Fully-human antibody and car-t cell targeting BAFF-r and use thereof

Abstract

A fully human single-domain antibody, a monoclonal antibody, a bispecific antibody, and a CAR-T cell targeting BAFF-R and a use thereof. Provided are a fully human antibody or antigen-binding fragment thereof targeting BAFF-R and obtained by means of screening a fully human phage library, a fully human chimeric antigen receptor (CAR) targeting BAFF-R, and an immune cell expressing the CAR. Further provided is a bispecific antibody targeting BAFF-R and CD3 in different combination configurations. Further provided is a use of the foregoing antibodies and the CAR-immune cell in the treatment of B cell tumors.

Description

Fully human antibodies, CAR-T cells targeting BAFF-R and their applications Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to fully human single-domain antibodies, monoclonal antibodies, bispecific antibodies, CAR-T cells, and their applications in CAR-T therapy targeting BAFF-R. Background Art

[0002] Lymphoma is a malignant tumor that originates in the lymph nodes and extranodal lymphoid tissues, accounting for 3%-4% of all malignant tumors in my country and the leading hematologic malignancy. The two most common subtypes of B-cell lymphoma are diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive B-NHL, accounting for approximately 30% of newly diagnosed NHL cases. Despite the standard first-line therapy, R-CHOP, 10% of initially treated DLBCL cases remain refractory, and 40% of those who respond to initial treatment relapse after 2-5 years. 1. Autologous hematopoietic stem cell transplantation (ASCT) after achieving a complete remission (CR) or partial remission (PR) after chemotherapy is currently the best treatment option for relapsed or refractory DLBCL, but the two-year disease-free survival rate after transplantation remains less than 15%. Follicular lymphoma (FL) is the most common indolent B-NHL, accounting for approximately 22% of newly diagnosed NHL cases. Although the disease progresses slowly, it is generally considered incurable, with an expected median survival of approximately 10 years. Within 10 years, approximately 15-28% of FL will transform into aggressive lymphoma.

[0003] Current CAR-T therapies for B-cell lymphomas are primarily based on the CD19 target. Four CD19-CAR-Ts are already on the market. Their primary indications are B-cell lymphoma (DLBCL) and follicular lymphoma (FL), with ORRs of approximately 70%-80% and CRs of 50-60%. Although CD19 CAR-T therapy for these two major types of B-NHL has achieved higher remission rates than standard therapy, follow-up studies have shown that some patients relapse due to antigen escape—that is, mutations or deletions in the CD19 protein expressed on the tumor cell surface that turn negative, rendering CAR-T cell therapy ineffective—accounting for approximately 10%-20% of patients. Therefore, there is an urgent need to explore new targets for CAR-T cell immunotherapy for patients with relapsed or refractory disease.

[0004] BAFF-R (B-cell activating factor receptor) is a member of the tumor necrosis factor (TNF) receptor superfamily. BAFF-R is expressed on the surface of peripheral B cells, excluding plasma cells and centroblasts located in the dark zone of germinal centers. Researchers examining 116 B-cell lymphoma clinical samples found that BAFF-R was highly expressed on various B lymphoma cells, including chronic lymphocytic leukemia (CLL) (21 / 21, 100%), mantle cell lymphoma (MCL) (7 / 7, 100%), FL (13 / 16, 81%), and DLBCL (14 / 18, 78%). This suggests that BAFF-R may serve as a novel therapeutic target for B-cell tumors, in addition to CD19.

[0005] 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.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to provide fully human single-domain antibodies, monoclonal antibodies, bispecific antibodies, CAR-T cells and their applications targeting BAFF-R.

[0008] The purpose of the present invention is to provide an antibody targeting BAFF-R, a CAR and a host cell expressing a CAR targeting BAFF-R, as well as their preparation methods and uses.

[0009] Another 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 its biological activity, and a method for evaluating its safety.

[0010] In a first aspect of the present invention, a single-domain antibody targeting B-cell activating factor receptor (BAFF-R) is provided, wherein the VHH chain of the single-domain antibody comprises the following complementarity determining regions (CDRs): CDR1 shown in SEQ ID NO.27, CDR2 shown in SEQ ID NO.28, and CDR3 shown in SEQ ID NO.29;

[0011] Any of the above amino acid sequences also includes derivative sequences that are optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain BAFF-R binding affinity.

[0012] In another preferred embodiment, the VHH chain of the single-domain antibody further includes a framework region (FR).

[0013] In another preferred embodiment, the framework region FR is of human, mouse, rabbit or camel origin.

[0014] In another preferred embodiment, the framework region FR includes a human FR region, a mouse FR region or a camel FR region.

[0015] In another preferred example, the VHH chain of the single-domain antibody targeting BAFF-R has an amino acid sequence as shown in SEQ ID NO. 20, 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.

[0016] In another preferred embodiment, the single-domain antibody targeting BAFF-R is a fully human antibody.

[0017] In a second aspect of the present invention, an antibody targeting B cell activating factor receptor (BAFF-R) is provided, wherein the heavy chain variable region of the antibody comprises the following complementarity determining regions (CDRs): CDR1 shown in SEQ ID NO.27, CDR2 shown in SEQ ID NO.28, and CDR3 shown in SEQ ID NO.29; or

[0018] The light chain variable region of the antibody comprises the following complementary determining regions (CDRs): CDR1 shown in SEQ ID NO. 36, CDR2 shown in AAS, and CDR3 shown in SEQ ID NO. 37; the heavy chain variable region of the antibody comprises the following complementary determining regions (CDRs): CDR1 shown in SEQ ID NO. 38, CDR2 shown in SEQ ID NO. 39, and CDR3 shown in SEQ ID NO. 40;

[0019] Any of the above amino acid sequences also includes derivative sequences that are optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain BAFF-R binding affinity.

[0020] In another preferred embodiment, the light chain variable region and / or heavy chain variable region further includes a framework region (FR).

[0021] In another preferred embodiment, the framework region FR is of human, mouse, rabbit or camel origin.

[0022] In another preferred embodiment, the framework region FR includes a human FR region, a mouse FR region or a camel FR region.

[0023] In another preferred embodiment, the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO. 25, 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.

[0024] In another preferred example, the light chain variable region has an amino acid sequence as shown in SEQ ID NO.30, 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 thereto, and the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO.32, 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 thereto.

[0025] In another preferred embodiment, the antibody is a single domain antibody.

[0026] In another preferred embodiment, the antibody is a monomer, a bivalent antibody, and / or a multivalent antibody.

[0027] In another preferred embodiment, the antibody is a fully human antibody, an animal antibody, a humanized antibody, a fully human chimeric antibody or a chimeric antigen receptor antibody (CAR).

[0028] In another preferred embodiment, the CDR region of the humanized antibody comprises 1, 2, or 3 amino acid changes.

[0029] In another preferred embodiment, the animal is a non-human mammal, preferably a mouse, sheep, rabbit, or camel.

[0030] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0031] In another preferred embodiment, the antibody is a monoclonal antibody.

[0032] In another preferred embodiment, the antibody is a fully human monoclonal antibody.

[0033] 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%, and more preferably 10%.

[0034] 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.

[0035] 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%.

[0036] In a third aspect of the present invention, a chimeric antigen receptor is provided, wherein the antigen binding domain of the chimeric antigen receptor comprises an antigen binding fragment that targets BAFF-R, and the heavy chain variable region of the antigen binding fragment comprises the following complementarity determining regions (CDRs): CDR1 set forth in SEQ ID NO. 27, CDR2 set forth in SEQ ID NO. 28, and CDR3 set forth in SEQ ID NO. 29;

[0037] Alternatively, the light chain variable region of the antigen-binding fragment comprises the following complementarity determining regions CDR: CDR1 shown in SEQ ID NO.36, CDR2 shown in AAS, and CDR3 shown in SEQ ID NO.37, and the heavy chain variable region of the antibody comprises the following complementarity determining regions CDR: CDR1 shown in SEQ ID NO.38, CDR2 shown in SEQ ID NO.39, and CDR3 shown in SEQ ID NO.40.

[0038] In another preferred embodiment, the antigen-binding fragment is the heavy chain variable region of a single-domain antibody.

[0039] In another preferred embodiment, the antigen-binding fragment has the amino acid sequence shown in SEQ ID NO. 25, 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.

[0040] In another preferred embodiment, the antigen-binding domain of the chimeric antigen receptor comprises one or more antigen-binding fragments.

[0041] In another preferred embodiment, the antigen-binding fragment is a single-chain antibody.

[0042] In another preferred embodiment, the antigen-binding fragment comprises a light chain variable region, a connecting peptide and a heavy chain variable region in sequence from segment N to segment C.

[0043] In another preferred embodiment, the antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO. 34, 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.

[0044] In another preferred embodiment, the CAR further comprises a transmembrane region derived from CD8a and / or an intracellular domain derived from CD3ζ.

[0045] In another preferred embodiment, the CAR comprises an intracellular co-stimulatory region derived from CD28.

[0046] In another preferred embodiment, the CAR comprises a co-stimulatory molecule derived from 4-1BB.

[0047] In another preferred embodiment, the structure of the chimeric antigen receptor is shown in Formula I below:

[0048] LVH-TM-C-CD3ζ (I)

[0049] Where,

[0050] Each "-" is independently a connecting peptide or a peptide bond;

[0051] L is the signal peptide sequence;

[0052] V is the antigen binding domain;

[0053] H is the hinge region;

[0054] TM is the transmembrane domain;

[0055] C is a co-stimulatory signal molecule;

[0056] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

[0057] In another preferred embodiment, the L is a signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

[0058] In another preferred embodiment, the L is a signal peptide derived from CD8.

[0059] In another preferred embodiment, the amino acid sequence of the CD8-derived signal peptide is shown as SEQ ID NO.2.

[0060] In another preferred embodiment, the anti-BAFF-R single-domain antibody includes fully human single-domain antibodies, humanized single-domain antibodies and camel-derived single-domain antibodies, preferably fully human single-domain antibodies.

[0061] In another preferred embodiment, the V is a fully human single-domain antibody.

[0062] In another preferred embodiment, the H is a hinge region derived from a protein selected from the group consisting of Fc, CD8, CD28, CD137, or a combination thereof.

[0063] In another preferred embodiment, the H is a hinge region derived from CD8, and the amino acid sequence is shown in SEQ ID NO.4.

[0064] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of ICOS, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0065] In another preferred embodiment, the TM is a transmembrane region derived from CD8.

[0066] In another preferred embodiment, the amino acid sequence of the transmembrane region derived from CD8 is shown as SEQ ID NO.6.

[0067] In another preferred embodiment, the C is a co-stimulatory signal molecule of a protein selected from the following group: ICOS, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.

[0068] In another preferred embodiment, the C is a co-stimulatory signal molecule derived from ICOS and / or 4-1BB.

[0069] In another preferred embodiment, the amino acid sequence of the 4-1BB-derived costimulatory signal molecule is shown in SEQ ID NO.8.

[0070] In another preferred embodiment, the amino acid sequence of CD3ζ is shown as SEQ ID NO.10.

[0071] In another preferred embodiment, the CAR (preferably the C-terminus) further includes a cell suicide element.

[0072] In another preferred embodiment, the CAR is connected to the cell suicide element through a self-cleavage element.

[0073] In another preferred embodiment, the cell suicide element is connected to the CD3ζ of the CAR via T2A or P2A.

[0074] In another preferred embodiment, the cell suicide element is selected from the group consisting of HSV-TK, iCasp9, ΔCD20, mTMPK, ΔCD19, RQR8, EGFRt, or a combination thereof.

[0075] In another preferred embodiment, the cell suicide element is tEGFR.

[0076] In another preferred embodiment, the amino acid sequence of tEGFR is shown as SEQ ID NO.16.

[0077] In another preferred example, the amino acid sequence of T2A is shown as SEQ ID NO.18.

[0078] In another preferred example, the amino acid sequence of the CAR is shown in SEQ ID NO.20.

[0079] In a fourth aspect of the present invention, a multispecific antibody is provided, 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.

[0080] 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, CD11a, 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.

[0081] In another preferred embodiment, the second antigen binding region is a fully human antibody, an animal antibody, a humanized antibody or a chimeric antibody.

[0082] In another preferred embodiment, the multispecific antibody comprises one or more second antigen binding regions.

[0083] In another preferred embodiment, the multispecific antibody contains or does not contain the Fc segment of an antibody.

[0084] In another preferred embodiment, the Fc segment of the antibody is of IgG1 subtype or IgG4 subtype.

[0085] In another preferred embodiment, the Fc segment of the antibody comprises L234A / L235A mutation or S228P / F234A / L235A mutation.

[0086] In another preferred embodiment, the Fc segment of the antibody comprises a knobs-into-holes structure; preferably, the knobs side has a S354C / T366W combined mutation, and the holes side has a Y349C / T366S / L368A / Y407V combined mutation.

[0087] In another preferred embodiment, the antigen binding region is an antibody or 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; and (vi) dAb fragment.

[0088] In a fifth aspect of the present invention, a bispecific antibody is provided, comprising:

[0089] targets the first antigen binding region of BAFF-R; and

[0090] Targeting the second antigen binding region of CD3; wherein,

[0091] The first antigen-binding region targeting BAFF-R comprises the following complementarity determining regions (CDRs): CDR1 shown in SEQ ID NO. 27, CDR2 shown in SEQ ID NO. 28, and CDR3 shown in SEQ ID NO. 29.

[0092] In another preferred example, the first antigen binding region targeting BAFF-R has an amino acid sequence as shown in SEQ ID NO. 25 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.

[0093] 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:

[0094] The CD3-targeting heavy chain variable region comprises the following complementarity determining regions (CDRs): CDR1 shown in SEQ ID NO.79, CDR2 shown in SEQ ID NO.80, and CDR3 shown in SEQ ID NO.81;

[0095] The CD3-targeting light chain variable region comprises the following complementarity determining regions (CDRs): CDR1 shown in SEQ ID NO.82, CDR2 with an amino acid sequence of GTN, and CDR3 shown in SEQ ID NO.83;

[0096] Any of the above amino acid sequences also includes derivative sequences that are optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and are capable of retaining CD3 binding affinity.

[0097] In another preferred embodiment, the amino acid sequence of the heavy chain variable region targeting CD3 is shown as SEQ ID NO.45, and the amino acid sequence of the light chain variable region targeting CD3 is shown as SEQ ID NO.47; or

[0098] The amino acid sequence of the heavy chain variable region targeting CD3 is shown in SEQ ID NO.65, and the amino acid sequence of the light chain variable region targeting CD3 is shown in SEQ ID NO.67.

[0099] In another preferred embodiment, the nucleotide sequence encoding the heavy chain variable region targeting CD3 is shown as SEQ ID NO.46, and the nucleotide sequence encoding the light chain variable region targeting CD3 is shown as SEQ ID NO.48; or

[0100] The nucleotide sequence encoding the heavy chain variable region targeting CD3 is shown in SEQ ID NO.66, and the nucleotide sequence encoding the light chain variable region targeting CD3 is shown in SEQ ID NO.68.

[0101] 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.

[0102] In another preferred embodiment, the second antigen binding region is an antibody or 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.

[0103] In another preferred example, the amino acid sequence of the CD3-targeting Fab fragment is shown in SEQ ID NO.49.

[0104] In another preferred embodiment, the nucleotide sequence encoding the CD3-targeting Fab fragment is shown as SEQ ID NO.50.

[0105] In another preferred embodiment, the bispecific antibody is a symmetric or asymmetric dimer.

[0106] In another preferred embodiment, the first antigen binding region is connected to the N segment or C segment of the second antigen binding region.

[0107] In another preferred embodiment, the first antigen binding region is connected to the N segment or C segment of the Fab fragment encoding the CD3-targeting protein.

[0108] 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, it is connected to the N segment of the heavy chain of the Fab fragment encoding targeting CD3.

[0109] In another preferred embodiment, the bispecific antibody further comprises an antibody Fc segment.

[0110] 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.

[0111] In another preferred embodiment, the bispecific antibody has the following structure:

[0112] (a) Two heavy chains, the first heavy chain comprising, from N-terminus to C-terminus, an anti-BAFF-R VH region and a human IgG Fc region; the second heavy chain comprising, from N-terminus to C-terminus, an anti-CD3 VH region, an antibody heavy chain constant region (CH1), and a human IgG Fc region;

[0113] (b) a light chain, comprising, from N-terminus to C-terminus, an anti-CD3 VL, an antibody light chain constant region (CL),

[0114] Wherein, a disulfide bond exists between the antibody heavy chain constant region (CH1) and the antibody light chain constant region (CL).

[0115] 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).

[0116] In another preferred embodiment, the anti-BAFF-R VH and the first antigen binding region targeting BAFF-R have the same meaning.

[0117] In another preferred embodiment, the antibody heavy chain constant region (CH1) is connected to the human IgG Fc region via a hinge region.

[0118] In another preferred embodiment, a disulfide bond exists between the two human IgG Fc regions contained in the bispecific antibody.

[0119] 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 the IgG4 subtype, and more preferably, contains S228P / F234A / L235A mutations.

[0120] In another preferred embodiment, the Fc segment of the antibody comprises a knobs-into-holes structure; preferably, the knobs side has a S354C / T366W combined mutation, and the holes side has a Y349C / T366S / L368A / Y407V combined mutation.

[0121] In another preferred embodiment, the bispecific antibody has the following structure:

[0122] (a) two heavy chains, wherein the first heavy chain comprises, from N-terminus to C-terminus, an anti-BAFF-R VH, an anti-CD3 VH, an antibody heavy chain constant region (CH1), and a human IgG Fc region; the second heavy chain comprises, from N-terminus to C-terminus, an anti-BAFF-R VH and a human IgG Fc region;

[0123] (b) a light chain, comprising, from N-terminus to C-terminus, an anti-CD3 VL, an antibody light chain constant region (CL),

[0124] Wherein, a disulfide bond exists between the antibody heavy chain constant region (CH1) and the antibody light chain constant region (CL).

[0125] 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).

[0126] In another preferred embodiment, the bispecific antibody has the following structure:

[0127] (a) two identical heavy chains, each comprising, from N-terminus to C-terminus, an anti-BAFF-R VH, an anti-CD3 VH, an antibody heavy chain constant region (CH1), and a human IgG Fc region;

[0128] (b) two identical light chains, each comprising, from N-terminus to C-terminus, an anti-CD3 VL, an antibody light chain constant region (CL),

[0129] Wherein, a disulfide bond exists between the antibody heavy chain constant region (CH1) and the antibody light chain constant region (CL).

[0130] 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).

[0131] In another preferred embodiment, the bispecific antibody, wherein,

[0132] The heavy chain comprises an amino acid sequence as shown in SEQ ID NO. 73 or 51, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto,

[0133] The light chain comprises the amino acid sequence shown in SEQ ID NO.71 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.

[0134] In another preferred embodiment, the bispecific antibody, wherein,

[0135] The heavy chain comprises an amino acid sequence as shown in SEQ ID NO. 75 or 51, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto,

[0136] The light chain comprises the amino acid sequence shown in SEQ ID NO.71 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.

[0137] In another preferred embodiment, the bispecific antibody, wherein,

[0138] The heavy chain comprises the amino acid sequence shown in SEQ ID NO. 77 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,

[0139] The light chain comprises the amino acid sequence shown in SEQ ID NO.71 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.

[0140] In a sixth aspect, the present invention provides a recombinant protein having:

[0141] (i) 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, the chimeric antigen receptor according to the third aspect of the present invention, the multispecific antibody according to the fourth aspect of the present invention, or the bispecific antibody according to the fifth aspect of the present invention; and

[0142] (ii) optionally a polypeptide molecule or fragment having therapeutic function; and / or

[0143] (iii) Optional functional domains that enhance the physicochemical properties or druggability of the protein.

[0144] In a seventh aspect, the present invention provides a nucleic acid molecule encoding a polypeptide selected from the group consisting of:

[0145] (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 chimeric antigen receptor as described in the third aspect of the present invention, the multispecific antibody as described in the fourth aspect of the present invention, or the bispecific antibody as described in the fifth aspect of the present invention; or

[0146] (2) The recombinant protein according to the sixth aspect of the present invention.

[0147] In another preferred embodiment, the nucleotide sequence of the nucleic acid molecule encoding the chimeric antigen receptor according to the second aspect of the present invention is shown as SEQ ID NO.26.

[0148] In another preferred embodiment, the polynucleotide includes RNA, DNA or cDNA.

[0149] The eighth aspect of the present invention provides a vector, wherein the vector contains the nucleic acid molecule described in the seventh aspect of the present invention.

[0150] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, or a combination thereof.

[0151] In another preferred embodiment, the vector is a lentiviral vector.

[0152] The ninth aspect of the present invention provides a host cell, wherein the host cell contains the vector described in the eighth aspect of the present invention, or the exogenous nucleic acid molecule described in the seventh aspect of the present invention integrated into the chromosome.

[0153] The tenth aspect of the present invention provides an engineered immune cell, which contains the vector described in the eighth aspect of the present invention, or has the exogenous nucleic acid molecule described in the seventh aspect of the present invention integrated into the chromosome, or expresses the chimeric antigen receptor described in the third aspect of the present invention.

[0154] In another preferred embodiment, the immune cells are T cells or NK cells.

[0155] In another preferred embodiment, the TCR gene expression of the T cells is silenced.

[0156] In another preferred embodiment, the immune cells express exogenous cell suicide elements.

[0157] In another preferred embodiment, the engineered immune cells are selected from the following group:

[0158] (i) chimeric antigen receptor T cells (CAR-T cells); or

[0159] (ii) Chimeric antigen receptor NK cells (CAR-NK cells).

[0160] In another preferred embodiment, CAR and cell suicide elements are co-expressed in the engineered immune cells.

[0161] The eleventh aspect of the present invention provides an immunoconjugate, wherein the immunoconjugate comprises:

[0162] (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 fourth aspect of the present invention, or a bispecific antibody as described in the fifth aspect of the present invention; and

[0163] (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.

[0164] In another preferred embodiment, the immunoconjugate is a single domain antibody drug conjugate.

[0165] In another preferred embodiment, the antibody portion and the coupling portion are coupled via a chemical bond or a linker.

[0166] In another preferred embodiment, the coupling moiety is a chemical label or a biological label.

[0167] In another preferred embodiment, the chemical label is an isotope, an immunotoxin and / or a chemical drug.

[0168] In another preferred embodiment, the biomarker is biotin, avidin or an enzyme label.

[0169] In another preferred embodiment, the coupling moiety is a drug or a toxin.

[0170] In another preferred embodiment, the drug is a cytotoxic drug.

[0171] 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.

[0172] 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.

[0173] In another preferred embodiment, the toxin is selected from the following group:

[0174] auristatins (e.g., auristatin E, auristatin F, MMAE, and MMAF), chlortetracycline, maytansinoids, ricin, ricin A-chain, combretastatin, duocarmycin, dolastatin, doxorubicin, 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, α-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, sapaonaria officinalis officinalis) inhibitors, glucocorticoids, or a combination thereof.

[0175] In another preferred embodiment, the coupling moiety is a detectable label.

[0176] In another preferred embodiment, the detectable marker comprises a radionuclide, and the radionuclide comprises:

[0177] (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

[0178] (ii) therapeutic isotopes 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.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] In another preferred embodiment, the detection is in vivo detection or in vitro detection.

[0183] In another preferred embodiment, the immunoconjugate is used for diagnosing and / or treating tumors expressing BAFF-R protein.

[0184] In another preferred embodiment, the immunoconjugate has the following molecular formula:

[0185] in:

[0186] nAbs are single-domain antibodies targeting BAFF-R, antibodies targeting BAFF-R, or multispecific antibodies;

[0187] LU is the linker (also called connector);

[0188] D is for medicine;

[0189] Furthermore, the subscript p is a value selected from 1-10.

[0190] 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 comprises 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).

[0191] In another preferred embodiment, the antibody portion is covalently bound to the linker by reacting with a portion selected from the group consisting of maleimidocaproyl (MC), maleimide (MAL), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (SMCC), and the like.

[0192] In another preferred embodiment, D is selected from the following group of compounds having anti-tumor activity:

[0193] (i) Tubulin inhibitors, such as maytansine derivatives (DM1, DM4), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF);

[0194] (ii) DNA-acting toxins, such as duocarmycin and pyrrolobenzodiazepine (PBD);

[0195] (iii) Topoisomerase inhibitors, such as camptothecin, SN38, exitecan, and Dxd.

[0196] The twelfth aspect of the present invention provides a pharmaceutical composition or preparation comprising:

[0197] (i) 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, the multispecific antibody according to the fourth aspect of the present invention, the bispecific antibody according to the fifth aspect of the present invention, the recombinant protein according to the sixth aspect of the present invention, the engineered immune cell according to the tenth aspect of the present invention, or the immunoconjugate according to the eleventh aspect of the present invention; and

[0198] (ii) a pharmaceutically acceptable carrier.

[0199] In another preferred embodiment, the pharmaceutical composition includes a single drug, a compound drug, or a synergistic drug.

[0200] In another preferred embodiment, the pharmaceutical composition further comprises other biologically active substances, such as drugs for treating tumors.

[0201] 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.

[0202] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of liquid, solid, and gel. In another preferred embodiment, the preparation is a liquid preparation.

[0203] In another preferred embodiment, the preparation is in the form of an injection.

[0204] The thirteenth 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 fourth aspect of the present invention, a bispecific antibody as described in the fifth aspect of the present invention, a recombinant protein as described in the sixth aspect of the present invention, an engineered immune cell as described in the tenth aspect of the present invention, or an immunoconjugate as described in the eleventh 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 BAFF-R-related diseases.

[0205] 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.

[0206] In another preferred embodiment, the BAFF-R related disease includes BAFF-R highly expressed tumors.

[0207] 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.

[0208] In another preferred embodiment, the tumor is a blood tumor.

[0209] In another preferred embodiment, the tumor is a plasma cell malignancy or a B cell malignancy.

[0210] A fourteenth aspect of the present invention provides a method for preparing an engineered immune cell, wherein the engineered immune cell expresses the chimeric antigen receptor according to the third aspect of the present invention, comprising the following steps:

[0211] The nucleic acid molecule described in the seventh aspect of the present invention or the vector described in the eighth aspect of the present invention is transduced into immune cells to obtain the engineered immune cells.

[0212] In another preferred embodiment, the immune cells are T cells or NK cells.

[0213] In the fifteenth aspect of the present invention, a kit is provided, which is used to prepare the engineered immune cells described in the tenth aspect of the present invention, and the kit contains a container, and the nucleic acid molecule described in the seventh aspect of the present invention or the vector described in the eighth aspect of the present invention located in the container.

[0214] In a sixteenth aspect, the present invention provides a method for detecting BAFF-R in a sample (including diagnostic or non-diagnostic) in vitro, the method comprising the steps of:

[0215] (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 eleventh aspect of the present invention;

[0216] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of BAFF-R in the sample.

[0217] In another preferred embodiment, the detection includes diagnostic or non-diagnostic.

[0218] A seventeenth aspect of the present invention provides a method for preparing a recombinant polypeptide, the method comprising:

[0219] (a) culturing the host cell according to the ninth aspect of the present invention under conditions suitable for expression; and

[0220] (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 fourth aspect of the present invention, a bispecific antibody as described in the fifth aspect of the present invention, or a recombinant protein as described in the sixth aspect of the present invention.

[0221] In the eighteenth 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, an antibody targeting BAFF-R as described in the second aspect of the present invention, a multispecific antibody as described in the fourth aspect of the present invention, a bispecific antibody as described in the fifth aspect of the present invention, a recombinant protein as described in the sixth aspect of the present invention, an engineered immune cell as described in the tenth aspect of the present invention, or an immunoconjugate as described in the eleventh aspect of the present invention, or a pharmaceutical composition or preparation as described in the twelfth aspect of the present invention, or a combination thereof.

[0222] In another preferred embodiment, the method further comprises: administering other drugs or treatment methods to a subject in need for combined treatment.

[0223] In another preferred embodiment, the other drugs or treatment methods include: anti-tumor immunotherapy drugs, tumor-targeted drugs, tumor chemotherapy drugs, and tumor radiotherapy.

[0224] In another preferred embodiment, the anti-tumor immunotherapy drugs include PD-1 monoclonal antibody and PD-L1 monoclonal antibody.

[0225] 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 described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0226] FIG1 shows the general process of screening specific antibodies targeting BAFF-R from a phage antibody library according to the present invention.

[0227] FIG2 shows the results of enzyme-linked immunosorbent assay (ELISA) of some of the selected phage monoclones with target antigen and control antigen.

[0228] FIG3 shows the flow cytometric analysis results of the binding of some phage monoclones to K562-BAFF-R and K562 cells.

[0229] Figures 4A-B show the flow cytometric analysis results (peak profiles) of the screened phage monoclonal binding to various BAFF-R-positive and -negative cell lines. Figure 4A shows the flow cytometric peak profiles of Clone 1, 5, and 14 binding to various cell lines, with the Negative Control being the negative control phage antibody clone. Figure 4B shows the flow cytometric peak profiles of Clone 77 and 80 binding to various cell lines, with VAY736 being the positive control phage antibody clone.

[0230] Figure 5 shows the results of enzyme-linked immunosorbent assays (ELISAs) using the screened phage monoclonal clones with BAFF-R antigen proteins from various companies and non-related antigens. Negative Controls are negative control phage antibody clones. Anti-M13 phage mouse Ab / anti-mouse HRP Ab are negative controls with only the primary antibody (Anti-M13 phage mouse Ab) and secondary antibody (Anti-mouse HRP Ab) added without phage; anti-mouse HRP Ab is a negative control with only the secondary antibody (Anti-mouse HRP Ab) added. VAY736 Ab / anti-human IgG HRP Ab are positive antibody controls for the target antigen (BAFF-R-his-Bio); and anti-human IgG HRP and anti-his HRP are positive antibody controls for detecting antigen tags. In FIG5 , the bar graphs corresponding to each test antibody and control group represent the test results of the reagents KACTUS-BAFF-R-his Bio, ACRO-BAFF-R-Fc, KACTUS-CD19-Fc, ACRO-CD70-his, and SA from left to right.

[0231] Figure 6 shows a schematic diagram of the structure of the BAFF-R CAR vector. CAR includes an extracellular signal peptide (SP), a ScFv (VL-linker-VH) or a single domain (V HH ), hinge region and transmembrane region between the cell membrane and extracellular binding region (CD8a hinge+TM), 4-1BB co-stimulatory molecule, CD3ζ intracellular domain, T2A self-cleaving peptide, truncated tEGFR.

[0232] FIG7 shows the expression of tEGFR in CAR-T cells of different clones.

[0233] Figure 8 shows the killing results of CAR-T cells of different clones against various target cells JEKO1-luc (Figure A), MEC1-luc (Figure B), RS4-luc (Figure C), NALM6-luc (Figure D), and MOLT4-luc (Figure E).

[0234] FIG9 shows the results of CD107a degranulation of CAR-T cells of different clones by tumor cells from different tissues.

[0235] Figure 10 shows the antitumor efficacy of CAR-T cells from different clones in JEKO1-luc tumor-bearing NCG mice. Panel A shows the change in photon count over time after CAR-T cell infusion, and Panel B shows the survival rate of mice after CAR-T cell infusion. Panel C shows the change in the T cell-to-lymphocyte ratio in mice after CAR-T cell infusion.

[0236] FIG11 shows IVIS images of CAR-T cells corresponding to different clones at different time points after transfusion into JEKO1-luc tumor-bearing NCG mice.

[0237] Figure 12 shows a schematic diagram of the α-BAFF-R × α-CD3 bispecific antibody 1+1 structure; Figure 12A shows a schematic diagram of the α-BAFF-R scFab × α-CD3 scFab bispecific antibody 1+1 structure; Figure 12B shows a schematic diagram of the α-BAFF-RVH × α-CD3 scFab bispecific antibody 1+1 structure.

[0238] FIG13 shows the activation of TDCC reporter cells mediated by K562-BAFF-R using α-BAFF-R×α-CD3 bispecific antibody.

[0239] FIG14 shows the activation of TDCC reporter cells by K562 mediated by α-BAFF-R×α-CD3 bispecific antibody.

[0240] FIG15 shows that α-BAFF-R×α-CD3 bispecific antibody mediates T cell killing of Nalm 6 cells.

[0241] FIG16 shows that α-BAFF-R×α-CD3 bispecific antibody mediates T cell killing of K562 cells.

[0242] FIG17 shows an amino acid comparison analysis of the extracellular regions of human, monkey and mouse BAFF-R.

[0243] FIG18 shows the structural schematic diagrams of different configurations of α-BAFF-R VH×α-CD3 Fab bispecific antibodies.

[0244] FIG18A shows a schematic diagram of the 1+1 structure of the α-BAFF-R VH×α-CD3 Fab bispecific antibody.

[0245] FIG18B shows a schematic diagram of the 2+1 structure of the α-BAFF-R VH×α-CD3 Fab bispecific antibody.

[0246] FIG18C shows a schematic diagram of the 2+2 structure of the α-BAFF-R VH×α-CD3 Fab bispecific antibody.

[0247] FIG19 shows that the α-BAFF-R VH×α-CD3 Fab bispecific antibody mediates the activation of TDCC reporter cells by K562-BAFF-R.

[0248] FIG20 shows that the α-BAFF-R VH×α-CD3 Fab bispecific antibody mediates the activation of TDCC reporter cells by Nalm6.

[0249] FIG21 shows that the α-BAFF-R VH×α-CD3 Fab bispecific antibody mediates the activation of TDCC reporter cells by Mec-1.

[0250] FIG22 shows the activation of TDCC reporter cells by Jeko-1 mediated by α-BAFF-R VH×α-CD3 Fab bispecific antibody.

[0251] FIG23 shows that the α-BAFF-R VH×α-CD3 Fab bispecific antibody mediates the activation of TDCC reporter cells by K562.

[0252] FIG24 shows that α-BAFF-R VH×α-CD3 Fab bispecific antibody mediates PBMC cells (Donor 301C) killing of Jeko-1 cells for 24 hours.

[0253] FIG25 shows that α-BAFF-R VH×α-CD3 Fab bispecific antibody mediates PBMC cells (Donor 301C) killing of Jeko-1 cells for 48 hours.

[0254] FIG26 shows that α-BAFF-R VH×α-CD3 Fab bispecific antibody mediates PBMC cells (Donor 605C) killing of Jeko-1 cells for 24 hours.

[0255] FIG27 shows that α-BAFF-R VH×α-CD3 Fab bispecific antibody mediates PBMC cells (Donor 605C) killing of Jeko-1 cells for 48 hours.

[0256] FIG28 shows that the α-BAFF-R VH×α-CD3 Fab bispecific antibody promotes the secretion of cytokine IFN-γ by PBMC cells (Donor 303C).

[0257] FIG28A shows the analysis of IFN-γ secretion by PBMCs after co-incubation of the bispecific antibody, Jeko-1, and PBMCs (Donor 303C) for 24 hours.

[0258] FIG28B shows the bispecific antibody, Jeko-1, and PBMC cells (Donor 303C) co-incubated for 48 hours, and the IFN-γ secretion of PBMC cytokines was analyzed.

[0259] FIG28C shows the analysis of IFN-γ secretion by PBMCs after co-incubation of the bispecific antibody and PBMCs (Donor 303C) for 24 hours.

[0260] FIG28D shows the bispecific antibody and PBMC cells (Donor 303C) were co-incubated for 48 hours, and the PBMC cytokine IFN-γ secretion was analyzed.

[0261] FIG29 shows that the α-BAFF-R VH×α-CD3 Fab bispecific antibody promotes the secretion of cytokine IFN-γ by PBMC cells (Donor 605C).

[0262] FIG29A shows the analysis of IFN-γ secretion by PBMCs after co-incubation of bispecific antibody, Jeko-1 and PBMCs (Donor 605C) for 24 hours.

[0263] FIG29B shows the bispecific antibody, Jeko-1, and PBMC cells (Donor 605C) co-incubated for 48 hours, and the IFN-γ secretion of PBMC cytokines was analyzed.

[0264] FIG29C shows the bispecific antibody and PBMC cells (Donor 605C) were co-incubated for 24 hours, and the PBMC cytokine IFN-γ secretion was analyzed.

[0265] FIG29D shows the bispecific antibody and PBMC cells (Donor 605C) were co-incubated for 48 hours, and the PBMC cytokine IFN-γ secretion was analyzed.

[0266] FIG30 shows that the α-BAFF-R VH×α-CD3 Fab bispecific antibody promotes the secretion of cytokine IL-2 by PBMC cells (Donor 303C).

[0267] FIG30A shows the bispecific antibody, Jeko-1, and PBMC cells (Donor 303C) co-incubated for 24 hours, and the cytokine IL-2 secretion analysis of PBMCs.

[0268] FIG30B shows the bispecific antibody, Jeko-1, and PBMC cells (Donor 303C) co-incubated for 48 hours, and the cytokine IL-2 secretion analysis of PBMCs.

[0269] FIG30C shows the bispecific antibody and PBMC cells (Donor 303C) were co-incubated for 24 hours, and the PBMC cytokine IL-2 secretion was analyzed.

[0270] FIG30D shows the bispecific antibody and PBMC cells (Donor 303C) were co-incubated for 48 hours, and the PBMC cytokine IL-2 secretion was analyzed.

[0271] FIG31 shows that the α-BAFF-R VH×α-CD3 Fab bispecific antibody promotes the secretion of cytokine IL-2 by PBMC cells (Donor 605C).

[0272] FIG31A shows the bispecific antibody, Jeko-1 and PBMC cells (Donor 605C) co-incubated for 24 hours, and the cytokine IL-2 secretion analysis of PBMC was performed.

[0273] FIG31B shows the bispecific antibody, Jeko-1, and PBMC cells (Donor 605C) co-incubated for 48 hours, and the cytokine IL-2 secretion analysis of PBMC was performed.

[0274] FIG31C shows the bispecific antibody and PBMC cells (Donor 605C) were co-incubated for 24 hours, and the PBMC cytokine IL-2 secretion was analyzed.

[0275] FIG31D shows the bispecific antibody and PBMC cells (Donor 605C) were co-incubated for 48 hours, and the PBMC cytokine IL-2 secretion was analyzed.

[0276] FIG32 shows that the α-BAFF-R VH×α-CD3 Fab bispecific antibody promotes the secretion of cytokine TNF-α by PBMC cells (Donor 303C).

[0277] FIG32A shows the analysis of TNF-α secretion by PBMCs after co-incubation of the bispecific antibody, Jeko-1, and PBMCs (Donor 303C) for 24 hours.

[0278] FIG32B shows the analysis of TNF-α secretion by PBMCs after co-incubation of the bispecific antibody, Jeko-1, and PBMCs (Donor 303C) for 48 hours.

[0279] FIG32C shows the analysis of TNF-α secretion by PBMCs after co-incubation of the bispecific antibody and PBMCs (Donor 303C) for 24 hours.

[0280] FIG32D shows the analysis of TNF-α secretion by PBMCs after co-incubation of the bispecific antibody and PBMCs (Donor 303C) for 48 hours.

[0281] FIG33 shows that the α-BAFF-R VH×α-CD3 Fab bispecific antibody promotes the secretion of cytokine TNF-α by PBMC cells (Donor 605C).

[0282] FIG33A shows the analysis of TNF-α secretion by PBMCs after co-incubation of the bispecific antibody, Jeko-1, and PBMCs (Donor 605C) for 24 hours.

[0283] FIG33B shows the bispecific antibody, Jeko-1, and PBMC cells (Donor 605C) co-incubated for 48 hours, and the TNF-α secretion of PBMC cytokines was analyzed.

[0284] FIG33C shows the analysis of TNF-α secretion by PBMCs after the bispecific antibody and PBMCs (Donor 605C) were co-incubated for 24 hours.

[0285] FIG33D shows the analysis of TNF-α secretion by PBMCs after the bispecific antibody and PBMCs (Donor 605C) were co-incubated for 48 hours.

[0286] FIG34 shows that the α-BAFF-R VH×α-CD3 Fab bispecific antibody promotes the secretion of cytokine IL-6 by PBMC cells (Donor 303C).

[0287] FIG34A shows the bispecific antibody, Jeko-1, and PBMC cells (Donor 303C) co-incubated for 24 hours, and the cytokine IL-6 secretion analysis of PBMCs.

[0288] FIG34B shows the bispecific antibody, Jeko-1, and PBMC cells (Donor 303C) co-incubated for 48 hours, and the cytokine IL-6 secretion analysis of PBMCs.

[0289] Figure 34C shows the bispecific antibody and PBMC cells (Donor 303C) were co-incubated for 24 hours, and the PBMC cytokine IL-6 secretion was analyzed.

[0290] FIG34D shows the bispecific antibody and PBMC cells (Donor 303C) were co-incubated for 48 hours, and the PBMC cytokine IL-6 secretion was analyzed.

[0291] FIG35 shows that the α-BAFF-R VH×α-CD3 Fab bispecific antibody promotes the secretion of cytokine IL-6 by PBMC cells (Donor 605C).

[0292] FIG35A shows the bispecific antibody, Jeko-1 and PBMC cells (Donor 605C) co-incubated for 24 hours, and the cytokine IL-6 secretion analysis of PBMCs.

[0293] FIG35B shows the bispecific antibody, Jeko-1, and PBMC cells (Donor 605C) co-incubated for 48 hours, and the cytokine IL-6 secretion analysis of PBMCs.

[0294] FIG35C shows the bispecific antibody and PBMC cells (Donor 605C) were co-incubated for 24 hours, and the PBMC cytokine IL-6 secretion was analyzed.

[0295] FIG35D shows the bispecific antibody and PBMC cells (Donor 605C) were co-incubated for 48 hours, and the PBMC cytokine IL-6 secretion was analyzed.

[0296] FIG36 shows the curve of the change in the concentration of bispecific antibody 0205-2 in SD rat serum over time after a single administration.

[0297] FIG37 shows the curve of the change in the concentration of bispecific antibody 0205-3 in SD rat serum over time after a single administration.

[0298] Figure 38 shows the subcutaneous tumor volume (mm) of mice treated with bispecific antibody 0205-2 and PBS control group after the first administration. 3 ) changes with the extension of observation time.

[0299] Figure 39 shows the subcutaneous tumor volume (mm) of mice in the PBS group after the first administration. 3 ) changes with the extension of observation time.

[0300] Figure 40 shows the subcutaneous tumor volume (mm) of mice in the bispecific antibody 0205-2 administration group at 1 mpk after the first administration. 3 ) changes with the extension of observation time.

[0301] Figure 41 shows the subcutaneous tumor volume (mm) of mice in the bispecific antibody 0205-2 10 mpk group after the first administration. 3 ) changes with the extension of observation time.

[0302] FIG42 shows a curve diagram showing changes in body weight (g) of mice in each group as the observation time increases after the first administration. DETAILED DESCRIPTION

[0303] After extensive and in-depth research, the inventors used a large-capacity phage antibody library to screen for fully human BAFF-R-specific antibodies. They then evaluated the specificity of these antibodies at the phage level using ELISA and FACS experiments. Ultimately, they obtained several fully human antibody clones with excellent specificity. Using fully human antibodies with lower immunogenicity, they have developed antibody drugs (including monoclonal antibodies, bispecific antibodies, ADCs, etc.) and cell therapy drugs (including CAR-T, CAR-NK, etc.).

[0304] Specifically, using different antibody libraries and panning with recombinant BAFF-R protein, a total of 182 monoclonal clones were selected for initial screening using enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS). Thirty-nine clones specifically bound to the BAFF-R-his-Bio antigen and BAFF-R-positive K562-BAFF-R cells, but not to the control protein SA or BAFF-R-negative K562 cells. Sequencing yielded five distinct monoclonal sequences. Subsequently, these five antibodies were identified by flow cytometry (FACS) with various BAFF-R-positive (K562-BAFF-R, NALM6) and negative cell lines (K562, Jurkat), and by enzyme-linked immunosorbent assay (ELISA) with BAFF-R proteins from different companies (KACTUSBAFF-R-his bio, ACRO-BAFF-R-Fc, SB-BAFF-R-Fc) and unrelated proteins (KACTUS-CD19-Fc, ACRO-CD70-his, SA). Four of the clones showed good binding and specificity across multiple cell lines and protein antigens. The overall project workflow is shown in Figure 1.

[0305] The candidate clones were further validated for their functionality in CAR-T cells. Clone 5 demonstrated the best in vivo antitumor efficacy and good mouse safety, making it a promising candidate for later-stage clinical development. The heavy chain variable region of clone 5 comprises the following complementarity-determining regions (CDRs): CDR1 of SEQ ID NO. 27, CDR2 of SEQ ID NO. 28, and CDR3 of SEQ ID NO. 29. More specifically, the heavy chain variable region of clone 5 has the amino acid sequence of SEQ ID NO. 25. Clone 1 also demonstrated good in vivo antitumor efficacy, making it a promising candidate. The light chain variable region of clone 1 comprises the following complementarity-determining regions (CDRs): CDR1 of SEQ ID NO. 36, CDR2 of AAS, and CDR3 of SEQ ID NO. 37. The heavy chain variable region of the antibody comprises the following complementarity-determining regions (CDRs): CDR1 of SEQ ID NO. 38, CDR2 of SEQ ID NO. 39, and CDR3 of SEQ ID NO. 40.

[0306] In addition, the present invention also provides fully human single-domain antibodies and bispecific antibodies targeting BAFF-R. These anti-BAFF-R and anti-CD3 bispecific antibodies in different configurations (e.g., 1+1, 2+1, and 2+2 configurations) can specifically bind to the B-cell activating factor receptor (BAFF-R) on the cell surface and the immune cell surface antigen cluster 3 (CD3) with high affinity, and can be used to treat tumors and autoimmune diseases in which BAFFR is expressed.

[0307] The present invention was completed on this basis.

[0308] the term

[0309] 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.

[0310] 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.

[0311] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0312] 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.

[0313] "Antibodies" refer to immunoglobulins secreted by plasma cells (effector B cells) and used by the body's immune system to neutralize foreign substances (peptides, viruses, bacteria, etc.). These foreign substances are correspondingly called antigens. The basic structure of an antibody molecule is a tetramer consisting of two identical heavy chains and two identical light chains. Based on conservative differences in their amino acid sequences, the heavy and light chains are divided into a variable region (V) at the amino terminus and a constant region (C) at the carboxyl terminus. The variable region of a heavy chain (HCVR, also known as VH) and the variable region of a light chain (LCVR, also known as VL) interact to form the antigen-binding site (Fv). Within the variable region, certain regions, known as hypervariable regions (HVRs), have a more variable composition and arrangement of amino acid residues than other regions within the variable region (framework regions, FRs). These regions are the key sites for antibody-antigen binding. Because these hypervariable regions are complementary to antigenic determinants, they are also known as complementarity-determining regions (CDRs). Both the heavy chain and the light chain have three complementary determining regions, referred to as HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, respectively. The heavy chain constant region comprises three constant domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, referred to as complementarity determining regions (CDRs), interspersed with more conserved regions, referred to as framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

[0314] Single-chain antibodies (scFv) are composed of a single peptide chain consisting of the variable regions of the heavy and light chains of an antibody, linked by a short peptide. Properly folded, the variable regions from the heavy and light chains interact through non-covalent bonds to form the Fv fragment, allowing scFv to better retain its affinity for the antigen.

[0315] "Single-domain antibody (sdAb, or VHH)" and "nanobody" have the same meaning, referring to cloning the variable region of the antibody heavy chain to construct a nanobody consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete function. Usually, an antibody that naturally lacks 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. As used herein, the terms "nanobody of the present invention", "anti-BAFF-R nanobody of the present invention", and "BAFF-R single-domain antibody of the present invention" are used interchangeably and all refer to single-domain antibodies that specifically recognize and bind to BAFF-R (including human BAFF-R). Particularly preferred is a single-domain antibody whose VHH chain amino acid sequence is shown in SEQ ID NO.25, i.e., clone No. 5 in the example.

[0316] "Murine antibodies" are antibodies produced by rodents against specific antigens, typically those produced by mouse B lymphocytes. In most cases, these murine antibodies are monoclonal antibodies produced by hybridoma cells. The fully human antibodies of the present invention are obtained by screening human phage antibody libraries. Compared to murine antibodies, they have reduced immunogenicity and are more suitable for therapeutic use in humans.

[0317] As used herein, "fully human antibodies or single-chain antibodies or fragments thereof" generally refer to any form of antigen-binding molecules that can bind to a target antigen, for example, the antigen-binding molecule can be a protein or polypeptide, including, for example, antibodies and antigen-binding fragments thereof, single-chain scFv antibodies, single-domain antibodies, various fusions and conjugates constructed based on scFv, such as scFv-Fc antibodies, immunoconjugates, antibody-drug conjugates (ADCs), multi- / bispecific antibodies, and chimeric antigen receptors (CARs).

[0318] "BAFF-R" or "BAFF-R" is the primary mediator of BAFF-regulated B cell differentiation. The BAFF / BAFF-R signaling pathway plays a crucial role in the proliferation of B-cell lymphoma cells. Upregulated BAFF-R expression is associated with the progression of B-cell lymphomas and pre-B-ALL. BAFF-R is highly specific, primarily expressed in B cells, and binds exclusively to BAFF, not other members of the TNF family. Therefore, BAFF-R is a promising therapeutic target for numerous cancers, autoimmune diseases, and a variety of other conditions characterized by BAFF-R expression.

[0319] As used herein, when referring to amino acid or nucleotide sequences, the term "sequence identity" (also referred to as "sequence identity") refers to the amount of consistency between two amino acid or nucleotide sequences (e.g., a query sequence and a reference sequence), generally expressed as a percentage. Typically, before calculating the percentage of identity between two amino acid or nucleotide sequences, the sequences are aligned and gaps (if any) are introduced. If the amino acid residues or bases in the two sequences are the same at a certain alignment position, the two sequences are considered to be consistent or matched at that position; if the amino acid residues or bases in the two sequences are different, they are considered to be inconsistent or mismatched at that position. In some algorithms, the number of matching positions is divided by the total number of positions in the alignment window to obtain sequence identity. In other algorithms, the number of gaps and / or the length of the gaps are also taken into account. For the purposes of the present invention, the publicly available alignment software BLAST (available on the webpage ncbi.nlm.nih.gov) can be used to obtain the best sequence alignment and calculate the sequence identity between two amino acid or nucleotide sequences using the default settings. In some embodiments, the "at least 90% sequence identity" of the present invention includes, but is not limited to, at least 95%, at least 98%, at least 99% or even 100% sequence identity.

[0320] In some embodiments, the fully human antibodies provided by the present invention further include an amino acid sequence having at least 90% sequence identity (e.g., at least 95%, at least 98%, at least 99% or even 100% sequence identity) with the sequence shown in SEQ ID NO.25.

[0321] Those skilled in the art will appreciate that, based on the specific sequences provided herein, a small number of amino acid substitutions, deletions, or additions can be made, and the resulting products can be verified or screened for binding to the corresponding antigen BAFF-R or biological activity, thereby obtaining corresponding variants of the BAFF-R-targeting antibodies provided herein, and these variants are also intended to be included within the scope of the present invention. For example, a fully human antibody, or single-chain antibody or antigen-binding fragment thereof, of the present invention can have at least one and no more than ten, or no more than five, four, three, two, or one amino acid alterations in the full-length or CDR sequence.

[0322] Those skilled in the art will also appreciate that, based on the specific heavy chain variable region sequences provided herein, light chain variable regions that match the heavy chain variable region and maintain BAFF-R binding ability can be obtained by screening an antibody light chain library (e.g., a human phage light chain library) using BAFF-R as an antigen. Anti-BAFF-R antibody molecules obtainable in this manner are also encompassed within the scope of the present invention.

[0323] In some embodiments, the antigen binding molecules of the present invention may further comprise post-translational modifications. Examples of post-translational protein modifications include phosphorylation, acetylation, methylation, ADP-ribosylation, ubiquitination, glycosylation, carbonylation, ubiquitination, biotinylation, or the addition of polypeptide side chains or hydrophobic groups. Thus, the modified soluble polypeptide may comprise non-amino acid components, such as lipids, polysaccharides or monosaccharides, and phosphates. A preferred form of glycosylation is sialylation, which binds one or more sialic acid groups to the polypeptide. Sialic acid groups improve the solubility and serum half-life of proteins, while also reducing the potential immunogenicity of proteins. See Raju et al. Biochemistry. 2001 31; 40(30): 8868-76.

[0324] "Epitope" refers to the portion of a molecule that is bound by an antigen binding protein (e.g., an antibody). An epitope can comprise non-contiguous portions of the molecule (e.g., amino acid residues in a polypeptide that are not contiguous in the primary sequence of the polypeptide but are sufficiently close to each other in the trivalent and tetravalent structures of the polypeptide to be bound by an antigen binding protein).

[0325] The present invention provides a kit comprising one or more containers containing a plurality of gene constructs encoding a polypeptide of the present invention and a pharmaceutically acceptable excipient. The kit may also include instructions for use. The kit may also include a notice in a form prescribed by a governmental agency regulating the production, use, or sale of pharmaceuticals or biological products, indicating that the kit has been licensed by the agency for the production, use, or sale of pharmaceuticals for human use.

[0326] As used herein, the terms "heavy chain variable region" and "V H ” can be used interchangeably.

[0327] As used herein, the terms "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0328] 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.

[0329] In a preferred embodiment of the present invention, the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region.

[0330] 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 to refer to polypeptides that specifically bind to BAFF-R protein, such as proteins or polypeptides having a heavy chain variable region. These may or may not contain an initial methionine.

[0331] The present invention also provides other proteins or fusion expression products comprising 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) comprising 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.

[0332] Generally, an antibody's antigen-binding properties are described by three specific regions within the heavy chain variable region, known as the variable region (CDR). This region is divided into four framework regions (FR). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a loop structure, spatially close to each other through the β-sheet formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antibody's antigen-binding site. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0333] 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 antigen binding. Thus, the present invention includes molecules having antibody heavy chain variable regions with CDRs that are 90% or more (preferably 95% or more, and most preferably 98% or more) homologous to the CDRs identified herein.

[0334] The antibodies of the present invention refer to polypeptides that have BAFF-R protein binding activity and include the aforementioned CDR regions. The term also encompasses variants of polypeptides that include the aforementioned CDR regions and have the same function as the antibodies of the present invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more (generally 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, as well as the addition of one or more (generally within 20, preferably within 10, and more preferably within 5) amino acids to the C-terminus and / or N-terminus. For example, substitutions with amino acids having similar or similar properties generally do not alter protein function. For another example, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter protein function. The term also encompasses active fragments and active derivatives of the antibodies of the present invention.

[0335] 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.

[0336] The present invention also provides other polypeptides, such as fusion proteins comprising single-domain antibodies or fragments thereof. In addition to substantially full-length polypeptides, the present invention also encompasses fragments of the single-domain antibodies of the invention. Typically, the fragment comprises at least about 50 contiguous amino acids of an antibody of the invention, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.

[0337] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides in which no more than 10, preferably no more than 8, more preferably no more than 5, and most preferably no more than 3 amino acids are replaced 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 making amino acid substitutions according to Table A.

[0338] Table A

[0339] The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies, fragments thereof, or fusion proteins thereof. The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0340] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0341] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and / or non-coding sequences.

[0342] The present invention also relates to polynucleotides that hybridize to the above-mentioned 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 hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0343] The full-length nucleotide sequence of the antibody of the present invention or its fragments can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis methods. One feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is relatively short. Generally, by first synthesizing multiple small fragments and then ligating them, very long fragments of sequence can be obtained. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0344] Bispecific antibodies

[0345] The present invention also includes a multispecific antibody containing the BAFF-R single domain antibody of the present invention, 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, CD11a, 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.

[0346] Preferably, the multispecific antibody comprises one or more second antigen-binding regions, or further comprises a third antigen-binding region.

[0347] A preferred bispecific antibody is an anti-BAFF-R and anti-CD3 bispecific antibody. The bispecific antibody of the present invention specifically binds to both the B-cell activating factor receptor (BAFF-R) on the cell surface and the immune cell surface antigen cluster 3 (CD3); the anti-BAFF-R antibody is a fully human single-domain antibody. The bispecific antibodies of the present invention are symmetrical or asymmetrical dimers, such as 1+1, 2+1, and 2+2 bispecific antibodies.

[0348] Preferably, the bispecific antibody has the following structure:

[0349] (a) two heavy chains, wherein the first heavy chain comprises, from N-terminus to C-terminus, an anti-BAFF-R VH region and a human IgG Fc region; the second heavy chain comprises, from N-terminus to C-terminus, an anti-CD3 VH region, an antibody heavy chain constant region (CH1), and a human IgG Fc region;

[0350] (b) a light chain, comprising, from N-terminus to C-terminus, an anti-CD3 VL, an antibody light chain constant region (CL),

[0351] The anti-BAFF-R VH is the heavy chain variable region of a single domain antibody targeting CD3,

[0352] 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 as set forth in SEQ ID NO. 51, a second heavy chain having an amino acid sequence as set forth in SEQ ID NO. 73, and a light chain having an amino acid sequence as set forth in SEQ ID NO. 71.

[0353] In another preferred embodiment, the bispecific antibody has the following structure:

[0354] (a) two heavy chains, wherein the first heavy chain comprises, from N-terminus to C-terminus, an anti-BAFF-R VH, an anti-CD3 VH, an antibody heavy chain constant region (CH1), and a human IgG Fc region; the second heavy chain comprises, from N-terminus to C-terminus, an anti-BAFF-R VH and a human IgG Fc region;

[0355] (b) a light chain, comprising, from N-terminus to C-terminus, an anti-CD3 VL, an antibody light chain constant region (CL),

[0356] The anti-BAFF-R VH is the heavy chain variable region of a single domain antibody targeting CD3,

[0357] 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. 51, a second heavy chain having an amino acid sequence such as SEQ ID NO. 75, and a light chain having an amino acid sequence such as SEQ ID NO. 71.

[0358] In another preferred embodiment, the bispecific antibody has the following structure:

[0359] (a) two heavy chains, each comprising, from N-terminus to C-terminus, an anti-BAFF-R VH, an anti-CD3 VH, an antibody heavy chain constant region (CH1), and a human IgG Fc region;

[0360] (b) two light chains, each comprising, from N-terminus to C-terminus, an anti-CD3 VL, an antibody light chain constant region (CL),

[0361] The anti-BAFF-R VH is the heavy chain variable region of a single domain antibody targeting CD3,

[0362] 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. 51, a second heavy chain having an amino acid sequence such as SEQ ID NO. 77, and a light chain having an amino acid sequence such as SEQ ID NO. 71.

[0363] Chimeric Antigen Receptor (CAR)

[0364] The chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen binding domain). The intracellular domain includes a costimulatory signaling region and a ζ chain portion. The costimulatory signaling region refers to a portion of the intracellular domain including costimulatory molecules. Costimulatory molecules are cell surface molecules required for the effective response of lymphocytes to antigens, rather than antigen receptors or their ligands.

[0365] Between the extracellular domain and the transmembrane domain of CAR, or between the cytoplasmic domain and the transmembrane domain of CAR, a linker can be incorporated. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane domain to the extracellular domain or cytoplasmic domain of a polypeptide chain. The linker may include 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

[0366] In a preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention includes an antigen binding domain targeting BAFF-R. When the CAR of the present invention is expressed in T cells, it is possible to perform antigen recognition based on antigen binding specificity. When it binds to its associated antigen, it affects tumor cells, causing them to not grow, be caused to die or be affected in other ways, and causing the patient's tumor load to shrink or be eliminated. The antigen binding domain is preferably fused with one or more intracellular domains from a costimulatory molecule and a ζ chain. Preferably, the antigen binding domain is fused with the intracellular domain of a combination of a 4-1BB signaling domain and a CD3ζ signaling domain.

[0367] As used herein, "antigen-binding domain" refers to a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or a single Fv fragment that has antigen-binding activity.

[0368] In the present invention, fully human phages including a single-domain library were used for antibody screening to obtain fully human single-domain antibodies with lower immunogenicity than humanized mouse antibodies. A CAR vector was constructed in which the antigen-binding domain contained one or more single-domain antibodies that specifically recognized BAFF-R, and CAR-T cells were then prepared.

[0369] carrier

[0370] The present invention includes a DNA construct comprising a CAR sequence, wherein the sequence includes a nucleic acid sequence of an antigen binding domain operably linked to a nucleic acid sequence of a signaling domain.

[0371] The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from a vector known to include the gene, or by directly isolating from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically.

[0372] The present invention also provides vectors into which the DNA of the present invention is inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of the transgene and its propagation in daughter cells.

[0373] In brief summary, the expression of the natural or synthetic nucleic acid encoding CAR is generally achieved by operably linking the nucleic acid encoding the CAR polypeptide or its portion to a promoter and incorporating the construct into an expression vector. The vector is suitable for replication and integration of eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

[0374] The nucleic acid can be cloned into many types of vectors. For example, the nucleic acid can be cloned into such vectors, which include but are not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors.

[0375] Furthermore, the expression vector can be provided to the cell in the form of a viral vector. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Typically, a suitable vector comprises an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers.

[0376] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0377] Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. Typically, these are located in the 30-110 bp region upstream of the start site, although recently it has been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that when an element is inverted or moved relative to another, promoter function is maintained. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50 bp before activity begins to decline. Depending on the promoter, it has been shown that individual elements can work together or independently to initiate transcription.

[0378] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive any polynucleotide sequence operably connected thereto for high-level expression. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr (Epstein-Barr) virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the present invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered to be part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0379] In order to evaluate the expression of the CAR polypeptide or its portion, the expression vector introduced into the cell may also include any one or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a cell population that is sought to be transfected or infected by a viral vector. In other aspects, selectable markers can be carried on a single DNA segment and used for co-transfection procedures. Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences to enable expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

[0380] Reporter gene is used to identify the cells of potential transfection and to evaluate the functionality of regulatory sequences. Generally, reporter gene is following gene: it is not present in or is expressed by receptor organism or tissue, and its coded polypeptide, the expression of this polypeptide is clearly represented by some easily detectable properties such as enzymatic activity. After DNA has been introduced into receptor cells, the expression of reporter gene is measured under the appropriate time. Suitable reporter gene can comprise the gene (for example, Ui-Tei etc., 2000FEBS Letters479:79-82) of coding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein gene. Suitable expression system is well known and can utilize known technology to prepare or obtain commercially. Generally, the construct with minimum 5 flanking regions that shows the highest level of reporter gene expression is identified as promoter. Such promoter region can be connected to reporter gene and be used to evaluate the ability of reagent regulating promoter-driven transcription.

[0381] Methods for introducing genes into cells and expressing genes in cells are known in the art. In the context of expression vectors, the vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0382] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0383] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others.

[0384] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0385] In the case of using non-viral delivery system, exemplary delivery means is liposome. Consider using lipid preparation, to introduce nucleic acid into host cell (in vitro, in vitro (ex vivo) or in vivo). On the other hand, this nucleic acid can be associated with lipid. The nucleic acid associated with lipid can be encapsulated in the aqueous interior of liposome, dispersed in the lipid bilayer of liposome, attached to liposome through the connecting molecule associated with liposome and oligonucleotide, trapped in liposome, compounded with liposome, dispersed in the solution comprising lipid, mixed with lipid, united with lipid, included in lipid as suspension, included in micelle or with micelle compound, or otherwise associated with lipid. The lipid, lipid / DNA or lipid / expression vector associated with composition are not limited to any specific structure in solution. For example, they can be present in bilayer structure, as micelle or have " collapsed (collapsed) " structure. They can also be simply dispersed in solution, may form aggregates of size or shape inhomogeneity. Lipid is a fatty substance, which can be a naturally occurring or synthetic lipid. For example, lipids include fat droplets that occur naturally in the cytoplasm as well as compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0386] In a preferred embodiment of the present invention, the vector is a lentiviral vector. The inventors have confirmed that the use of the lentiviral vector to construct the CAR of the present invention has a high transfection efficiency for T cells and is highly reproducible.

[0387] In a preferred embodiment of the present invention, the vector further comprises a signal peptide sequence. Preferably, the signal peptide sequence is connected upstream of the antigenic tuberculosis domain nucleic acid sequence.

[0388] Antibody preparation

[0389] 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 heavy chain can be fused together to form a single-chain antibody.

[0390] 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.

[0391] In addition, the sequences can also be synthesized by artificial synthesis, especially when the fragment length is shorter. Usually, a long fragment can be obtained by synthesizing multiple small fragments and then connecting them.

[0392] Currently, DNA sequences encoding the antibodies (or fragments thereof, or derivatives thereof) of the present invention can be obtained entirely through chemical synthesis. This 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 sequences of the present invention through chemical synthesis.

[0393] 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.

[0394] 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 and HEK-293 cells.

[0395] Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the present invention. The antibodies of the present invention are then purified using conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, among other conventional separation and purification methods well known to those skilled in the art.

[0396] The resulting monoclonal antibodies can be characterized using conventional methods. For example, the binding specificity of the monoclonal antibodies can be determined using 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, using the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0397] The antibodies of the present invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be separated and purified by various separation methods utilizing 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 a protein precipitant (salting out method), centrifugation, osmotic shock, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0398] Immunoconjugates

[0399] The present invention also provides an immunoconjugate (ADC) based on the antibody of the present invention, preferably a single-domain antibody-drug conjugate (NDC).

[0400] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, preferably chemically conjugated. The effector molecule is preferably a therapeutically active drug. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug, or a radionuclide.

[0401] The antibody of the present invention and the effector molecule can be coupled via a coupling agent. Examples of the coupling agent may include 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 may include any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (where the coupling site is an acylhydrazone).

[0402] 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 reagents, stabilizers) are coupled (covalently linked) to antibodies. Functional agents can be directly or indirectly connected to antibodies through linkers.

[0403] Single-domain antibodies can be conjugated to drugs to form antibody-drug conjugates (NDCs). Typically, NDCs contain a linker positioned between the drug and the antibody. The linker can be a degradable or non-degradable linker. Degradable linkers typically readily degrade in the intracellular environment, for example, at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, including linkers containing peptidyl groups that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronide-containing linkers that can be degraded by glucuronidases. 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 (e.g., linkers that hydrolyze at a pH below 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0404] 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 via the active reactive group. Thiol-specific active reactive groups are preferred and include, for example, maleimides, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethylketones (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, where the counter ion is acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. Linkers may include, for example, maleimides attached to the antibody via thiosuccinimide.

[0405] The drug can be any cytotoxic, cytostatic, or immunosuppressive drug. In embodiments, a linker connects the antibody and the drug, and the drug has a functional group capable of forming a bond with the linker. For example, the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group capable of forming a bond with the linker. In cases where the drug is directly attached to the linker, the drug has a reactive group prior to attachment to the antibody.

[0406] 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.

[0407] In the present invention, drug-linkers can be used to form NDCs in a single step. In other embodiments, bifunctional linker compounds can be used to form NDCs 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.

[0408] Typically, the functional group on the linker is selected to facilitate specific reaction with an appropriate reactive group on the drug moiety. As a non-limiting example, an azide-based moiety can be used to specifically react with a reactive alkynyl group on the drug moiety. The drug is covalently attached to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. 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 linking 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 between the drug moiety and the linker, each member of the complementary pair can be used for both the linker and the drug.

[0409] The present invention also provides a method for preparing NDC, which may further comprise: combining an antibody and a drug-linker compound under conditions sufficient to form an antibody conjugate (NDC).

[0410] In certain embodiments, the methods of the present invention comprise conjugating an antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the present invention further comprise conjugating the antibody-linker conjugate to a drug moiety under conditions sufficient to covalently attach the drug moiety to the antibody via the linker.

[0411] In some embodiments, the structure of the immunoconjugate, preferably a single domain antibody drug conjugate (NDC), is shown in the following molecular formula:

[0412] in:

[0413] nAb is a single-domain antibody targeting BAFF-R, an antibody targeting BAFF-R, or a multispecific antibody.

[0414] LU is the linker / connector;

[0415] D is for medicine;

[0416] And the subscript p is a value selected from 1 to 10.

[0417] application

[0418] The present invention also provides uses of the antibodies of the present invention, such as for preparing diagnostic agents or medicaments for preventing and / or treating BAFF-R-related diseases. The BAFF-R-related diseases include tumor development, growth, and / or metastasis, diseases associated with tumor drug resistance, autoimmune diseases, inflammation, and metabolic diseases.

[0419] The uses of the antibodies or ADCs of the present invention include (but are not limited to):

[0420] (i) Diagnosis, prevention, and / or treatment of tumor development, growth, and / or metastasis, particularly tumors with high BAFF-R expression. Such tumors include (but are not limited to): leukemia, lymphoma, myeloma, breast cancer (e.g., triple-negative breast cancer), lung cancer (e.g., 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.; particularly triple-negative breast cancer, non-small cell lung cancer, pancreatic cancer, and malignant glioma, more preferably triple-negative breast cancer and / or non-small cell lung cancer.

[0421] (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.

[0422] (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, and idiopathic pulmonary fibrosis.

[0423] (iv) diagnosing, preventing and / or treating metabolic diseases, including but not limited to diabetes, diet-induced obesity and adipose inflammation.

[0424] Pharmaceutical composition

[0425] 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 formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0426] The antibody of the present invention can also be expressed in cells by nucleotide sequences for cell therapy, for example, the antibody is used in chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.

[0427] The pharmaceutical composition of the present invention can be used to directly 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.

[0428] The pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned monoclonal antibody of the present invention (or its conjugate) 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, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. 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 μg / kg body weight to about 5 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used in conjunction with other therapeutic agents.

[0429] 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 generally at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight. Preferably, the dose is about 10 μg / kg body weight to about 20 mg / kg body weight. Of course, the specific dose should also take into account factors such as the route of administration and the patient's health status, which are all within the skill of a skilled physician.

[0430] 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 cell surface protein (antigen), thereby releasing the drug 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. The above-mentioned antibody-drug conjugate can be administered to a subject (e.g., a human) in a therapeutically effective amount through an appropriate route. The subject in need of treatment can 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 through routine physical examinations.

[0431] When treating with the single domain antibody-drug conjugates of the present invention, delivery can be performed by conventional methods in the art. For example, it can be introduced into cells 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.

[0432] The main advantages of the present invention include

[0433] (1) The present invention uses fully human phage for antibody screening, directly obtaining a series of fully human monoclonal antibodies with excellent performance. Compared with traditional hybridoma technology, the difficult step of humanizing mouse antibodies is eliminated. In addition, fully human antibodies have lower immunogenicity than humanized mouse antibodies, and have great application potential in the development of antibody drugs (including monoclonal antibodies, bispecific antibodies, ADCs, etc.) and cell therapy drugs (including CAR-T, CAR-NK, etc.).

[0434] (2) The high-affinity specific antibodies provided by the present invention can also be used in the development of detection reagents.

[0435] (3) 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.

[0436] (4) The bispecific antibody targeting BAFF-R and CD3 of the present invention can specifically and effectively mediate the killing of BAFF-R positive target cells by PBMC and has good in vivo biological activity and safety.

[0437] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which detailed conditions are not specified, were generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0438] Example 1. Enrichment of specific antibody clones targeting BAFF-R protein from phage antibody library by affinity panning

[0439] Appropriate negative and positive panning strategies were used to enrich the specific antibody clones we desired from the phage antibody library.

[0440] Construction of phage antibody library

[0441] The phage antibody libraries we construct include natural, semi-synthetic, and single-domain libraries. Semi-synthetic phage antibody libraries, used in conjunction with natural libraries, address the potential lack of high-affinity BAFF-R antibody clones in natural libraries. Single-domain phage antibody libraries, composed solely of amino acids from the variable regions of heavy-chain antibodies, have a molecular weight of only 12-15 kDa but exhibit similar or higher specificity and affinity than traditional antibodies. Furthermore, single-domain antibodies are attracting significant attention due to their stable physicochemical properties, high affinity, ease of recombinant expression, and ease of combination with antibodies targeting other targets or epitopes.

[0442] BAFF-R protein panning

[0443] Using BAFF-R-his-Bio as the positive panning protein, multiple rounds of panning were performed to obtain a phage pool enriched for the target antibody clone. The experimental steps are briefly described as follows:

[0444] 1) Block the SA magnetic beads with blocking solution for 2 hours, then bind the target antigen (BAFF-R-his-Bio) to the blocked SA magnetic beads;

[0445] 2) Add phage library (containing 5x10 12 phage particles) and a clean SA magnetic bead were incubated together to remove the phage antibody clones that non-specifically bound to the SA magnetic beads;

[0446] 3) After incubation, the supernatant is transferred to SA magnetic beads bound to the target antigen and incubated again to allow the phage to bind to the target antigen;

[0447] 4) Wash the magnetic beads with washing solution to remove unbound phages;

[0448] 5) Elute the positive phage from the target antigen with an elution solution and neutralize it with a neutralization solution;

[0449] 6) Re-infect the host bacteria XL1-blue with the eluted phage and amplify the recovered phage. A small amount of sample is retained for serial dilution, infected with the host bacteria, and plated on an Amp-resistant plate to count the recovered phage.

[0450] 7) Repeat steps 1) to 6), usually 3 to 4 rounds of panning are required, until a significant increase in the phage recovery rate (number of eluted phages / number of input phages) is observed.

[0451] The enriched phage pool can be used for subsequent single clone selection and ELISA / FACS screening.

[0452] Main materials and reagents:

[0453] Fully human phage antibody library, including natural library, semi-synthetic library and single domain library;

[0454] Helper phage KO7, Thermo / Invitrogen, 18311019;

[0455] Recombinant biotinylated Human BAFF-R Protein,KACTUS,BAFF-R-HM401;

[0456] BeaverBeads TM Streptavidin, Beaver Bio, 22307-10;

[0457] High binding ELSIA plate,Costar,#3590

[0458] Blocking solution: PBS + 3% BSA

[0459] Rinse solution: PBS + 0.1% Tween20

[0460] Eluent: 0.2 M Glycine, pH 2.2

[0461] Neutralization solution: 1M Tris, pH 9.1

[0462] Experimental results:

[0463] Using different antibody libraries, four rounds of protein panning were performed, and a significant increase in recovery rate was observed in each panning (Table 1), demonstrating that the antibody clones were effectively enriched.

[0464] Table 1 Protein panning experiment results

[0465] It can be seen that after 4 rounds of panning, different antibody libraries have been enriched (the recovery rate in the 4th round was significantly higher than that in the previous round).

[0466] Example 2. Screening of specific clones from the enriched phage pool using enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS)

[0467] Purpose and Principle: The phage pool enriched by affinity panning contains a variety of phage antibodies: specific clones, nonspecific clones, and negative clones. To obtain specific clones, we isolate single clones, package them into monoclonal phage, and screen a large number of these clones using enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS). From these clones, we select clones that specifically bind to both the BAFF-R protein and the BAFF-R-positive cell line K562-BAFF-R. DNA sequencing is then performed to confirm the unique antibody sequence contained in these specific clones.

[0468] In the ELISA primary screening, the binding of streptavidin to biotin makes the biotinylated target antigen (BAFF-R-his-Bio) closer to the native antigen conformation in the reaction solution. Clones that only bind to BAFF-R-his-Bio and not to the control protein SA are identified as specific clones. The FACS primary screening is performed using the BAFF-R-positive cell line K562-BAFF-R with high BAFF-R expression and the BAFF-R-negative cell line K562. Clones that only bind to K562-BAFF-R and not to K562 cells are identified as specific clones. Through both ELISA and FACS primary screening, we can obtain candidate antibodies that can bind to recombinantly expressed BAFF-R protein and recognize the native BAFF-R molecules on the cell surface for subsequent further screening.

[0469] Brief steps of ELISA experiment:

[0470] 1) Cultivate and package monoclonal phage using deep-well 96-well plates;

[0471] 2) Dilute Strepavidin to 2 μg / mL with PBS and add 100 μL / well to the high-binding ELISA plate. Allow to bind for 2 h at room temperature.

[0472] 3) Discard the coating solution, add 250 μL of blocking solution to each well, and block overnight at 4°C;

[0473] 4) Wash the plate twice with 250 μL of rinse buffer;

[0474] 5) Dilute the biotin-tagged target protein and control protein to 2 μg / mL with PBS and add 100 μg / well to the Strepavidin-precoated ELISA plate for binding at room temperature for 1 hour.

[0475] 6) Wash the plate twice with 250 μL of rinse buffer;

[0476] 7) Add 100 μL of the phage supernatant cultured in step 1) to the wells coated with the target antigen and allow to bind for 2 h at room temperature;

[0477] 8) Wash the plate 4 times with 250 μL of rinse buffer;

[0478] 9) Add 1:2000 diluted mouse anti-M13 primary antibody, 100 μL / well, and incubate at room temperature for 45 min;

[0479] 10) Wash the plate 4 times with 250 μL of rinse buffer;

[0480] 11) Add 1:2000 diluted HRP Donkey anti-mouse IgG, 100 μL / well, and incubate at room temperature for 45 min;

[0481] 12) Wash the plate 6 times with 250 μL of rinse buffer;

[0482] 13) Add 100 μL TMB chromogenic substrate and allow color development for 5 to 10 minutes;

[0483] 14) Add 100 μL of 2M H2SO4 to terminate the reaction and read the results on a microplate reader.

[0484] Brief steps of FACS screening experiment:

[0485] 1) Cultivate and package monoclonal phage using deep-well 96-well plates;

[0486] 2) Wash Raji and Jurkat cells twice with PBS and resuspend in PBS to 1x10 7 / mL concentration, and 50 μL was dispensed into 96-well deep-well plates;

[0487] 3) Add 50 μL of packaged monoclonal phage to each well, mix well, and allow to bind at 4°C for 2 h.

[0488] 4) Wash twice with 200 μL PBS;

[0489] 5) Add 1:2000 diluted mouse anti-M13 primary antibody, 100 μL / well, pipette to mix, and incubate at room temperature for 45 minutes;

[0490] 6) Wash twice with 200 μL PBS;

[0491] 7) Add 1:300 diluted FITC horse anti-mouse-IgG (H+L) at 100 μL / well, mix thoroughly by pipetting, and incubate at room temperature for 45 min.

[0492] 8) Wash twice with 200 μL PBS; finally, resuspend the cells in 200 μL PBS;

[0493] 9) Detect the fluorescence intensity of the sample in the FITC channel on a flow cytometer and analyze the results.

[0494] Main materials and reagents:

[0495] Helper phage KO7, Thermo / Invitrogen, 18311019

[0496] Streptavidin,Pierce,21125

[0497] Recombinant biotinylated Human BAFF-R Protein,KACTUS,BAFF-R-HM401;

[0498] High binding ELSIA plate,Costar,#3590

[0499] Corning 96Well Clear Round Bottom TC-Treated Microplate,Costar,#3799

[0500] Blocking solution: PBS + 3% BSA

[0501] Rinse solution: PBS + 0.1% Tween20

[0502] Soluble one-component TMB substrate solution, Tiangen, PA-107-02

[0503] Anti-M13 Bacteriophage Coat Protein g8p antibody,abcam,ab9225

[0504] HRP Goat anti-mouse IgG(minimal x-reactivity)Antibody,Biolegend,405306

[0505] FITC horse anti mouse-IgG(H+L),Vector,FI2000

[0506] Experimental results:

[0507] Monoclonal clones were randomly selected from the enriched phage antibody pool and packaged into phage. Phage ELISA was then used to test the binding of these monoclonal phage to the BAFF-R-his-Bio protein and the control protein SA, identifying BAFF-R-specific phage antibody clones. The ELISA results for some clones are shown in Figure 2. As can be seen, clones H1-H2, H4-H7 bind well to the target antigen BAFF-R (BAFF-R-Bio-his) and do not bind to the control antigen SA, demonstrating good specificity. Clone H3 binds neither to the target antigen BAFF-R-his-Bio nor to the control protein SA, indicating a negative clone.

[0508] The initial FACS screening results of some clones are shown in Figure 3. The negative control phage antibody clones are shown. Clone numbers H1-H2, H4-H7 do not bind to K562 but bind to K562-BAFF-R cells, indicating they are specific clones. Clone H3 does not bind to either K562-BAFF-R or K562, indicating it is a negative clone.

[0509] Through ELISA detection and FACS screening, we obtained a total of 39 specific clones.

[0510] Example 3. Identification of monoclonal specificity by FACS using multiple cell lines

[0511] Purpose and Principle of the Experiment: Antibodies used for therapeutic purposes must possess excellent target specificity, binding only to the target antigen and not to any unrelated antigens. Furthermore, the amino acid sequence of the same antigen on different cell lines may vary (isomers or mutants) or bind to different ligands, necessitating the ability of our antibodies to bind to cells expressing a variety of target proteins. To further analyze the specificity and universality of these monoclonal antibodies and identify the best candidate clones, we used flow cytometry to further evaluate the specificity of the initial screening clones. In this experiment, we used multiple BAFF-R-positive and BAFF-R-negative cell lines to react with these monoclonal phage antibodies, analyzing whether these clones could bind to BAFF-R antigens on different cell lines and whether there was any non-specific binding to other cell lines that did not express BAFF-R. Through this experiment, we obtained several clones with excellent specificity.

[0512] Experimental method: Same as FACS primary screening;

[0513] Main samples and reagents:

[0514] K562-BAFF-R cell line, BAFF-R positive cell line;

[0515] K562 cell line, BAFF-R negative cell line;

[0516] NALM6 cell line, BAFF-R positive cell line;

[0517] Jurkat cell line, BAFF-R negative cell line;

[0518] The remaining reagents were the same as those for the FACS primary screening.

[0519] Experimental results:

[0520] Antibodies used for therapeutic purposes must possess excellent target specificity. To further analyze the specificity of these monoclonal antibodies, we characterized the unique clone obtained in Example 2 using enzyme-linked immunosorbent assay (ELISA) and flow cytometry on a wider range of antigens and cell lines. The results are shown in Figures 4A-B. Figure 4A shows the flow cytometric peak profiles of Clone 1, 5, and 14 binding to various cell lines, with the Negative Control being a negative control phage antibody clone. Figure 4B shows the flow cytometric peak profiles of Clone 77 and 80 binding to various cell lines, with VAY736 being a positive control phage antibody clone. The VAY736 antibody bound to all BAFF-R-positive cell lines but not to BAFF-R-negative cell lines, indicating that the four selected cell lines expressed BAFF-R antigen at normal levels. Clone 5, 77, and 80 all bound to BAFF-R-positive cell lines, while Clones 1 and 14 only bound to the BAFF-R-overexpressing cell line K562-BAFF-R and did not bind to NALM6. They also did not bind to BAFF-R-negative cell lines, demonstrating good specificity.

[0521] Example 4. Identification of monoclonal specificity by ELISA using antigens from different companies

[0522] Purpose and Principle of the Experiment: Antibodies used for therapeutic purposes must have excellent target specificity, binding only to the target antigen and not to any unrelated antigens. On the other hand, the amino acid sequences of the same antigen produced by different companies may vary (isomers or mutants), and it is also necessary to examine whether our antibodies can bind to various target proteins. In order to further analyze the specificity and universality of these monoclonal clones and identify the best candidate clones, we further evaluated the specificity of the initial screening clones using enzyme-linked immunosorbent assay (ELISA). In this experiment, we used BAFF-R antigens purchased from different companies and a variety of BAFF-R-unrelated antigens to react with these monoclonal phage antibodies to analyze whether these clones can bind to different BAFF-R antigens and whether there is any non-specific binding to other BAFF-R-unrelated antigens. Through this experiment, we obtained several clones with excellent specificity.

[0523] Experimental method: Same as ELISA primary screening;

[0524] Main samples and reagents:

[0525] Table 2

[0526] The remaining reagents are the same as those for the ELISA primary screening.

[0527] Experimental results:

[0528] Antibodies used for treatment must have very good target specificity. In order to further analyze the specificity of these monoclonal antibodies, we identified the multiple clones obtained in Example 2 using enzyme-linked immunosorbent assay (ELISA) on multiple antigens. The results are shown in Figure 5. Negative control is a negative control phage antibody clone. Anti-M13 phage mouse Ab / anti-mouse HRP Ab is a negative control without adding phage, only adding the first antibody (Anti-M13 phage mouse Ab) and the second antibody (anti-mouse HRP Ab); anti-mouse HRP Ab is a negative control with only adding the second antibody (anti-mouse HRP Ab); VAY736 Ab / anti-human IgG HRP Ab is a positive control with the addition of BAFF-R detection antibody; anti-human IgG HRP Ab / anti-his HRP Ab is a positive control for detecting antigen tag antibodies. Clone 1, 5, 77, and 80 bound to both BAFF-R antigens, but not to the unrelated antigens KACTUS-CD19-Fc, Acro-CD70-his, and SA. This suggests that these four clones can bind to different conformations of BAFF-R with good specificity. Clone 14 showed weak binding to the unrelated antigen Acro-CD70-his, and its specificity requires further confirmation.

[0529] Example 5. Detection of EGFRt expression on the surface of CAR-T cells

[0530] In order to verify the function of candidate clones on CAR-T cells, we constructed a CAR vector with the structure shown in Figure 6 based on the candidate antibody sequence. The CAR structure contains CD8α signal peptide, V HHOr scFv, CD8α hinge region, CD8α transmembrane region, 4-1BB costimulatory molecule and CD3ζ, and connected to a truncated EFGR molecule (EGFRt) with T2A can be used as a safety switch during clinical translation. The CAR vector was then packaged with lentivirus, and EGFRt expression was detected 5 to 7 days after T cells were transfected with the CAR lentivirus (Figure 7). Since EGFRt is co-expressed with the CAR molecule, it can be used as an indirect detection indicator for the distribution of the CAR molecule on the T cell surface without affecting the structure and function of the CAR.

[0531] The brief experimental steps for CAR-T / EGFRt expression detection are as follows:

[0532] 1) Take 1×10 6 Each well of CAR-T / T cells was washed once with PBS, centrifuged at 300 g for 5 minutes, and the supernatant was discarded.

[0533] 2) Resuspend the cell pellet in 100 μL PBS, add 5 μL APC-CD5 antibody and 5 μL APC-EGFR antibody, respectively, and incubate at 4°C in the dark for 15 minutes.

[0534] 3) Wash twice with PBS and centrifuge at 300g for 5 minutes.

[0535] 4) Resuspend in 200 μL PBS and detect on flow cytometry.

[0536] Main materials and reagents:

[0537] APC anti-human EGFR Antibody,Clone AY13,BioLegend,Cat.No.352906;

[0538] Fetal bovine serum (FBS), Gibco, Cat. No. 10099141;

[0539] Experimental results:

[0540] As shown in FIG7 , on the seventh day, the expression of EGFR in T cells of each group was ≥30%.

[0541] Example 7. CD107a degranulation experiment

[0542] Experimental principle and purpose:

[0543] CD107a is a marker for intracellular microvesicles. When microvesicles loaded with granzymes fuse with the cell membrane, CD107a levels on the cell membrane increase. Blocking their recovery with monesin (purchased from BioLegend) can quantitatively reflect the intensity of microvesicle release. When CAR-T cells are stimulated by target antigens on target cells, granzymes are released, and flow cytometry can be used to detect increases in CD107a to assess T cell activation.

[0544] Brief experimental steps of CD107a degranulation:

[0545] 1) The CAR-T cells and target cells to be tested were centrifuged at 300g for 5 minutes at room temperature, the supernatant was discarded, and the cells were resuspended in 1640 medium + 10% FBS to a concentration of 2x10 6 cells / mL;

[0546] 2) Add 100 μL of the CAR-T cells to be tested and 100 μL of the target cells to a 96-well plate and mix well.

[0547] 3) Add 1.5 μL of PE / Cy7 anti-human CD107a antibody and 0.2 μL of monensin to each well of cells, and then incubate in a cell culture incubator at 37°C, 5% CO2 for 4 h;

[0548] 4) After incubation, centrifuge at 300 g for 5 min at 4°C, discard the supernatant, and wash the cells twice with 200 μL PBS.

[0549] 5) Resuspend the cells in 100 μL PBS and add 1.5 μL BV421 anti-human CD8 and 1.5 μL APC anti-human EGFR antibodies, mix well, and incubate on ice in the dark for 20 minutes.

[0550] 6) After incubation, wash the cells three times with 200 μL PBS; resuspend in 100 μL PBS and analyze by flow cytometry.

[0551] Main samples and reagents:

[0552] Target cells NALM6-luc (BAFF-R+), RS4-luc (BAFF-R+), MEC1-luc (BAFF-R+)

[0553] JEKO1-luc(BAFF-R+), THP1-luc(BAFF-R-), Molm13-luc(BAFF-R-), CCRF-luc(BAFF-R-), MOLT4-luc(BAFF-R-);

[0554] Fetal bovine serum (FBS), Gibco, Cat. No. 10099141;

[0555] Monensin,BioLegend,Cat.No.420701;

[0556] PE / Cy7 mouse anti-human CD107a, BD, Cat.No.561348;

[0557] BV421 mouse anti-human CD8,BD,Cat.No.301036;

[0558] APC anti-human EGFR Antibody, Clone AY13, BioLegend, Cat. No. 35290.

[0559] Experimental results:

[0560] CAR-T cells were obtained by lentiviral transduction and cultured in vitro for 9-12 days before CD107a degranulation assay. The CAR-T cells to be tested were incubated with target cells, monensin and CD107a antibody for 4 hours. The cell density of CAR-T cells and target cells was 2×10 5 cells / mL. The samples were then labeled with CD8 and EGFR antibodies and analyzed by flow cytometry. Analysis was performed using FlowJo software. The live cell gate (P1) was selected from the scatter plot to remove cell debris. Cells in gate P1 were then analyzed and gated to single, scattered cells (P2). CD8-positive cells were further selected in gate P2 (P3). Finally, the proportion of CD107a-positive cells among cells positive for EGFR antibodies (i.e., CAR-positive cells) was analyzed in gate P3. The results are shown in Table 3. Degranulation levels were significantly increased only after co-incubation with BAFF-R-positive cells, whereas no significant increase was observed after co-incubation with negative cell lines. Degranulation levels were higher in BAFF-R-positive cells from clones 1, 5, 14, 77, and 80, and were higher than those of the control Vay736 CAR (the CAR scFV sequence is derived from lanalumab).

[0561] Table 3 Results of CD107a degranulation effects of different target cells on CAR-T cells in each group.

[0562] Example 8. In vitro cell killing experiment

[0563] Experimental purpose and principle:

[0564] In vitro cell killing experiments were conducted using human B-cell acute lymphoblastic leukemia (NALM6) and RS4 cells, mucoepidermoid carcinoma (MEC1) cells, and mantle cell lymphoma (JEKO1) cells as BAFF-R-positive target cells, and MOLT4 cells as BAFF-R-negative target cells to evaluate the antigen-specific killing ability of BAFF-R CAR-T cells. These target cells were stably expressed in firefly luciferase using lentiviral transduction. Therefore, luciferase activity in the sample can reflect the number of target cells. CAR-T cells and target cells were co-incubated and cultured. When target cells are killed by CAR-T cells, luciferase is released and quickly inactivated (the half-life of firefly luciferase is approximately 0.5 hours). If target cells are not killed or inhibited by CAR-T cells, more luciferase will be produced as the target cells proliferate and luciferase expression continues. Therefore, luciferase activity can be used to monitor target cell killing by CAR-T cells.

[0565] Brief experimental steps for in vitro cell killing:

[0566] 1) The target cells were centrifuged at 500 g for 5 min at room temperature, the supernatant was discarded, and the cells were resuspended in 1640+10% FBS medium to a volume of 1x10 5 cells / mL; 100 μL of target cells were added to each well of a 96-well plate;

[0567] 2) Based on the CAR-T cell positivity and effector-target ratio of the CAR-T sample to be tested, 100 μL of CAR-T cells were added to each well of a 96-well plate and mixed with the target cells. The cells were then incubated in a CO2 incubator for 24 hours.

[0568] 3) Use a luciferase detection kit to detect the luciferase activity in each well of the sample.

[0569] Main samples and reagents:

[0570] BAFF-R positive target cells: NALM6-luc, RS4-luc, MEC1-luc, JEKO1-luc

[0571] BAFF-R negative target cells Raji-luc, CCRF-luc;

[0572] Steady-Glo Luciferase Assay System, Promega, Cat. No. E2520.

[0573] Experimental results:

[0574] CAR-T cell samples and a fixed number of target cells (1x10 4 After mixing the cells at different effector-target ratios (E:T), the cells were incubated for 24 hours, and the luciferase activity (RLU) in the samples was measured. Since luciferase activity reflects the number of target cells in the sample, the CAR-T cell's ability to kill or inhibit target cells can be determined by changes in luciferase activity in the sample. The lower the luciferase activity reading (RLU), the more target cells were killed.

[0575] As shown in Figure 8, all CAR-T cell samples were able to effectively kill BAFF-R-positive target cells, with a killing ability similar to that of the control CAR Vay736. Clone 14 showed a certain degree of killing against MOLT4-negative cells. Clones 1, 5, 77, and 80 showed no nonspecific killing against negative cells.

[0576] Through CD107a degranulation experiments and in vitro cell killing experiments, it was found that clones 1, 5, 77, and 80 had similar in vitro functions to the control CAR Vay736. The CAR specificity and in vivo efficacy of these three clones were further evaluated below.

[0577] Example 9. Determination of the performance of organ-derived tumor cells stimulating CAR cell degranulation

[0578] CD107a is a marker for intracellular microvesicles. When microvesicles loaded with granzymes fuse with the cell membrane, the amount of CD107a on the cell membrane increases. Blocking their recovery with monesin (purchased from BioLegend) can quantitatively reflect the intensity of microvesicle release. When CAR-T cells are stimulated by target antigens on target cells, granzymes are released, and flow cytometry can be used to detect the increase in CD107a to determine the activation of T cells by target cells.

[0579] Brief experimental steps of CD107a degranulation:

[0580] 1) The CAR-T cells and target cells to be tested were centrifuged at 300g for 5 minutes at room temperature, the supernatant was discarded, and the cells were resuspended in 1640 medium + 10% FBS to a concentration of 2x10 6 cells / mL;

[0581] 2) Add 100 μL of the CAR-T cells to be tested and 100 μL of the target cells to a 96-well plate and mix well.

[0582] 3) Add 1.5 μL of PE / Cy7 anti-human CD107a antibody and 0.2 μL of monensin to each well of cells, and then place them in a cell culture incubator (37°C, 5% CO2 and incubate for 4 hours);

[0583] 4) After incubation, centrifuge at 300 g for 5 min at 4°C, discard the supernatant, and wash the cells twice with 200 μL PBS.

[0584] 5) Resuspend the cells in 100 μL PBS and add 1.5 μL BV421 anti-human CD8 and 1.5 μL APC anti-human EGFR antibodies, mix well, and incubate on ice in the dark for 20 minutes.

[0585] 6) After incubation, wash the cells three times with 200 μL PBS; resuspend in 100 μL PBS and analyze by flow cytometry.

[0586] Main samples and reagents:

[0587] Target cells: NALM6, 293T, A375, HepG2, NCHI460, OVCAR-3, Raji, HCT116, Panc-1, MDA-MB-468, KATOIII

[0588] Fetal bovine serum (FBS), Gibco, Cat. No. 10099141;

[0589] Monensin,BioLegend,Cat.No.420701;

[0590] PE / Cy7 mouse anti-human CD107a, BD, Cat.No.561348;

[0591] BV421 mouse anti-human CD8,BD,Cat.No.301036;

[0592] APC anti-human EGFR Antibody, Clone AY13, BioLegend, Cat. No. 35290.

[0593] Experimental results:

[0594] CAR-T cells were obtained by lentiviral transduction and cultured in vitro for 9-12 days before CD107a degranulation assay. The CAR-T cells to be tested were incubated with target cells, monensin and CD107a antibody for 4 hours. The cell density of CAR-T cells and target cells was 2×10 6cells / mL. The samples were then labeled with CD8 and EGFR antibodies and analyzed by flow cytometry. Analysis was performed using FlowJo software. The live cell gate (P1) was selected from the scatter plot to remove cell debris. Cells in gate P1 were analyzed and gated for single, dispersed cells (P2). CD8-positive cells (P3) were further selected in gate P2. Finally, the proportion of CD107a-positive cells among cells positive for EGFR antibodies (i.e., CAR-positive cells) was analyzed in gate P3. The analysis results are shown in Figure 13. BAFF-R CAR-T clones 1, 5, 77, and 80 showed a significant increase in degranulation levels only after co-incubation with BAFF-R-positive cells, whereas no significant increase in degranulation was observed after co-incubation with organ-derived tumor cell lines. This suggests that BAFF-R CAR-T cells are not easily activated by normal tissue and organ cells, demonstrating a high safety profile.

[0595] The above in vitro experiments demonstrated that CAR-T cells from candidate clones 1, 5, 77, and 80 effectively recognized the BAFF-R antigen on tumor cells and produced effective killing activity. In vitro negative cell validation demonstrated the absence of nonspecific activation and killing activity, demonstrating a good clinical safety profile. Repeated stimulation experiments demonstrated that the four candidate clones could efficiently expand in response to target antigen stimulation in vitro and possess the potential to maintain long-term, effective tumor cell killing.

[0596] Example 10. Antitumor efficacy experiment on NCG tumor-bearing mice

[0597] To further validate the antitumor efficacy of the four candidate clones in mice, we identified JEKO1-Luc, a tumor cell line expressing firefly luciferase (ffLuc), as a naturally occurring cell line with high BAFF-R expression. JEKO1-luc cells were inoculated via the tail vein into immunodeficient NCG mice. After tumors formed in the mice, the candidate clones were inoculated with CAR-T cells to verify their antitumor activity.

[0598] Brief test process:

[0599] a) Establishing a firefly luciferase (ffLuc) overexpressing cell line JEKO1-luc and verifying that it can normally express firefly luciferase;

[0600] b) culturing the cells to the logarithmic growth phase and collecting the cells;

[0601] c) 48 NCG mice were purchased, raised for 1 week, and injected with JEKO1-luc cells into the tail vein at a rate of 5×10 cells per mouse. 6cells / 200 μL PBS, and the general condition of the mice was observed every day after injection;

[0602] d) On day 10, mice were divided into groups and injected via the tail vein with CAR-T cells (3E+06CAR+ cells / mouse), control untransfected Mock-T cells (2E+07 cells / mouse), and PBS buffer.

[0603] e) Regular imaging observation, weighing, observing and recording the general condition of the mice, and regularly measuring the proliferation of T cells in the mouse blood.

[0604] Experimental results:

[0605] Figures 10 and 11 show the in vivo antitumor efficacy of candidate clones in mice. As shown in Figures 10A and 11, tumor burden continued to increase after infusion of PBS, mockT, 1500 (Vay CAR), 1499 (city of hope CAR), clones 77, and 80 CAR-T cells. Tumor burden after infusion of clone 1 CAR-T cells was slightly lower than that of PBS and mockT, but the inhibitory effect on tumor growth was weaker. Clone 5 significantly suppressed tumor burden. As shown in Figure 10B, the 35-day survival rate of clone 5 mice exceeded 80%. Figure 10C shows T cell proliferation in mouse blood. In vivo T cell proliferation was not significant for BS, mockT, 1500 (Vay CAR), 1499 (city of hope CAR), and clone 77 CAR. Clone 80 T cell proliferation was limited to the first two weeks, with a rapid decline in the latter stages of proliferation, which may contribute to its poor efficacy. Clones 1 and 5 maintained sustained and effective expansion in vivo, significantly outperforming the other clones, and clone 5 initiated expansion earlier than clone 1.

[0606] In summary, Clone 5 has the best in vivo antitumor efficacy and a good safety profile, making it a promising candidate for later clinical development. Clone 1 also has some antitumor efficacy and a good safety profile, making it a viable candidate.

[0607] Example 11 Screening of specific antibodies targeting BAFF-R protein

[0608] A fully human phage antibody library (including a fully human single-chain antibody library and a fully human single-domain antibody library) was used to enrich antibodies that specifically bind to BAFF-R by affinity panning. 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 Bio). Streptavidin-coupled magnetic beads were used as negative antigen to remove nonspecifically binding phage clones from the phage library. Phage clones that bind to BAFF-R antigen were then enriched using magnetic beads bound to BAFF-R antigen as positive antigen. This enrichment process was repeated 3-4 times. As shown in Table 4, 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.

[0609] Table 4. Recovery rate and enrichment factor of phage antibody library after each round of panning

[0610] Selected phage monoclonal clones were identified by ELISA using BAFFR-His-biotin as the positive antigen and Streptavidin (21125, Pierce) as the negative antigen to identify specific phage clones that bind only to BAFFR-His-biotin but not to Streptavidin. Furthermore, phage clones that bind specifically to K562-BAFF-R but not to K562 were identified by FCM using the BAFF-R-overexpressing cell line K562-BAFF-R as the positive cell line and K562 as the negative cell line. Phage clones exhibiting bispecific binding were designated as candidate positive clones based on the ELISA and FCM results. The amino acid sequences of candidate positive clones were determined after sequencing.

[0611] Example 12 Analysis of Cytotoxic Activity of Candidate Clones

[0612] To identify clones with superior T-cell dependent cellular cytotoxicity (TDCC) activity, candidate clones were combined with the CD3 antibody OKT3 to create a 1+1 bispecific antibody. This means that both the BAFF-R and CD3 antibodies are monovalent within the bispecific antibody molecule. The molecular configuration design is shown in Figure 12: the OKT3 molecular structure is scFab, while the BAFF-R candidate clones have a VH or scFab structure (single-domain antibodies are constructed as VH, single-chain antibodies are constructed as scFab). To facilitate antibody purification, the Fc of the antibody was retained. To eliminate ADCC, ADCP, and CDC effects, the Fc used the IgG4 subtype combined with S228P / F234A / L235A mutations. To reduce the risk of Fc mispairing, a knobs-into-holes structure was introduced, with knobs on the CD3 antibody side and holes on the BAFF-R antibody side. After codon optimization, the antibody amino acid sequences were constructed and incorporated into pcDNA3.4 plasmids (OKT3 scFab-Fc PAA Ks, α-BAFF-R VH-Fc PAA Hs, and α-BAFF-R scFab-Fc PAA Hs). The expression plasmids expressing the OKT3 scFab antibody and either the BAFF-R VH or scFab antibody were co-transfected into CHOS cells. After 7 days of suspension culture, the expression supernatant was harvested. The supernatant was centrifuged at 10,000 g for 30 minutes to remove debris and then filtered through a 0.22 μm filter for protein A affinity chromatography. The purified antibody was concentrated and exchanged with PBS for TDCC activity analysis.

[0613] α-BAFF-R × The activity analysis of α-CD3 bispecific antibody was carried out in two steps.

[0614] First, the expression of α-BAFF-R was evaluated based on the TDCC reporter gene cell line (Jurkat-NFAT-luc2, Nanjing IASO Biotechnology Co., Ltd.). × α-CD3 bispecific antibody activates CD3 signaling pathway.

[0615] T cells were used as effector cells to evaluate α-BAFF-R × The killing activity of α-CD3 bispecific antibody on BAFF-R positive target cells. ×α-CD3 bispecific antibodies were sorted to identify α-BAFF-R clones with better activity. The CD3 signaling pathway activation assay based on the TDCC reporter gene cell line was briefly described as follows: K562 cells stably overexpressing BAFF-R (K562-BAFF-R) were used as positive target cells, K562 cells were used as negative control cells, and Jurkat-NFAT-luc2 cells were used as effector cells. 2E4 positive target cells or control cells were added to each well of a 96-well plate, and an equal number of effector cells were added to each well. The bispecific antibodies were diluted 5-fold and added to the 96-well plate. The final dilution concentration of the antibodies 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) was added. TM EX Luciferase Assay System, E8120, Promega) was used to detect the luminescence intensity, and the EC50 curve of luminescence intensity changing with antibody concentration was calculated.

[0616] As shown in Figure 13 , the bispecific antibodies formed by combining clone 02, clone 04, clone 05, clone 06, and clone 07 can all mediate the activation of the CD3 signaling pathway in K562-BAFF-R target cells, among which clone 02, clone 05, and clone 06 have stronger activation activity. As shown in Figure 14 , when BAFF-R is not expressed on the surface of the target cells, these five bispecific antibodies cannot cause activation of the CD3 signaling pathway.

[0617] The brief experimental method of the T cell-based target cell killing assay is as follows: Nalm6 stably overexpressing luciferase (Nalm6-luc) is used as the positive target cell, and K562 stably overexpressing luciferase (K562-luc) is used as the negative control cell; T cells are used as effector cells. PBMCs are positively sorted 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 serial dilution of the bispecific antibody, it is added to the 96-well plate. The final dilution concentration of the antibody is 10nM, 2nM, 0.4nM, 0.08nM, 0.016nM, 0.0032nM, 0.00064nM, and 0.00nM. After incubation at 37°C for 14-16 hours, the luciferase substrate (ONE-Glo TMEX 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 versus concentration.

[0618] As shown in Figure 15, the bispecific antibodies formed by combining clones 02, 04, 05, 06, and 07 can mediate cytotoxicity against target cells called Nalm6 by PBMCs. Clone 05 showed the strongest activity, while clone 07 showed weaker activity. No activity was observed with clone 02. As shown in Figure 16, when BAFF-R was not expressed on the target cells, none of the five bispecific antibodies induced cytotoxicity against the target cells.

[0619] Example 13 Study on Antibody Cross-species Reactivity

[0620] According to the BAFF-R protein information provided by Uniprot, analysis and comparison showed that the amino acid similarities between the extracellular region of human BAFF-R and those of cynomolgus monkey and mouse were 96.16% and 48.57%, respectively, and the amino acid similarity between the extracellular region of cynomolgus monkey BAFF-R and mouse was 47.14% (Table 5, Figure 17).

[0621] Table 5. Comparison of amino acid similarities between the extracellular regions of BAFF-R in humans, monkeys, and mice

[0622] To determine the cross-binding activity of BAFF-R antibodies against monkey and mouse, an enzyme-linked immunosorbent assay (ELISA) was used to test the binding strength of the antibodies to mouse and monkey BAFF-R antigens. A monoclonal antibody was constructed from clone 5, with the Fc residues S228P / L235E. The antibody was expressed in CHOS and used for cross-species reactivity studies. BAFF-R antigens from different species (BAFFR-human-bio-his, BAF-HM40RB, KACTUS; BAFFR-cyno-His, BAF-CM10R, KACTUS; and BAFFR-mouse-rFc, 50353-M31H, Sino) were diluted to 2 μg / mL in PBS and coated onto a 96-well ELISA plate at 100 μL / well overnight at 4°C. The plates were blocked with 3% BSA at 37°C for 2 hours. The antibody was serially diluted 5-fold (500 nM, 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, 0.0064 nM, and 0.00 nM) at 100 μL / well and incubated at 37°C for 2 hours. HRP-conjugated anti-human IgG antibody (HRP Donkey anti-human IgG, 410902, Biolegend) was diluted 1:2000 at 100 μL / well and incubated at 37°C for 1 hour. After washing three times with 0.01% PBST, 100 μL / well of TMB substrate was added and incubated in the dark for 3 minutes. The reaction was terminated with 50 μL / well of 2 M H2SO4 and A450 was measured using a microplate reader.

[0623] Calculate the maximum binding value and binding EC according to the change of A450 with concentration gradient 50 As shown in Table 6, clone 5 binds to the EC of human, monkey and mouse BAFF-R. 50 The values ​​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.

[0624] Table 6. Cross-binding data of Antibody No. 5 with human, monkey, and mouse BAFF-R

[0625] Example 14 Preferred cloned bispecific antibody structure design and preparation

[0626] The preferred BAFF-R single-domain antibody clone was combined with the CD3 antibody SP34 to construct a bispecific antibody. The three configurations are shown in Figure 18. Configuration 1 is a 1+1 structure, that is, in one bispecific antibody molecule, both the BAFF-R antibody and the CD3 antibody are monovalent (Figure 18A); Configuration 2 is a 2+1 structure, that is, in one bispecific antibody molecule, the BAFF-R antibody is bivalent, while the CD3 antibody is monovalent (Figure 18B); Configuration 3 is a 2+2 structure, that is, in one bispecific antibody molecule, both the BAFF-R antibody and the CD3 antibody are bivalent (Figure 18C). In the three configurations, the BAFF-R antibody is a single-domain antibody, while the CD3 antibody SP34 is a Fab structure.

[0627] Similarly, to enhance the half-life of the antibody and facilitate antibody purification, the Fc portion of the antibody was retained. At the same time, to eliminate ADCC, ADCP, and CDC effects, the Fc used the IgG4 subtype combined with S228P / F234A / L235A mutations. In the asymmetric structure, 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. The plasmids were named SP34-H PAA Ks, 05VH-SP34-H PAA Ks, 05VH-SP34-H PAA, SP34-L and 05VH-hFc PAA Hs, respectively. 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 7).

[0628] CHOS cells were co-transfected with the plasmid combinations listed in Table 7, and expression supernatants were collected after 7 days of suspension culture. The supernatants were centrifuged at 10,000 g for 30 minutes to remove debris and filtered through a 0.22 μm filter membrane before purification by Protein A affinity chromatography. The purified antibodies were concentrated and the buffer exchanged for cationic chromatography using MES buffer. The Protein A-purified antibodies were further purified using a cationic chromatography column to remove mismatched antibodies. The fully purified antibodies were used for analyses of binding kinetics, in vitro and in vivo biological activity, and other analyses.

[0629] Table 7. Combination of bispecific antibody expression plasmids

[0630] Example 15 Study on the binding kinetics of antibodies and antigens

[0631] To investigate the binding kinetics of the α-BAFF-R × α-CD3 bispecific antibody to human BAFF-R and CD3 proteins, the dissociation constants and affinities of the bispecific antibody binding to BAFF-R and CD3 proteins were analyzed using Bio-Layer Interferometry (BLI) based on the ForteBio Octet molecular interaction analysis system.

[0632] His-tagged human BAFF-R antigen (BAR-H52H3, Acro) was diluted to 5 μg / mL in binding buffer (1×PBS containing 0.02% Tween and 0.1% BSA, pH = 7.4) and bound to a biosensor HIS1K (18-0038, ForteBio) with a thickness of 0.25 nm. The antigen-bound sensor was equilibrated in sample diluent (1×PBS containing 0.02% Tween and 0.1% BSA, pH = 7.4) for 180 seconds, placed in a gradient dilution solution of antibody (200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM) for 60 seconds, and then placed in sample dilution solution for dissociation for 60-180 seconds. Finally, the sensor was regenerated stepwise using 10 mM glycine and binding buffer (1×PBS containing 0.02% Tween and 0.1% BSA, pH = 7.4). Data were analyzed using the Octet Molecular Interaction Analysis System's built-in data analysis system to determine binding and dissociation constants and affinity data. As shown in Table 8, the 05 monoclonal antibody has an affinity for human BAFF-R of 2.63 nM, while the affinities of the bispecific antibodies 0205-1, 0205-2, and 0205-3 for human BAFF-R are 1.78 nM, 1.12 nM, and 1.27 nM, respectively.

[0633] Table 8. Kinetic parameters of α-BAFF-R monoclonal antibody and α-BAFF-R × α-CD3 bispecific antibody binding to BAFF-R

[0634] 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 bound to a biosensor HIS1K (18-0038, ForteBio) with a thickness of 0.4 nm. After binding to the antigen, the sensor was equilibrated in 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 antibody (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM) for 60 sec, and then placed in sample dilution solution 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 BSA in 1× PBS (pH 7.4). Data were analyzed using the Octet Molecular Interaction Analysis System's built-in data analysis system to obtain binding and dissociation constants and affinity data. As shown in Table 9, the affinities of monoclonal antibody SP34 for human CD3E&D and human CD3E&G were 6.38 nM and 7.87 nM, respectively; the affinities of bispecific antibody 0205-1 for CD3E&D and human CD3E&G were 10.7 nM and 6.68 nM, respectively; the affinities of bispecific antibody 0205-2 for CD3E&D and human CD3E&G were 10.3 nM and 11.1 nM, respectively; and the affinities of bispecific antibody 0205-3 for CD3E&D and human CD3E&G were 10.6 nM and 9.03 nM, 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.

[0635] Table 9. Kinetic parameters of α-CD3 monoclonal antibody and α-BAFF-R×α-CD3 bispecific antibody binding to CD3E&D and CD3E&G

[0636] Example 16 Study on the activation activity of bispecific antibodies on CD3 signaling pathway

[0637] α-BAFF-R ×The first step for α-CD3 bispecific antibody to exert its biological function is bispecific antibody-mediated T cell activation, and T cell activation occurs in the presence of BAFF-R positive target cells, while in the absence of BAFF-R positive cells, T cells are not activated. Based on the TDCC reporter gene cell line (Jurkat-NFAT-luc2), the α-BAFF-R × Activation activity and specificity of α-CD3 bispecific antibody on CD3 signaling pathway. Human chronic myeloid leukemia cell line K562 (K562-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 as negative control cells, and Jurkat-NFAT-luc2 as effector cells. 2E4 positive target cells or negative control cells were added to each well of a 96-well plate, and an equal number 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 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 the EC50 curve of luminescence intensity changing with antibody concentration was calculated.

[0638] As shown in Figures 19-20 and Tables 10-13, the target cells were K562-BAFF-R with high BAFF-R expression. 0205-1, 0205-2, and 0205-3 could 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 showed significantly different activities in TDCC reporter gene cell activation, with 0205-3 having the best activity and EC 50 Between 0.02nM and 0.065; 0.205-2 is second, EC 50 The activity ranged from 0.06nM to 0.5; 0205-1 had the worst activity.

[0639] As shown in Figure 23 and Table 14, the three bispecific antibodies mediated low levels of TDCC reporter gene activation in BAFF-R-negative cells. Only at the highest concentrations, 0205-1 and 0205-3, did TDCC reporter gene cells show slight activation. Because 0205-3 is a bivalent CD3 antibody, this result suggests that 0205-3 may have certain safety risks, which require further evaluation in subsequent experiments.

[0640] Table 10. α-BAFF-R VH × Parameters of α-CD3 Fab bispecific antibody-mediated activation of K562-BAFF-R on TDCC reporter cells

[0641] Table 11. α-BAFF-R VH × Parameters of Nalm6-mediated activation of TDCC reporter cells by α-CD3 Fab bispecific antibody

[0642] Table 12. α-BAFF-R VH × Parameters of Mec-1 activation of TDCC reporter cells mediated by α-CD3 Fab bispecific antibody

[0643] Table 13. α-BAFF-R VH × Parameters of α-CD3 Fab bispecific antibody-mediated activation of TDCC reporter cells by Jeko-1

[0644] Table 14. α-BAFF-R VH × Parameters of K562-mediated activation of TDCC reporter cells by α-CD3 Fab bispecific antibody

[0645] Example 17 Study on the Cytotoxicity of PBMC to Target Cells Mediated by Bispecific Antibodies

[0646] 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 serial dilution of the antibody, it was added to the 96-well plate. The final dilution concentration 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, luciferase substrate (ONE-Glo TM Luminescence intensity was measured using the EX Luciferase Assay System (E8120, Promega), and the target cell killing efficiency was calculated. The EC50 curve, showing the killing efficiency as a function of concentration, was also calculated. In this assay, Regeneron's bispecific antibody REGN1979 targeting CD20 and CD3 was used as a positive control.

[0647] As shown in Figures 24-27 and Tables 15 and 16, the activity response rankings of the three bispecific antibodies in PBMCs from two donors were consistent. No cytotoxic activity against BAFF-R-positive tumor cells was observed for 0205-1 in either donor. The cytotoxic activity of 0205-2 and 0205-3 against tumor cells was relatively weak at 24 hours, but both achieved complete elimination of tumor cells at 48 hours.

[0648] Comprehensively comparing the activation activity on TDCC reporter gene cells and the clearance activity on BAFF-R tumor cells, the activity ranking of α-BAFF-R VH×α-CD3 Fab bispecific antibody was 0205-3≥0205-2>0205-1.

[0649] Table 15: α-BAFF-R VH × α-CD3 Fab bispecific antibody-mediated killing parameters of PBMC cells (Donor 301C) against Jeko-1 cells

[0650] Table 16: α-BAFF-R VH × α-CD3 Fab bispecific antibody-mediated killing parameters of PBMC cells (Donor 605C) against Jeko-1 cells

[0651] Example 18 Study on Bispecific Antibody-Mediated Cytokine Release from PBMC

[0652] 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 target cells was also designed. 2E4 positive target cells or control cells were added to each well of a 96-well plate. A 10-fold increase in the number of effector cells was also added to each well to achieve an effector-target ratio of 10:1. An equal number of effector cells were added to the solvent control group. The bispecific antibody was serially diluted 5-fold and added to the 96-well plate. The final dilution concentration of the antibody was 1 nM, 0.4 nM, 0.016 nM, and 0.0 nM. After incubation at 37°C for 24 and 48 hours, the cells were centrifuged at 500 g for 10 minutes, and the supernatants were carefully removed. The levels of cytokines IFN-γ (62HIFNGPEG, cisbio), IL-2 (62HIL02PEG, cisbio), TNFα (62HTNFAPEG, cisbio), and IL-6 (62HIL06PEG, cisbio) in the supernatants were measured by time-delayed fluorescence to assess the potency of bispecific antibody-mediated cytokine release from PBMCs. In this experiment, Regeneron's bispecific antibody REGN1979 targeting CD20 and CD3 was used as a positive reference, and SP34 monoclonal antibody was used as a negative reference antibody.

[0653] As shown in Figures 28-33, when co-incubated with BAFF-R-positive target cells and PBMCs, bispecific antibodies 0205-2 and 0205-3 can significantly induce PBMCs to release IFN-γ, IL-2, and TNFα, with levels slightly lower than those of the positive reference antibodies; in the absence of BAFF-R-positive target cells, the secretion of IFN-γ, IL-2, and TNFα was significantly reduced.

[0654] As shown in Figures 34 and 35 , 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.

[0655] Example 19 Antibody Pharmacokinetics Study

[0656] To study the metabolic kinetics of the antibody, 6-8 week old female SD rats, weighing 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 was intravenous. Before and after administration (5 min, 30 min, 2 hr, 6 hr, 24 hr, 72 hr, 120 hr, 168 hr, 240 hr, 336 hr), 200 μL of blood was collected from the jugular vein. After standing at room temperature for about 1 hour, the serum was collected by centrifugation and stored at -70 ° C. Serum antibody concentrations were determined using a sandwich ELISA method. Goat anti-human IgG Fc antibody (609-101-017, Rockland) was diluted to 2.5 μg / mL and coated on an ELISA plate. Serum was diluted 20-fold for quantitative detection. Peroxidase-conjugated secondary antibody, anti-human IgG (Fab specific)-peroxidase (A0293, Sigma), was diluted 1:5000 and bound to the Fab end of the serum antibody. Serum antibody concentrations at each time point were calculated using a standard curve and the dilution factor.

[0657] After administration, no abnormal reactions were observed in any SD rats. The serum antibody concentrations of bispecific antibody 0205-2 at each time point are shown in Table 17 and Figure 36 , and the antibody half-life was approximately 13.7 days. The serum antibody concentrations of bispecific antibody 0205-3 at each time point are shown in Table 18 and Figure 37 , and the antibody half-life was approximately 10.3 days.

[0658] Table 17. Concentrations and pharmacokinetic parameters of bispecific antibody 0205-2 in SD rat serum

[0659] Table 18. Concentrations and pharmacokinetic parameters of bispecific antibody 0205-3 in SD rat serum

[0660] Example 20 Antibody in vivo efficacy study

[0661] Considering the safety of CD3 antibodies, we selected bispecific antibody 0205-2 containing monovalent CD3 antibodies and reconstructed a subcutaneous tumor model in NCG mice based on PBMC for in vivo anti-tumor efficacy evaluation. As shown in Table 19, on Day 0, Jeko-1 tumor cells were subcutaneously inoculated at a 1:1 ratio with a seeding dose of 5E6 / mouse; on Day 3, PBMC cells were intraperitoneally infused at a dose of 1E7 / mouse; and the tumor volume reached 110mm. 3The 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 when the tumor volume reached the standard.

[0662] Table 19. Bispecific antibody efficacy test design based on PBMC-based reconstruction of NCG mouse subcutaneous tumor model

[0663] As shown in Figure 38, the tumor volume reached 110 mm 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. In the 1 mpk dose group and the PBS group, the changes in the tumor volume of mice were relatively consistent, while in the 10 mpk dose group, the tumor volume of one mouse reached 650 mm 3 , it can still disappear quickly and completely (Figures 39-41).

[0664] As shown in Figure 42, during the experimental observation period, no mouse deaths were found in the 1mpk dose group and the 10mpk dose group. No obvious weight loss, decreased activity, arched back, ruffled hair, or other phenomena were observed in the mice, indicating that the mice tolerated the drug well.

[0665] References

[0666] 1.Wang S,Wang L,Hu J,Qian W,Zhang X,Hu Y,Zhu Q,Chen B,Wu D,Chang CH et al:Outcomes in refractory diffuse large B-cell lymphoma:results from a multicenter real-world study in China.Cancer Commun(Lond)2021,41(3):229-239.

[0667] 2.Spiegel JY,Patel S,Muffly L,Hossain NM,Oak J,Baird JH,Frank MJ,Shiraz P,Sahaf B,Craig J et al:CAR T cells with dual targeting of CD19 and CD22 in adult patients with recurrent or

[0668] refractory B cell malignancies:a phase 1trial. Nat Med 2021,27(8):1419-1431.

[0669] 3. Rodig SJ, Shahsafaei A, Li B, Mackay CR, Dorfman DM: BAFF-R, the major B cell-activating factor receptor, is expressed on most mature B cells and B-cell lymphoproliferative disorders. Hum Pathol 2005, 36(10): 1113-1119.

[0670] Sequences of the present invention

[0671] CD8α signal peptide nucleic acid sequence SEQ ID NO.1

[0672] CD8α signal peptide protein sequence SEQ ID NO.2

[0673] CD8α hinge region nucleic acid sequence SEQ ID NO.3

[0674] CD8α hinge region protein sequence SEQ ID NO.4

[0675] CD8α transmembrane region nucleic acid sequence SEQ ID NO.5

[0676] CD8α transmembrane protein sequence SEQ ID NO.6

[0677] 4-1BB intracellular domain nucleic acid sequence SEQ ID NO.7

[0678] 4-1BB intracellular domain protein sequence SEQ ID NO.8

[0679] CD3z intracellular signaling domain nucleic acid sequence SEQ ID NO.9

[0680] CD3z intracellular signaling domain protein sequence SEQ ID NO.10

[0681] Cleavage peptide T2A nucleic acid sequence SEQ ID NO.11

[0682] Cleavage peptide T2A protein sequence SEQ ID NO.12

[0683] CSF2RA signal nucleic acid sequence SEQ ID NO.13

[0684] CSF2RA signal protein sequence SEQ ID NO.14 MLLLVTSLLLCELPHPAFLLIP

[0685] EGFRt nucleic acid sequence SEQ ID NO.15

[0686] EGFRt protein sequence SEQ ID NO.16

[0687] P2A nucleic acid sequence SEQ ID NO. 17

[0688] P2A protein sequence SEQ ID NO.18

[0689] BAFF-R-5 CAR VHH nucleic acid sequence SEQ ID NO.19

[0690] BAFF-R-5 CARVHH amino acid sequence SEQ ID NO.20

[0691] Vay736 nucleic acid sequence SEQ ID NO.21

[0692] Vay736 amino acid sequence SEQ ID NO.22

[0693] City of hope CAR scFV nucleic acid sequence SEQ ID NO.23

[0694] City of hope CAR scFV amino acid sequence SEQ ID NO.24

[0695] 05VH amino acid sequence SEQ ID NO.25

[0696] 05VH nucleotide sequence SEQ ID NO.26

[0697] 05VH CDR1 SEQ ID NO.27 GFTFSTSG

[0698] 05VH CDR2 SEQ ID NO.28 ISSSSSTI

[0699] 05VH CDR3 SEQ ID NO.29 VTQSCGAGKCYPGV

[0700] 01VL amino acid sequence SEQ ID NO.30

[0701] 01VL nucleotide sequence SEQ ID NO.31

[0702] 01VH amino acid sequence SEQ ID NO.32

[0703] 01VH nucleotide sequence SEQ ID NO.33

[0704] The amino acid sequence of the scFv (VL-VH) contained in the CAR constructed by clone 1 (01) is SEQ ID NO.34

[0705] The nucleotide sequence of the scFv (VL-VH) contained in the CAR constructed by clone 1 (01) is SEQ ID NO.35

[0706] 01VL CDR1 SEQ ID NO.36 QKISSY

[0707] 01VL CDR2 AAS

[0708] 01VL CDR3 SEQ ID NO.37 QQSYSPPTT

[0709] 01VH CDR1 SEQ ID NO.38 GYTFTSYG

[0710] 01VH CDR2 SEQ ID NO.39 ISAYNGNT

[0711] 01VH CDR3 SEQ ID NO.40 ARNTYYYDSSGYPNDAFDI

[0712] SEQ ID NO.41 hFc PAA Hs amino acid sequence

[0713] SEQ ID NO.42 hFc PAA Hs nucleotide sequence

[0714] SEQ ID NO.43 hFc PAA Ks amino acid sequence

[0715] SEQ ID NO.44 hFc PAA Ks nucleotide sequence

[0716] SEQ ID NO.45 OKT3 VH amino acid sequence

[0717] SEQ ID NO.46 OKT3 VH nucleotide sequence

[0718] SEQ ID NO.47 OKT3 VK amino acid sequence

[0719] SEQ ID NO.48 OKT3 VK nucleotide sequence

[0720] SEQ ID NO.49 OKT3 scFab amino acid sequence

[0721] SEQ ID NO.50 OKT3 scFab nucleotide sequence

[0722] SEQ ID NO.51 05VH-hFc PAA Hs amino acid sequence

[0723] SEQ ID NO.52 05VH-hFc PAA Hs nucleotide sequence

[0724] SEQ ID NO.53 OKT3 scFab-H PAA Ks amino acid sequence

[0725] SEQ ID NO.54 OKT3 scFab-H PAA Ks nucleotide sequence

[0726] SEQ ID NO.55 hFc PE amino acid sequence

[0727] SEQ ID NO.56 hFc PE nucleotide sequence

[0728] SEQ ID NO.57 05VH-hFc PE amino acid sequence

[0729] SEQ ID NO.58 05VH-hFc PE nucleotide sequence

[0730] SEQ ID NO.59 human BAFF-R amino acid sequence

[0731] SEQ ID NO.60 Cynomolgus BAFF-R amino acid sequence

[0732] SEQ ID NO.61 mouse BAFF-R amino acid sequence

[0733] SEQ ID NO.62 human BAFF-R extracellular domain amino acid sequence

[0734] SEQ ID NO.63 Cynomolgus BAFF-R extracellular domain amino acid sequence

[0735] SEQ ID NO.64 Mouse BAFF-R extracellular domain amino acid sequence

[0736] SEQ ID NO.65 SP34 VH amino acid sequence

[0737] SEQ ID NO.66 SP34 VH nucleotide sequence

[0738] SEQ ID NO.67 SP34 VL amino acid sequence

[0739] SEQ ID NO.68 SP34 VL nucleotide sequence

[0740] SEQ ID NO.69 SP34-H PE amino acid sequence

[0741] SEQ ID NO.70 SP34-H PE nucleotide sequence

[0742] SEQ ID NO.71 SP34-L amino acid sequence

[0743] SEQ ID NO.72 SP34-L nucleotide sequence

[0744] SEQ ID NO.73 SP34-H PAA Ks amino acid sequence

[0745] SEQ ID NO.74 SP34-H PAA Ks nucleotide sequence

[0746] SEQ ID NO.75 05VH-SP34-H PAA amino acid sequence

[0747] SEQ ID NO.76 05VH-SP34-H PAA nucleotide sequence

[0748] SEQ ID NO.77 05VH-SP34-H PAA Ks amino acid sequence

[0749] SEQ ID NO.78 05VH-SP34-H PAA Ks nucleotide sequence

[0750] SEQ ID NO.79 SP34 H-CDR1

[0751] SEQ ID NO.80 SP34 H-CDR2

[0752] SEQ ID NO.81 SP34 H-CDR3

[0753] SEQ ID NO.82 SP34 L-CDR1

[0754] SP34 L-CDR2

[0755] SEQ ID NO.83 SP34 L-CDR3

[0756] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A single domain antibody targeting 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.27, CDR2 shown in SEQ ID NO.28, and CDR3 shown in SEQ ID NO.29; 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. An antibody targeting B cell activating factor receptor (BAFF-R), characterized in that The heavy chain variable region of the antibody comprises the following complementarity determining regions CDR: CDR1 shown in SEQ ID NO.27, CDR2 shown in SEQ ID NO.28 and CDR3 shown in SEQ ID NO.29; or The light chain variable region of the antibody comprises the following complementary determining regions CDR: CDR1 shown in SEQ ID NO.36, CDR2 shown in AAS, and CDR3 shown in SEQ ID NO.37, and the heavy chain variable region of the antibody comprises the following complementary determining regions CDR: CDR1 shown in SEQ ID NO.38, CDR2 shown in SEQ ID NO.39, and CDR3 shown in SEQ ID NO.40; 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.

3. The antibody according to claim 2, characterized in that The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO.25 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.

4. The antibody according to claim 2, characterized in that The light chain variable region has an amino acid sequence as shown in SEQ ID NO.30, 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, and the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO.32, 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.

5. A chimeric antigen receptor, characterized in that The antigen binding domain of the chimeric antigen receptor comprises an antigen binding fragment targeting BAFF-R, and the heavy chain variable region of the antigen binding fragment comprises the following complementary determining regions CDR: CDR1 shown in SEQ ID NO.27, CDR2 shown in SEQ ID NO.28, and CDR3 shown in SEQ ID NO.29; Or the light chain variable region of the antigen-binding fragment comprises the following complementary determining regions CDR: CDR1 shown in SEQ ID NO.36, CDR2 shown in AAS, and CDR3 shown in SEQ ID NO.37, and the heavy chain variable region of the antibody comprises the following complementary determining regions CDR: CDR1 shown in SEQ ID NO.38, CDR2 shown in SEQ ID NO.39, and CDR3 shown in SEQ ID NO.

40.

6. A multispecific antibody, characterized in that: The multispecific antibody comprises: the single domain antibody targeting BAFF-R as claimed in claim 1 or the antibody targeting BAFF-R as claimed in claim 2.

7. 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.27, CDR2 shown in SEQ ID NO.28, and CDR3 shown in SEQ ID NO.

29.

8. The bispecific antibody according to claim 7, 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).

9. A recombinant protein, characterized in that The recombinant protein has: (i) the single domain antibody targeting BAFF-R according to claim 1, the antibody targeting BAFF-R according to any one of claims 2 to 4, the chimeric antigen receptor according to claim 5, the multispecific antibody according to claim 6, or the bispecific antibody according to claim 7; 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.

10. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes a polypeptide selected from the group consisting of: (1) the single domain antibody targeting BAFF-R as described in claim 1, the antibody targeting BAFF-R as described in any one of claims 2 to 4, the chimeric antigen receptor as described in claim 5, the multispecific antibody as described in claim 6, or the bispecific antibody as described in claim 7; or (2) The recombinant protein according to claim 9.

11. A carrier, characterized in that The vector contains the nucleic acid molecule according to claim 10.

12. A host cell, characterized in that The host cell contains the vector according to claim 11, or the exogenous nucleic acid molecule according to claim 10 is integrated into the chromosome.

13. An engineered immune cell, characterized in that: The immune cell contains the vector according to claim 11, or has the exogenous nucleic acid molecule according to claim 10 integrated into its chromosome, or expresses the antibody according to claim 1 or the chimeric antigen receptor according to claim 5.

14. An immunoconjugate, characterized in that: The immunoconjugate contains: (a) an antibody portion, wherein the antibody portion is a single domain antibody targeting BAFF-R as described in claim 1, an antibody targeting BAFF-R as described in any one of claims 2 to 4, a multispecific antibody as described in claim 6, or a bispecific antibody as described in claim 7; 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.

15. A pharmaceutical composition or formulation comprising: (i) the single domain antibody targeting BAFF-R as described in claim 1, the antibody targeting BAFF-R as described in any one of claims 2 to 4, the multispecific antibody as described in claim 6, or the bispecific antibody as described in claim 7, the recombinant protein as described in claim 9, the engineered immune cell as described in claim 13, or the immunoconjugate as described in claim 14; and (ii) a pharmaceutically acceptable carrier.

16. 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 claim 1, an antibody targeting BAFF-R as described in any one of claims 2-4, a multispecific antibody as described in claim 6, or a bispecific antibody as described in claim 7, a recombinant protein as described in claim 9, an engineered immune cell as described in claim 13, or an immunoconjugate as described in claim 14, 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 BAFF-R related diseases.

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