 0205-1 against BAFF-R positive tumor cells was observed in either donor. The killing activities of 0205-2 and 0205-3 against tumor cells were relatively weak at 24 hours, but they could achieve complete elimination of tumor cells at 48 hours.

[0376] Comprehensively comparing the activation activity on TDCC reporter gene cells and the clearance activity on BAFF-R tumor cells, the activity of α-BAFF-R VH×α-CD3 Fab bispecific antibody was ranked as 0205-3≥0205-2﹥0205-1.

[0377] Table 12: α-BAFF-R VH×α-CD3 Fab bispecific antibody-mediated killing parameters of PBMC cells (Donor 301C) against Jeko-1 cells

[0378]

[0379] Table 13: α-BAFF-R VH×α-CD3 Fab bispecific antibody-mediated killing parameters of PBMC cells (Donor 605C) against Jeko-1 cells

[0380]

[0381] Example 8 Study on bispecific antibody-mediated PBMC cytokine release

[0382] PBMCs were used as effector cells, and Jeko-1 stably overexpressing luciferase (Jeko-1-luc) was used as positive target cells. A solvent control without adding target cells was also designed. 2E4 positive target cells or control cells were added to each well of a 96-well plate, and 10-fold amount of effector cells were added to each well to make the effector-target ratio 10:1. The solvent control group was added with an equal amount of effector cells. The bispecific antibody was diluted 5-fold and added to the 96-well plate. The final dilution concentration gradient of the antibody was 1nM, 0.4nM, 0.016nM, and 0.0nM. After incubation at 37°C for 24 hours and 48 hours, the supernatant was carefully removed after centrifugation at 500g for 10 minutes. The contents of cytokines IFN-γ (62HIFNGPEG, cisbio), IL-2 (62HIL02PEG, cisbio), TNFα (62HTNFAPEG, cisbio), and IL-6 (62HIL06PEG, cisbio) in the supernatant were detected based on the time-delayed fluorescence method to evaluate the strength of bispecific antibody-mediated PBMC cytokine release. In this experiment, Regeneron's bispecific antibody REGN1979 analog targeting CD20 and CD3 was used as a positive reference, and SP34 monoclonal antibody was used as a negative reference antibody.

[0383] like Figure 17-22 As shown in the figure, when co-incubated with BAFF-R positive target cells and PBMC, bispecific antibodies 0205-2 and 0205-3 can significantly induce PBMC to release IFN-γ, IL-2 and TNFα, and the levels are slightly lower than those of the positive reference antibodies; when there are no BAFF-R positive target cells, the secretion of IFN-γ, IL-2 and TNFα is significantly reduced.

[0384] like Fig.21 , 22 As shown, regardless of whether BAFF-R positive target cells were provided, the three bispecific antibodies and the positive reference antibody induced low levels of IL-6 secretion from PBMCs.

[0385] Example 9 Antibody Pharmacokinetics Study

[0386] To study the metabolic kinetics of antibodies, 6-8 week old female SD rats, 220-240 g, were used. SD rats were purchased from Shanghai Jihui Laboratory Animal Care Co. LTD. The antibody dose was 1 mg / kg, and the administration method was intravenous administration. Before and after administration at each time point (5min, 30min, 2hr, 6hr, 24hr, 72hr, 120hr, 168hr, 240hr, 336hr), 200 μL of blood was collected from the jugular vein, and after being placed at room temperature for about 1 hour, the serum was collected by centrifugation and stored at -70°C. The serum antibody concentration was detected by sandwich ELISA method. The Goat anti-human IgG Fc antibody (609-101-017, Rockland) was diluted to 2.5 μg / mL and coated on the ELISA plate. The serum was diluted 20 times for quantitative detection. The peroxidase-labeled secondary antibody Anti-Human IgG (Fab specific)-peroxidase (A0293, Sigma) was diluted 1:5000 and bound to the Fab end of the serum antibody. The serum antibody concentration at each time point was calculated based on the standard curve and the dilution factor.

[0387] After administration, no abnormal reaction was observed in all SD rats. The serum antibody concentration of bispecific antibody 0205-2 at each time point is shown in Table 14 and Fig.25 As shown in Table 15 and Table 16, the half-life of the antibody is about 13.7 days; the serum antibody concentration of the bispecific antibody 0205-3 at each time point is shown in Table 15 and Table 16. Fig.26 As shown, the antibody half-life is approximately 10.3 days.

[0388] Table 14. Serum concentrations and pharmacokinetic parameters of bispecific antibody 0205-2 in SD rats

[0389]

[0390]

[0391] Table 15. Serum concentrations and pharmacokinetic parameters of bispecific antibody 0205-3 in SD rats

[0392]

[0393] Example 10 In vivo efficacy study of antibodies

[0394] Considering the safety of CD3 antibody, we selected bispecific antibody 0205-2 containing monovalent CD3 antibody, and reconstructed the subcutaneous tumor model of NCG mice based on PBMC for in vivo antitumor efficacy evaluation. As shown in Table 16, on Day 0, Jeko-1 tumor cells were subcutaneously inoculated with gelatin at a ratio of 1:1, with an inoculation volume of 5E6 / mouse; on Day 3, PBMC cells were intraperitoneally infused at 1E7 / mouse; the tumor volume reached 110mm 3 The mice were randomly divided into groups: the first group was the PBS control group, the second group was the 0205-2 low-dose group with a dose of 1 mpk, and the third group was the 0205-2 high-dose group with a dose of 10 mpk; the administration method was intraperitoneal administration; the administration frequency was twice a week, for a total of two weeks; the mouse weight was recorded and the mouse tumor volume was measured twice a week; in accordance with the ethical regulations of animal experiments, the mice were euthanized after the tumor volume reached the standard.

[0395] Table 16. Design of bispecific antibody efficacy test based on PBMC-reconstructed NCG mouse subcutaneous tumor model

[0396]

[0397]

[0398] like Fig. 27 As shown, the tumor volume reached 110mm 3 At 14:00 pm, the drug was started. In both dose groups (1 mpk and 10 mpk), the tumor volume of mice decreased rapidly and gradually disappeared completely, and there was no difference between the two dose groups. The changes in the tumor volume of mice in the 1 mpk dose group and the PBS group were relatively consistent, while in the 10 mpk dose group, the tumor volume of one mouse reached 650 mm 3 , can still disappear completely quickly ( Figure 28-30 ).

[0399] like Fig.31 As shown, during the experimental observation period, no mouse deaths were found in the 1mpk dose group and the 10mpk dose group, and no obvious weight loss, decreased activity, hunched back, ruffled hair and other phenomena were observed in the mice, indicating that the mice tolerated the drug well.

[0400] Sequences of the present invention

[0401] SEQ ID NO.1 05VH amino acid sequence

[0402] EVQLVESGGGVVQPGRSLRLTCAASGFTFSTSGMYWIRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTQSCGAGKCYPGVWGQGTTVTVSS

[0403] SEQ ID NO.2 05VH nucleotide sequence

[0404] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCACCTGTGCAGCCTCTGGATTCACGTTCAGTACCTCTGGCATGTACTGGATTCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTAGTAGTAGTACCATATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTATTGTGTGACTCAGTCGTGTGGTGCTGGTAAATGCTACCCGGGGGTCTGGGGCCAAGGGACCACGGTCACTGTCTCCTCA

[0405]

[0406] ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0407] SEQ ID NO.7 hFc PAA Hs nucleotide sequence

[0408] GAGAGCAAGTACGGCCCCCCCTGCCCCCCCTGCCCCGCCCCCGAGGCCGCCGGCGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAGGAGGACCCCGAGGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGCGAGGAGCAGTTCAACAGCACCTACCGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGGCCTGCCCAGCAGCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTGtgcACCCTGCCCCCCAGCCAGGAGGAGATGACCAAGAACCAGGTGAGCCTGagcTGCgccGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGgtgAGCagaCTGACCGTGGACAAGAGCCGCTGGCAGGAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCTGGGCAAG

[0409] Amino acid sequence of SEQ ID NO.8 hFc PAA Ks

[0410] ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0411] SEQ ID NO. 9 hFc PAA Ks nucleotide sequence

[0412] GAGAGCAAGTACGGCCCCCCCTGCCCCCCCTGCCCCGCCCCCGAGGCCGCCGGCGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAGGAGGACCCCGAGGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGCGAGGAGCAGTTCAACAGCACCTACCGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGGCCTGCCCAGCAGCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTGTACACCCTGCCCCCCtgcCAGGAGGAGATGACCAAGAACCAGGTGAGCCTGtggTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCCGCTGGCAGGAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCTGGGCAAG

[0413] SEQ ID NO. 10 OKT3 VH amino acid sequence

[0414] QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSS

[0415] SEQ ID NO.11 OKT3 VH nucleotide sequence

[0416] CAGGTGCAGCTGGTGCAGAGCGGCGGCGGCGTGGTGCAGCCCGGCCGCAGCCTGCGCCTGAGCTGCAAGGCCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCGCCTTCCTGCAGATGGACAGCCTGCGCCCCGAGGACACCGGCGTGTACTTCTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCCCCGTGACCGTGAGCAGC

[0417] SEQ ID NO.12 OKT3 VK amino acid sequence

[0418] DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITR

[0419] SEQ ID NO.13 OKT3 VK nucleotide sequence

[0420] GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCAGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGACCCCCGGCAAGGCCCCCAAGCGCTGGATCTACGACACCAGCAAGCTGGCCAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTACACCTTCACCATCAGCAGCCTGCAGCCCGAGGACATCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCTTCACCTTCGGCCAGGGCACCAAGCTGCAGATCACCCGC

[0421] SEQ ID NO.14 OKT3 scFab amino acid sequence

[0422] DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV

[0423] SEQ ID NO.15 OKT3 scFab nucleotide sequence

[0424]

[0425] SEQ ID NO.16 Amino acid sequence of 05VH-hFc PAA Hs

[0426] EVQLVESGGGVVQPGRSLRLTCAASGFTFSTSGMYWIRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTQSCGAGKCYPGVWGQGTTVTVSSESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0427] SEQ ID NO.17 Nucleotide sequence of 05VH-hFc PAA Hs

[0428]

[0429] SEQ ID NO.18 OKT3 scFab-H PAA Ks Amino Acid Sequence

[0430] DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0431] SEQ ID NO.19 OKT3 scFab-H PAA Ks Nucleotide Sequence

[0432]

[0433] SEQ ID NO.20 hFc PE amino acid sequence

[0434] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0435] SEQ ID NO.21 hFc PE nucleotide sequence

[0436] GAGAGCAAGTACGGCCCTCCCTGCCCACCCTGCCCCGCTCCCGAGTTTGAAGGCGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAGGAGGACCCCGAGGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGCGAGGAGCAGTTCAACAGCACCTACCGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGGCCTGCCCAGCAGCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTGTACACCCTGCCCCCCAGCCAGGAGGAGATGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCCGCTGGCAGGAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCTGGGCAAG

[0437] SEQ ID NO.22 05VH-hFc PE amino acid sequence

[0438] EVQLVESGGGVVQPGRSLRLTCAASGFTFSTSGMYWIRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTQSCGAGKCYPGVWGQGTTVTVSSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0439] SEQ ID NO.23 05VH-hFc PE nucleotide sequence

[0440]

[0441] SEQ ID NO.24 Amino acid sequence of human BAFF-R

[0442] MRRGPRSLRGRDAPAPTPCVPAECFDLLVRHCVACGLLRTPRPKPAGASSPAPRTALQPQESVGAGAGEAALPLPGLLFGAPALLGLALVLALVLVGLVSWRRRQRRLRGASSAEAPDGDKDAPEPLDKVIILSPGISDATAPAWPPPGEDPGTTPPGHSVPVPATELGSTELVTTKTAGPEQQ

[0443] SEQ ID NO.25 Amino acid sequence of cynomolgus BAFF-R

[0444] MKRGPRSLRGRDAPAPTPCVPAECFDLLVRHCVACGLLRTPRPKPAPASSPAPRTALQPQESVGAGAGEAALSLPGLLFGAPALLGLALVLALVLVGLVSWRRRQRRLRGASSAEAPDGDKDKDEPLDKVIILSPGISDAAAPAWPPPGEDPGTTPPGHSVPVPATELGSTELVTTKTAGPEQQ

[0445] SEQ ID NO.26 Amino acid sequence of mouse BAFF-R

[0446] MGARRLRVRSQRSRDSSVPTQCNQTECFDPLVRNCVSCELFHTPDTGHTSSLEPGTALQPQEGSALRPDVALLVGAPALLGLILALTLVGLVSLVSWRWRQQLRTASPDTSEGVQQESLENVFVPSSETPHASAPTWPPLKEDADSALPRHSVPVPATELGSTELVTTKTAGPEQ

[0447] SEQ ID NO.27 Amino acid sequence of the extracellular region of human BAFF-R

[0448] MRRGPRSLRGRDAPAPTPCVPAECFDLLVRHCVACGLLRTPRPKPAGASSPAPRTALQPQESVGAGAGEAALPLPGLL

[0449] SEQ ID NO.28 Amino acid sequence of cynomolgus BAFF-R extracellular region

[0450] MKRGPRSLRGRDAPAPTPCVPAECFDLLVRHCVACGLLRTPRPKPAPASSPAPRTALQPQESVGAGAGEAALSLPGLL

[0451] SEQ ID NO.29 Amino acid sequence of mouse BAFF-R extracellular region

[0452] MGARRLRVRSQRSRDSSVPTQCNQTECFDPLVRNCVSCELFHTPDTGHTSSLEPGTALQPQEGSALRPDVALL

[0453] SEQ ID NO.30 Amino acid sequence of SP34 VH

[0454] EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

[0455] SEQ ID NO.31 Nucleotide sequence of SP34 VH

[0456] GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCAAGGGCAGCCTGAAGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAACACCTACGCCATGAACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCCGCATCCGCAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGACCGCTTCACCATCAGCCGCGACGACAGCCAGAGCATCCTGTACCTGCAGATGAACAACCTGAAGACCGAGGACACCGCCATGTACTACTGCGTGCGCCACGGCAACTTCGGCAACAGCTACGTGAGCTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC

[0457] SEQ ID NO.32 Amino acid sequence of SP34 VL

[0458] QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL

[0459] SEQ ID NO.33 SP34 VL nucleotide sequence

[0460] CAGGCCGTGGTGACCCAGGAGAGCGCCCTGACCACCAGCCCCGGCGAGACCGTGACCCTGACCTGCCGCAGCAGCACCGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGGAGAAGCCCGACCACCTGTTCACCGGCCTGATCGGCGGCACCAACAAGCGCGCCCCCGGCGTGCCCGCCCGCTTCAGCGGCAGCCTGATCGGCGACAAGGCCGCCCTGACCATCACCGGCGCCCAGACCGAGGACGAGGCCATCTACTTCTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGCGGCACCAAGCTGACCGTGCTG

[0461] SEQ ID NO.34 SP34-H PE amino acid sequence

[0462] EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0463] SEQ ID NO.35 SP34-H PE nucleotide sequence

[0464]

[0465] SEQ ID NO.36SP34-L amino acid sequence

[0466] QAVVTQESALTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLT VLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS

[0467] SEQ ID NO.37SP34-L nucleotide sequence

[0468] CAGGCCGTGGTGACCCAGGAGAGCGCCCTGACCACCAGCCCCGGCGAGACCGTGACCCTGACCTGCCGCAGCAGCACCGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGGAGAAGCCCGACCACCTGTTCACCGGCCTGATCGGCGGCACCAACAAGCGCGCCCCCGGCGTGCCCGCCCGCTTCAGCGGCAGCCTGATCGGCGACAAGGCCGCCCTGACCATCACCGGCGCCCAGACCGAGGACGAGGCCATCTACTTCTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGCGGCACCAAGCTGACCGTGCTGGGTCAGCCCAAGGCTGCCCCTAGCGTGACACTGTTCCCACCTTCTAGCGAGGAACTGCAAGCGAATAAGGCTACACTGGTGTGCCTGATTAGCGACTTCTACCCTGGCGCCGTGACCGTAGCATGGAAAGCGGACTCTAGCCCCGTGAAGGCCGGCGTGGAGACCACAACCCCATCTAAGCAGTCTAACAACAAGTACGCCGCCTCTTCCTACCTGTCCCTCACCCCTGAGCAGTGGAAGTCTCACAAGTCTTACTCTTGCCAGGTGACACACGAGGGCTCTACAGTGGAAAAGACAGTGGCCCCTACAGAGTGCAGC

[0469] SEQ ID NO.38 Amino acid sequence of SP34-H PAA Ks

[0470] EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0471] SEQ ID NO.39 SP34-H PAA Ks nucleotide sequence

[0472]

[0473] SEQ ID NO.40 Amino acid sequence of 05VH-SP34-H PAA

[0474] EVQLVESGGGVVQPGRSLRLTCAASGFTFSTSGMYWIRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTQSCGAGKCYPGVWGQGTTVTVSSGGGGSGGGGSEVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0475] SEQ ID NO.41 Nucleotide sequence of 05VH-SP34-H PAA

[0476]

[0477] SEQ ID NO.42 Amino acid sequence of 05VH-SP34-H PAA Ks

[0478] EVQLVESGGGVVQPGRSLRLTCAASGFTFSTSGMYWIRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTQSCGAGKCYPGVWGQGTTVTVSSGGGGSGGGGSEVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0479] SEQ ID NO.43 Nucleotide sequence of 05VH-SP34-H PAA Ks

[0480]

[0481]

[0482] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A single domain antibody against B cell activating factor receptor (BAFF-R), characterized in that The VHH chain of the single-domain antibody comprises the following complementary determining regions CDR: CDR1 shown in SEQ ID NO.3, CDR2 shown in SEQ ID NO.4, and CDR3 shown in SEQ ID NO.5; Any of the above amino acid sequences also includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and is able to retain BAFF-R binding affinity.

2. The single domain antibody according to claim 1, characterized in that The VHH chain of the single domain antibody further comprises a framework region (FR).

3. An antibody targeting BAFF-R, characterized in that: The antibody comprises one or more VHH chains of the single domain antibody targeting BAFF-R according to claim 2.

4. A multispecific antibody, characterized in that: The multispecific antibody comprises: the single-domain antibody targeting BAFF-R according to claim 1 or the antibody targeting BAFF-R according to claim 3.

5. A bispecific antibody, characterized in that: The bispecific antibody comprises: Targeting the first antigen binding region of BAFF-R; and Targeting the second antigen binding region of CD3; wherein, The first antigen binding region targeting BAFF-R comprises the following complementary determining regions CDR: CDR1 shown in SEQ ID NO.3, CDR2 shown in SEQ ID NO.4 and CDR3 shown in SEQ ID NO.

5.

6. The bispecific antibody according to claim 5, characterized in that The bispecific antibody has the following structure: (a) two heavy chains, the first heavy chain comprises, from N-terminus to C-terminus: anti-BAFF-R VH, anti-CD3 VH, antibody heavy chain constant region (CH1), human IgG Fc region; the second heavy chain comprises, from N-terminus to C-terminus: anti-BAFF-R VH, human IgG Fc region; (b) a light chain, comprising, from N-terminus to C-terminus, an anti-CD3 VL, an antibody light chain constant region (CL), Wherein, there is a disulfide bond between the antibody heavy chain constant region (CH1) and the antibody light chain constant region (CL); Or, the bispecific antibody has the following structure: (a) two identical heavy chains, wherein the heavy chains comprise, from N-terminus to C-terminus, anti-BAFF-R VH, anti-CD3 VH, antibody heavy chain constant region (CH1), and human IgG Fc region; (b) two identical light chains, wherein the light chains comprise, from N-terminus to C-terminus, anti-CD3 VL, antibody light chain constant region (CL), Wherein, a disulfide bond exists between the antibody heavy chain constant region (CH1) and the antibody light chain constant region (CL).

7. A recombinant protein, characterized in that The recombinant protein has: (i) the single domain antibody targeting BAFF-R according to claim 1 or 2, the antibody targeting BAFF-R according to claim 3, or the bispecific antibody according to claim 5; and (ii) optional polypeptide molecules or fragments having therapeutic functions; and / or (iii) Optional functional domains that enhance the physicochemical properties or druggability of the protein.

8. An immunoconjugate, characterized in that: The immunoconjugate contains: (a) an antibody portion, wherein the antibody portion is the single domain antibody targeting BAFF-R according to claim 1 or 2, the antibody targeting BAFF-R according to claim 3, or the bispecific antibody according to claim 5; and (b) a conjugated moiety conjugated to the antibody portion, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, an enzyme, or a combination thereof.

9. A use of an active ingredient, wherein the active ingredient is selected from the group consisting of the single domain antibody targeting BAFF-R as claimed in claim 1 or 2, the antibody targeting BAFF-R as claimed in claim 3, the multispecific antibody as claimed in claim 4, the bispecific antibody as claimed in claim 5, the recombinant protein as claimed in claim 7, the immunoconjugate as claimed in claim 8, or a combination thereof, characterized in that: The active ingredient is used for (a) preparing a detection reagent, a detection plate or a test kit; and / or (b) preparing a drug for preventing and / or treating BAFF-R related diseases.

10. A polynucleotide, characterized in that The polynucleotide encodes a polypeptide selected from the group consisting of: (1) the single domain antibody targeting BAFF-R according to claim 1 or 2, the antibody targeting BAFF-R according to claim 3, the multispecific antibody according to claim 4, or the bispecific antibody according to claim 5; or (2) The recombinant protein according to claim 7.

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