Chimeric antigen receptor molecule that specifically recognizes BAFF-R and its use

Abstract

This application discloses a chimeric antigen receptor molecule that specifically recognizes BAFF-R, a nucleic acid encoding the receptor molecule, an engineered immune effector cell containing the receptor molecule, a pharmaceutical composition, and uses thereof. The receptor molecule comprises a domain that specifically recognizes BAFF-R and an activation stimulatory domain, and can activate downstream signaling pathways after specifically recognizing and binding to BAFF-R, thereby triggering, promoting, or enhancing immune response reactions against BAFF-R, ultimately achieving the purpose of treating or preventing diseases caused by imbalance of the B cell BAFF-BAFF-R signaling pathway.

Description

Submission of the sequence list in ASCII text format.

[0001] The entire contents submitted in the following ASCII text file are incorporated herein by reference: Sequence listing in computer-readable format (CRF) (filename: TPF02324-sequence listing.txt, date: May 12, 2022, size: 12KB). [Technical Field]

[0002] This application relates to an engineered specific antigen-recognizing receptor molecule and engineered immunoeffector cells containing the receptor molecule. [Background technology]

[0003] In recent decades, conventional tumor treatments (surgery, chemotherapy, radiation therapy, etc.) have advanced significantly, but major challenges remain in terms of survival rates, recurrence, and metastasis. With the rapid advancements in tumor biology and immunology, immunotherapy has become a new star in tumor treatment in recent years. Among these, chimeric antigen receptor T-cell (CAR-T) therapy is currently one of the most promising tumor immunotherapies.

[0004] T lymphocytes play a major role in the tumor immune response and possess extremely potent killing ability against tumor cells, but this role is MHC-restricted. Using genetic engineering techniques, single-chain antibodies that recognize target antigens without MHC restriction are assembled into a CAR structure by linking them with hinge regions, transmembrane domains, and T cell activation motifs. This CAR structure is then incorporated into T cells using vectors such as viruses, ultimately causing the T cells to specifically bind to tumor cell surface-associated antigens and become activated, i.e., CAR-T cells. CAR-T cells directly kill tumor cells by releasing perforin, granzyme B, etc., but activated CAR-T cells also release cytokines, mobilizing endogenous immune cells in the human body to kill tumor cells and effectively inhibit the tumor. Simultaneously, immune memory T cells are formed, enabling the acquisition of a specific long-lasting antitumor mechanism.

[0005] Generally, chimeric antigen receptors consist of an extracellular domain, a transmembrane domain, and an activation-stimulating domain. Typically, the extracellular domain consists of an antigen recognition domain and a hinge region that plays a linking role. The antigen recognition domain is the basis for the CAR's specific binding to tumor antigens, and its main structure is scFv, which is usually composed of the light chain (VL) and heavy chain (VH) of a monoclonal antibody linked via polypeptides, maintaining the antibody's specificity and affinity for the antigen. The hinge region links the scFv to the transmembrane domain, and the hinge region of most CARs is derived from the IgG hinge or the CD8α / CD28 extracellular domain. The transmembrane domain links the CAR's extracellular domain to the intracellular activation-stimulating domain, anchoring the receptor to the T cell membrane. Commonly used transmembrane domains include those derived from CD4, CD8α, CD28, and CD3ζ. The activation-stimulating domain consists of an intracellular co-stimulatory domain and a signaling domain. The co-stimulatory domain is typically derived from the CD28 receptor family (CD28, ICOS) or the tumor necrosis factor receptor family (4-1BB, OX40, CD27), while the signaling domain is typically a T cell receptor TCR / CD3ζ chain containing immune receptor tyrosine-based activation motifs (ITAMs). The activation-stimulating domain mediates T cell signaling, playing a crucial role in activating and proliferating T cells until they ultimately complete tumor death.

[0006] B-cell malignancies are highly prevalent, with approximately 300,000 new cases worldwide annually, representing an annual growth rate of 5%–7%. Chimeric antigen receptor T cells (CAR-T) are widely used to treat B-cell malignancies, and CD19-targeted CAR-T cell therapy is highly effective against refractory B-cell malignancies, with high response rates of 80%–90% in patients with acute lymphoblastic leukemia (ALL) and various non-Hodgkin lymphoma subtypes. However, only about 40%–50% of patients show long-term efficacy, and CD19 downregulation / negative relapse occurs in about 30% of patients. Downregulation of CD19 expression caused by gene modification, or even complete loss, or unrecognizable CD19 CAR-T cells due to shearing into other forms, all present significant challenges to CD19 CAR-T therapy. While the clinical prognosis for B-cell malignancies has improved significantly in recent years due to advances in immunotherapy and molecular targeted drugs, high recurrence rates remain a problem. Therefore, exploring other antigenic targets highly expressed on the surface of B cells is currently an important research direction for resolving relapsed and refractory B-cell malignancies.

[0007] B lymphocyte stimulator (BLyS), a member of the tumor necrosis factor (TNF) superfamily, is also known as TNF- and ApoL-related leukocyte-expressed ligand 1 (TALL-1). It is a TNF homolog that activates apoptosis, nuclear factor-κB, and c-Jun NH2-terminal kinase (THANK), a TNF superfamily member 13B (TNFSF13B), a B cell-activating factor (BAFF), and zTNF4. These are type II transmembrane proteins that can specifically bind to B cells and play crucial roles in B cell survival, proliferation, development, and differentiation. BAFF has three receptors: the BAFF receptor (BAFF-R), the B cell maturation antigen (BCMA), and the TNFR homology transmembrane activator and calcium modulator and cyclophilin ligand intergrator (TACI), all of which are type III transmembrane proteins. BCMA and TACI can bind not only to BAFF but also to a proliferation-inducing ligand (APRIL), another member of the TNF ligand family. BAFF-R, however, is the specific receptor for BAFF and plays a more important role in B lymphocyte regulation than the other two receptors.

[0008] BAFF-R (B-cell activating factor receptor), also known as tumor necrosis factor receptor superfamily member 13C (TNFRSF13C), is a type III transmembrane protein lacking a signal peptide and is a member of the tumor necrosis factor receptors (TNFR) superfamily on the B cell membrane. The publicly available human BAFF-R amino acid sequence consists of 184 amino acids in total length, with amino acids 1-78 being the extracellular domain, amino acids 79-99 being the transmembrane domain, and amino acids 100-184 being the intracellular domain. Its coding gene is located in the chromosome 22q13.1 region. BAFF-R is primarily expressed in B cells and promotes B cell survival and proliferation by activating the NF-κB pathway, and is highly expressed on the surface of various B cell malignant tumor cells.

[0009] Imbalances in the BAFF-BAFF-R signaling pathway can lead to immune imbalances in the body, including a range of lesions caused by abnormal or normal high expression of BAFF-R, such as autoimmune diseases, graft-versus-host diseases, and tumors. This target covers a wide range of stages in the B cell maturation process and can target all B cell malignancies except plasma cell diseases (multiple myeloma), making it highly applicable. Furthermore, since this target is not expressed in stem cells or B progenitor cells, it does not cause permanent damage to the recovery and functional reconstruction of normal B cells, making it very safe.

[0010] While groundbreaking advances in immunotherapy and molecularly targeted drugs have significantly improved the clinical prognosis of B-cell malignancies, high recurrence rates remain a problem. Therefore, to address relapsed and refractory B-cell malignancies, it is necessary to explore other antigen targets and therapeutic agents highly expressed on the surface of B cells. [Disclosure of the Invention]

[0011] This application provides a chimeric antigen receptor molecule (CAR molecule) that specifically recognizes BAFF-R, and engineered immune effector cells (e.g., chimeric antigen receptor T cells (CAR-T)) for diseases caused by imbalances in the B-cell BAFF-BAFF-R signaling pathway, such as B-cell malignancies. Studies have demonstrated that the BAFF-R targeted chimeric antigen receptor T cells of this application have extremely potent killing and in vivo antitumor effects against tumor cells and have potential for development.

[0012] Specifically, this application relates to the following:

[0013] 1. A chimeric antigen receptor molecule that specifically recognizes BAFF-R, comprising a specific recognition domain, an activation stimulus domain, and a transmembrane domain located between the specific recognition domain and the activation stimulus domain, wherein the specific recognition domain comprises an amino acid sequence selected from SEQ ID NOs. 6, SEQ ID NOs. 7, SEQ ID NOs. 8, SEQ ID NOs. 10, SEQ ID NOs. 11, and SEQ ID NOs. 12, or an amino acid sequence having approximately 70% or more sequence identity with them, for example, an amino acid sequence having approximately 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity with them.

[0014] 2. The specific recognition domain is A heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or an amino acid sequence having approximately 70% or more sequence identity with them, for example, an amino acid sequence having approximately 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity with them, A light chain variable region containing an amino acid sequence selected from SEQ ID NOs. 10, SEQ ID NOs. 11, and SEQ ID NOs. 12, or an amino acid sequence having approximately 70% or more sequence identity with them, for example, an amino acid sequence having approximately 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity with them. A chimeric antigen receptor molecule that specifically recognizes BAFF-R as described in item 1, including the above.

[0015] In some embodiments, the present application relates to a chimeric antigen receptor molecule that specifically recognizes BAFF-R, comprising a specific recognition domain, an activation-stimulating domain, and a transmembrane domain located between the specific recognition domain and the activation-stimulating domain, wherein the specific recognition domain comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising three complementarity-determining regions CDR H1, CDR H2, and CDR H3, where CDR H1 comprises the amino acid sequence shown in SEQ ID NO: 6, CDR H2 comprises the amino acid sequence shown in SEQ ID NO: 7, and CDR H3 comprises the amino acid sequence shown in SEQ ID NO: 8, the light chain variable region comprising CDR L1, CDR L2, and CDR L3, where CDR L1 comprises the amino acid sequence shown in SEQ ID NO: 10, CDR L2 comprises the amino acid sequence shown in SEQ ID NO: 11, and CDR L3 comprises the amino acid sequence shown in SEQ ID NO: 12. The aforementioned CDR H1, CDR H2, CDR H3 and CDR L1, CDR L2, and CDR L3 were determined according to Kabat et al. (Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3).

[0016] 3. A chimeric antigen receptor molecule that specifically recognizes BAFF-R as described in item 2, wherein the heavy chain variable region and the light chain variable region further comprise a human antibody framework region. In some embodiments, the human antibody framework region is a human universal framework region. In some embodiments, the human universal framework region comprises a kappa I subgroup (κ I subgroup) framework sequence of VL and a VH III subgroup (VH III subgroup) framework sequence. The framework region was determined according to Kabat et al. (Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3). In some embodiments, the human universal framework region comprises 1-15, 1-10, 2-9, 3-8, 4-7, or 5-6 amino acid changes.

[0017] 4. A chimeric antigen receptor molecule that specifically recognizes BAFF-R as described in item 2 or 3, wherein the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having approximately 70% or more sequence identity thereto, for example, an amino acid sequence having approximately 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity thereto, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having approximately 70% or more sequence identity thereto, for example, an amino acid sequence having approximately 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity thereto.

[0018] 5. The heavy chain variable region is linked to the light chain variable region (e.g., linked via a peptide chain such as a linker or a connector consisting of (G)n(S)m, where n or m can be any integer from 1 to 10) to form a scFv, and the scFv contains the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence having at least about 70% sequence identity thereto, for example, an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity thereto. The chimeric antigen receptor molecule specifically recognizing BAFF-R according to item 4.

[0019] 6. The activation-stimulating domain contains a signal transduction domain containing an immunoreceptor tyrosine activation motif. The chimeric antigen receptor molecule specifically recognizing BAFF-R according to any one of items 1 to 5.

[0020] 7. The signal transduction domain is derived from the intracellular region of a CD3ζ, CD3γ, CD3δ, CD3ε, FcεRIγ, FcεR1β, CD79α, CD79β, FcγRIIa, DAP10, or DAP12 molecule. The chimeric antigen receptor molecule specifically recognizing BAFF-R according to item 6. In some embodiments, the signal transduction domain contains the signal transduction domain of the intracellular region of a CD3ζ, CD3γ, CD3δ, CD3ε, FcεRIγ, FcεR1β, CD79α, CD79β, FcγRIIa, DAP10 or DAP12 molecule, or a variant thereof retaining the same function. In some embodiments, the signal transduction domain contains the signal transduction domain of the intracellular region of CD3ζ or FcεRIγ, or a variant thereof retaining the same function.

[0021] 8. The signal transduction domain contains the amino acid sequence shown in SEQ ID NO: 20, or an amino acid sequence having at least about 70% sequence identity thereto, for example, an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity thereto. The chimeric antigen receptor molecule specifically recognizing BAFF-R according to item 7.

[0022] 9. The chimeric antigen receptor molecule that specifically recognizes BAFF-R according to any one of items 6 to 8, wherein the activation stimulation domain further comprises one, two, or more co-stimulatory domains. In some embodiments, the co-stimulatory domain is derived from a co-stimulatory domain of the CD28 receptor family or the tumor necrosis factor receptor family.

[0023] 10. The chimeric antigen receptor molecule that specifically recognizes BAFF-R according to item 9, wherein the co-stimulatory domain comprises the intracellular region of one or more molecules selected from CD27, CD28, 4-1BB, OX40, CD30, CD40, CD2, LFA-1, LIGHT, NKG2C, B7-H3, PD-1, ICOS, CDS, ICAM-1, GITR, BAFFR, LIGHTR, SLAMF7, CD7, NKp80 (KLRF1), CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, CD229, CD160, PSGL1, CD100, CD69, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, a ligand that specifically binds to CD83, and variants thereof that retain the same function, preferably the intracellular region of 4-1BB, the intracellular region of CD28, the co-stimulatory domain of the intracellular region of OX40, or variants thereof that retain the same function.

[0024] 11. A chimeric antigen receptor molecule that specifically recognizes BAFF-R as described in item 10, wherein the co-stimulatory domain includes the amino acid sequence shown in SEQ ID NO: 18, or an amino acid sequence having approximately 70% or more sequence identity thereto, for example, an amino acid sequence having approximately 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity thereto.

[0025] 12. The transmembrane domains are TCRα, TCRβ, TCRγ, CD3ζ, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1, ICOS (CD278), 4-1BB, CTLA-4, GITR, CD40, BAFFR, LIGHTR, SLAMF7, NKp80, C D160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49α, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, CD226, SLAMF4, CD84, CD96, CEACAM1, CRT A chimeric antigen receptor molecule that specifically recognizes BAFF-R as described in any one of items 1 to 11, comprising a transmembrane domain of a molecule selected from the group consisting of AM, CD229, CD160, PSGL1, CDIOO, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, NKG2C, or a variant thereof having the same function, preferably comprising a transmembrane domain of CD4, CD8α, CD28, CD3ζ, PD1, or 4-1BB, or a variant thereof having the same function.

[0026] 13. A chimeric antigen receptor molecule that specifically recognizes BAFF-R as described in item 12, wherein the transmembrane domain comprises the transmembrane domain of CD8α or a variant thereof that retains the same function. In some embodiments, the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 16, or an amino acid sequence having about 70% or more sequence identity thereto, for example, an amino acid sequence having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity thereto.

[0027] 14. A chimeric antigen receptor molecule that specifically recognizes BAFF-R as described in any one of items 1 to 13, wherein the transmembrane domain is directly linked to the specific recognition domain.

[0028] 15. A chimeric antigen receptor molecule that specifically recognizes BAFF-R as described in any one of items 1 to 13, wherein the transmembrane domain and the specific recognition domain are linked via a hinge region. In some embodiments, the hinge region includes an amino acid sequence selected from the group consisting of human CD8α or an antibody Fc fragment or its functional equivalent, fragment or derivative, human CD8α or an antibody hinge region or its functional equivalent, fragment or derivative, an antibody CH2 region, an antibody CH3 region, an artificial spacer sequence, and combinations thereof. In some embodiments, the hinge region includes an amino acid sequence of an IgG, IgD, CD8α, or CD28 hinge region, or a variant thereof having the same function.

[0029] 16. A chimeric antigen receptor molecule that specifically recognizes BAFF-R as described in item 15, wherein the hinge region comprises the amino acid sequence shown in SEQ ID NO: 14, or an amino acid sequence having approximately 70% or more sequence identity thereto, for example, an amino acid sequence having approximately 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity thereto.

[0030] 17. A chimeric antigen receptor molecule that specifically recognizes BAFF-R as described in any one of items 1 to 16, further comprising a signal peptide selected from the signal peptide sequence of any secreted protein or membrane protein. In some embodiments, the signal peptide sequence is the signal peptide of CD8α.

[0031] 18. A chimeric antigen receptor molecule that specifically recognizes BAFF-R as described in item 17, wherein the signal peptide sequence includes the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having approximately 70% or more sequence identity thereto, for example, an amino acid sequence having approximately 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity thereto.

[0032] 19. A nucleic acid molecule encoding a chimeric antigen receptor molecule that specifically recognizes BAFF-R as described in any one of items 1 to 18.

[0033] 20. A nucleic acid molecule according to item 19, comprising the polynucleotide sequence shown in Sequence ID No. 3, or a polynucleotide sequence having about 70% or more sequence identity therewith, for example, a polynucleotide sequence having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity therewith. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence obtained by codon-optimizing the nucleotide sequence of Sequence ID No. 3 based on a specific cell or tissue.

[0034] 21. A nucleic acid molecule according to item 19 or 20, further comprising a coding sequence for a membrane-localized signal peptide molecule. In some embodiments, the coding sequence for the membrane-localized signal peptide molecule comprises the polynucleotide sequence shown in SEQ ID NO: 1, or a polynucleotide sequence having about 70% or more sequence identity therewith, for example, a polynucleotide sequence having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity therewith.

[0035] 22. A nucleic acid molecule according to item 21, comprising, from the 5' end to the 3' end, in this order: the polynucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19, or polynucleotide sequences having approximately 70% or more sequence identity with them, for example, polynucleotide sequences having approximately 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity with them.

[0036] 23. Manipulated immune effector cells comprising a chimeric antigen receptor molecule that specifically recognizes BAFF-R as described in any one of items 1 to 18 and / or a nucleic acid molecule as described in any one of items 19 to 22.

[0037] 24. Manipulated immunoeffector cells as described in Section 23, which are CAR-T and / or CAR-NK cells.

[0038] 25. A modified T cell receptor that specifically recognizes BAFF-R, comprising the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or amino acid sequences having approximately 70% or more sequence identity with them, for example, amino acid sequences selected from amino acid sequences having approximately 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity with them.

[0039] 26. The T cell receptor comprises an α chain variable region (Vα) and a β chain variable region (Vβ), The Vα comprises an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or an amino acid sequence having approximately 70% or more sequence identity with them, and the Vβ comprises an amino acid sequence selected from SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or an amino acid sequence having approximately 70% or more sequence identity with them, or The Vβ comprises an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, or an amino acid sequence having approximately 70% or more sequence identity with them, and the Vα comprises an amino acid sequence selected from SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, or an amino acid sequence having approximately 70% or more sequence identity with them. The T cell receptor described in item 25.

[0040] 27. A nucleic acid molecule encoding a T cell receptor as described in item 25 or 26.

[0041] 28. Manipulated immune effector cells comprising the T cell receptor described in item 25 or 26 and / or the nucleic acid molecule described in item 27.

[0042] 29. TCR-T cells, which are the modified immune effector cells described in section 28.

[0043] 30. A pharmaceutical composition comprising a therapeutically effective amount of a chimeric antigen receptor molecule that specifically recognizes BAFF-R as described in any one of claims 1 to 18, a T cell receptor as described in claim 25 or 26, a nucleic acid molecule as described in any one of claims 19 to 22 or 27, or an engineered immune effector cell as described in claim 23, 24, 28 or 29.

[0044] 31. A method for treating or preventing a disease in a test subject requiring such treatment, comprising administering a pharmaceutical composition described in item 30 to the test subject, wherein the disease is selected from diseases caused by an imbalance in the B cell BAFF-BAFF-R signaling pathway.

[0045] 32. The method according to item 31, wherein the disease includes autoimmune diseases, graft-versus-host diseases, and tumors.

[0046] The chimeric antigen receptor molecule provided by this application, which specifically recognizes BAFF-R, can specifically recognize and bind to BAFF-R, and after doing so, activate downstream signaling pathways, thereby inducing, promoting, or enhancing an immune response to BAFF-R, for example, by specifically killing target cells expressing BAFF-R in vitro or in vivo, and ultimately achieving the objective of treating or preventing diseases caused by imbalances in the B cell BAFF-BAFF-R signaling pathway. [Brief explanation of the drawing]

[0047] [Figure 1] This is an example of the structure of the BAFF-R chimeric antigen receptor of the present invention. [Figure 2] This is a plasmid map of an exemplary anti-BAFF-R chimeric antigen receptor lentiviral expression vector, pCDH-EF1a-H90-11CAR-4-1BB-EGFRt. [Figure 3] This is the plasmid map of the lentiviral expression vector pCDH-EF1a-EGFRt-AT-Free(delete T2A) as a control. [Figure 4] This figure shows the flow cytometry results used to detect the CAR positivity rate using FITC-EGFRt antibody. [Figure 5] This figure shows the results of flow cytometry used to detect CAR positivity using the BAFF-R protein. [Figure 6] This figure shows the results of H90-11 CAR-T cells specifically killing Nalm6-luciferase target cells. [Figure 7A] This figure shows the secretion results of IL-2 (Figure A) and IFN-γ (Figure B) in the supernatant after co-culturing H90-11 CAR-T cells and Nalm6-luciferase target cells. [Figure 7B] This figure shows the secretion results of IL-2 (Figure A) and IFN-γ (Figure B) in the supernatant after co-culturing H90-11 CAR-T cells and Nalm6-luciferase target cells. [Figure 8A]This figure shows the in vivo antitumor effect of H90-11 CAR-T in mice. Figure 8A shows the results of in vivo imaging of mice at different time points, Figure 8B is a graph plotting the in vivo absolute luminescence of mice at different time points, and Figure 8C is a graph plotting the change in body weight of mice within 22 days. [Figure 8B] This figure shows the in vivo antitumor effect of H90-11 CAR-T in mice. Figure 8A shows the results of in vivo imaging of mice at different time points, Figure 8B is a graph plotting the in vivo absolute luminescence of mice at different time points, and Figure 8C is a graph plotting the change in body weight of mice within 22 days. [Figure 8C] This figure shows the in vivo antitumor effect of H90-11 CAR-T in mice. Figure 8A shows the results of in vivo imaging of mice at different time points, Figure 8B is a graph plotting the in vivo absolute luminescence of mice at different time points, and Figure 8C is a graph plotting the change in body weight of mice within 22 days.

[0048] Details of the invention To further improve and enhance the therapeutic effects of B-cell malignancies, this application designs a novel chimeric antigen receptor molecule that recognizes BAFF-R, a protein specifically and highly expressed on the surface of B cells, and provides a nucleic acid molecule encoding the receptor, engineered immune effector cells containing the receptor, a pharmaceutical composition, and the use of these for the treatment or prevention of diseases caused by imbalances in the B-cell BAFF-BAFF-R signaling pathway, such as B-cell malignancies.

[0049] definition For the purposes of interpreting this Spec., the following definitions apply, with singular terms also including plural forms where applicable, and vice versa. In the event of any conflict between the following definitions and any documents incorporated herein by reference, the following definitions shall prevail.

[0050] As used herein, “receptor” refers to a biomolecule that can bind to hormones, neurotransmitters, drugs, or intracellular signaling molecules, thereby causing changes in cellular function. Depending on the location of the receptor within the cell, receptors can be classified into two categories: cell membrane receptors and intracellular receptors. The receptor itself contains at least two active sites: one active site that recognizes and binds to a ligand (referred to herein as the “specific recognition domain”), and the other a functional active site responsible for generating a response (referred to herein as the “activation-stimulation domain”). The activation-stimulation domain can only trigger a response by binding to the receptor and ligand to form a binary complex and becoming allosteric, thereby initiating a series of biochemical reactions that ultimately cause a biological effect on the effector cell in which the receptor is located. As used herein, the “activation-stimulation domain” includes a signaling domain, or includes one or more signaling domains and one or more co-stimulatory domains. As used herein, unless otherwise specified, the “signaling domain” provides a first signal that activates lymphocytes such as T cells or NK cells, and the “co-stimulatory domain” provides a second signal that activates lymphocytes. In some embodiments, a transmembrane domain is further included between the antigen recognition region and the activation stimulation domain. In some embodiments, the antigen recognition region and the transmembrane domain are connected by a hinge region.

[0051] As used herein, "intracellular domain" and "intracellular region" may be used interchangeably and may refer to a domain of a receptor molecule located inside a cell that plays a role in signal transduction after the receptor has bound to a ligand.

[0052] As used herein, “chimeric antigen receptor (CAR)” is a type of engineered cell surface receptor that is generally expressed on immune cells and mediates the killing of engineered immune cells against specific target tumor cells or other diseased cells. As a receptor, a CAR also includes a specific recognition domain and an activation-stimulating domain. The specific recognition domain of a CAR is also called the antigen-recognition region because it can specifically recognize an antigen. Typically, the antigen-recognition region of a CAR is located outside the cell membrane. In some embodiments, the antigen-recognition region is a single-chain variable fragment (scFv) of Ig, where “scFv” includes one heavy-chain variable region (VH) and one light-chain variable region (VL) of Ig. In some specific embodiments, the VL and VH regions are linked by a peptide chain.

[0053] The terms "heavy chain" ("CH"), "light chain" ("CL"), "light chain variable region" ("VL"), "heavy chain variable region" ("VH"), and "framework region" ("FR") refer to the domains of naturally occurring immunoglobulins and the corresponding domains of synthetic (e.g., recombinant) binding proteins (e.g., humanized antibodies). The basic structural unit of naturally occurring immunoglobulins (such as IgG) is a tetramer with two light chains and two heavy chains. The amino-terminus ("N") portion of each chain contains a variable region of approximately 100-110 or more amino acids, which is primarily involved in antigen recognition. The carboxyl-terminus ("C") portion of each chain defines a constant region; the light chain has a single constant domain, while the heavy chain typically has three constant domains and one hinge region. Thus, the naturally occurring light chain structure of the IgG molecule is N-VL-CL-C, and the IgG heavy chain structure is N-VH-CH1-H-CH2-CH3-C (where H is the hinge region). Here, CH1, CH2, and CH3 are components of the constant region of the antibody heavy chain. The CH3 region is involved in cell membrane surface receptor binding, and CH2 is involved in the complement activation pathway and is the complement binding site. CH1 contains a genetic marker for the Ig allotype. The variable region of IgG consists of a complementation-determining region (CDR) and a non-CDR fragment (called the framework region). Here, the CDR contains residues that come into contact with the antigen, and the framework region is used to maintain the structure of the variable region and determine the position of the CDR loop. Thus, the VL domain and VH domain have the structure N-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-C.

[0054] In some embodiments, the specific recognition domain is a polypeptide or protein ligand other than scFv. For example, a modified interleukin-13 (IL-13) molecule can be used to prepare the specific recognition domain, thereby obtaining an IL-13Rα2 specific CAR for the treatment of glioblastoma.

[0055] As used herein, the terms “single-chain variable fragment,” “single-chain antibody variable fragment,” or “scFv” antibody refer to an antibody form that contains only the variable regions of the heavy chain (VH) and light chain (VL) linked by a linker peptide. scFv can be expressed as a single-chain polypeptide. scFv retains the specificity of the complete antibody from which it is derived. The light and heavy chains may be in any order, for example, VH-linker-VL or VL-linker-VH, as long as the specificity of scFv to the target antigen is maintained. In some special embodiments, the linker may also be omitted.

[0056] As used herein, the term “linker” refers to an oligopeptide or polypeptide region of approximately 1 to 100 amino acids in length that links together any domains / regions of the CAR of the present invention. The linker is composed of flexible residues (such as glycine or serine) that allow adjacent protein domains to move freely between them. Longer linkers can be used if it is desired that two adjacent domains not sterically interfere with each other. Selectable linkers are known to those skilled in the art and can be used in conjunction with alternative embodiments of the present invention.

[0057] As used herein, the term “antibody” refers to a complete immunoglobulin, or a monoclonal or polyclonal antigen-binding fragment having an Fc (crystallizable fragment) region or an FcRn-binding fragment of the Fc region (referred herein to as an “Fc fragment” or “Fc domain”). Antigen-binding fragments can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of an intact antibody. Antigen-binding fragments include, among other things, Fab, Fab', F(ab')2, FV, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (scFv), single-domain antibodies, chimeric antibodies, diabodies, and polypeptides containing at least a portion of immunoglobulin sufficient to provide specific antigen binding. The Fc domain comprises two heavy chain CH2 and CH3 portions and can be produced by recombinant DNA technology or by enzymatic (e.g., papain cleavage) or chemical cleavage of an intact antibody. The term "antibody fragment" refers to a protein fragment that contains only a portion of an intact antibody, usually including the antigen-binding site of the intact antibody, and therefore retaining its ability to bind to the antigen.Examples of antibody fragments described herein include: (i) Fab fragments having VL, CL, VH, and CH1 domains; (ii) Fab' fragments, i.e., Fab fragments having one or more cysteine ​​residues at the C-terminus of the CH1 domain; (iii) Fd fragments having VH and CH1 domains; (iv) Fd' fragments having VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (v) Fv fragments having VL and VH domains on one arm of the antibody; (vi) dAb fragments consisting of a VH domain (Ward et al., Nature 341, 544-546 (1989)); (vii) F(ab')2 fragments, which are bivalent fragments containing an isolated CDR region and (viii) two Fab' fragments crosslinked by disulfide bonds in the hinge region; and (ix) single-chain antibody molecules (e.g., single-chain Fv; scFv) (Bird et al., Science 242:423-426 (1988); and Huston et al., PNAS (USA) 85:5879-5883 (1988)), (x) a "diabody" having two antigen-binding sites including a heavy chain variable domain (VH) linked to a light chain variable domain (VL) within the same polypeptide chain (see, e.g., EP404, 097; WO93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)), (xi) a "linear antibody" containing a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions together with a complementary light chain polypeptide (Zapata et al., Protein This includes, but is not limited to, Eng.8(10):1057-1062(1995); and U.S. Patent No. 5,641,870.

[0058] As used herein, the term “specifically binds” means that an antibody binds to a particular antigen but not to any other antigens. The specific antigen may be one or more, and in some embodiments, the specific antigens include the same or similar antigenic epitopes. In some embodiments, the specific binding is at least 10 degrees of contact between the antibody and the antigen. -6 It has binding affinity for M. In certain embodiments, the antibody is at least about 10 -7 M, preferably 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 It binds due to its affinity for M.

[0059] In this application, the terms “polynucleotide,” “nucleic acid,” and “nucleic acid molecule” may be used interchangeably and include, but are not limited to, DNA, RNA, cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA), shRNA (small hairpin RNA), snRNA (nuclear small RNA), snoRNA (nucleolar small RNA), miRNA (microRNA), genomic DNA, synthetic DNA, synthetic RNA, and / or tRNA.

[0060] As used herein, “vector,” “cloning vector,” and “expression vector” refer to vectors that can introduce a polynucleotide sequence (e.g., an exogenous gene) into a host cell to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, phages, viruses, and the like.

[0061] As used herein, the “signaling domain” generally comprises an immune-receptor tyrosine-based activation motif (ITAM), the basic composition of which is YXXL / V, where Y is tyrosine, L / V is leucine or valine, and X is any amino acid. When a receptor binds to its corresponding ligand, the tyrosine within the ITMA linked to it is phosphorylated by a PTK, a type of protein tyrosine kinase linked to the cell membrane, thereby recruiting other free protein kinases or adapter proteins within the cell to transmit an activation signal into the cell. In some embodiments, the “signaling domain” is selected from the intracellular signaling domains of TCRξ(CD3ξ) or FcεRIγ.

[0062] As used herein, the “costimulatory domain” is also referred to as the “costimulatory signaling domain” and is primarily used to provide a costimulatory signal to enhance the capabilities of immune cells, such as promoting the proliferation, survival, and / or development of memory cells. In some embodiments, the “costimulatory domain” is selected from CD28, 4-1BB (CD137), OX40 (CD134), and the like.

[0063] As used herein, the “transmembrane domain,” also known as a “transmembrane region,” refers to a thermodynamically stable protein structural region anchored to the cell membrane. Transmembrane domains can be obtained from native proteins, such as those derived from T cell receptors (TCRs). In some embodiments, the transmembrane domain is selected from the transmembrane domains of CD4, CD8α, CD28, and CD3ζ.

[0064] As used herein, the “hinge region” is a series of peptide chains linking an antigen recognition region to a transmembrane domain, and is typically elastic. In some embodiments, the hinge region is derived from the hinge of IgG or the extracellular region of CD8α / CD28. The “hinge” of IgG refers to the region between the CH1 and CH2 functional regions of IgG, and typically contains a large amount of proline.

[0065] As used herein, “anti-BAFF-R chimeric antigen receptor” refers to one of the “receptors that specifically recognize BAFF-R.” Specifically, it refers to a chimeric antigen receptor that includes the heavy chain variable region and light chain variable region of an anti-BAFF-R antibody in its antigen recognition region. The chimeric antigen receptor can specifically recognize BAFF-R and activate downstream pathways of cells in which the receptor resides through signal transduction. In some embodiments, the anti-BAFF-R chimeric antigen receptor is located on the surface of immune cells selected from NK cells, macrophages, neutrophils, T cells, etc., and after specifically recognizing BAFF-R, activates the immune effector cells, thereby activating immune effects such as humoral immunity, cellular immunity, and / or cytotoxicity, or activating and further proliferating the immune cells.

[0066] As used herein, “immune effector cells” refers to cells, such as T cells and natural killer (NK) cells, that are capable of achieving immune effects and immune responses, such as immunokilling effects and immune response effects, against target antigens or target cells.

[0067] As used herein, "CAR-T" refers to chimeric antigen receptor T cells, which are T cells that express a chimeric antigen receptor molecule on their cell surface and can recognize target antigens on the cell surface. Currently, CAR-T cells have been developed up to the fourth generation. The CAR molecule of the first-generation CAR-T is formed by the linkage and fusion of the signaling domains of the CD3ζ chain or FcεRIγ with the antigen recognition region, and does not contain a costimulatory domain. First-generation CAR-T cells have limited proliferative capacity in the body and are prone to apoptosis. Second-generation CAR-T cells have added costimulatory domains such as CD28 or 4-1BB (CD137). CD28 has potent antitumor activity, and the advantage of 4-1BB is that it extends the survival time of T lymphocytes and maintains the antitumor effect. Second-generation CAR-T cells have stronger proliferative capacity than the first generation and can secrete more cytokines and anti-apoptotic proteins. Third-generation CAR-T cells can not only simultaneously express two costimulatory signaling molecules but also secrete more IFN-γ, exhibiting a higher antitumor cytotoxic effect. Fourth-generation CAR-T cells can also secrete specific cytokines (such as IL-12) within the tumor, thereby altering the tumor microenvironment, influencing and activating other immune cells, and triggering an immune response.

[0068] As used herein, “signal peptide” refers to a short peptide chain, typically 5 to 30 amino acids in length, that induces the transmembrane movement of a newly synthesized protein into the secretory pathway. In some embodiments, the signal peptide is a membrane-localized signal peptide, i.e., an amino acid sequence used to direct the transmembrane movement (localization) of a protein. In most cases, the signal peptide is located at the N-terminus of the amino acid sequence. In mRNA, the coding sequence for the signal peptide is usually located after the start codon and is an RNA region encoding a hydrophobic amino acid sequence. After the signal peptide has induced the protein to complete its localization, it is usually excised by the action of a signal peptidase.

[0069] As used herein, a "variant" of a protein or nucleic acid refers to a protein or nucleic acid that has the same function as a particular protein or nucleic acid but has one or more mutations in its sequence. For example, a "variant" of a protein is a protein that has the same function as the protein and at least 70% sequence identity, obtained by artificial or natural mutation causing one or more amino acid insertions, deletions, substitutions, or other mutations in the amino acid sequence of the protein.

[0070] As used herein, "sequence identity" refers to the degree of similarity between amino acid sequences or nucleotide sequences as determined by sequence alignment software such as BLAST.

[0071] Those skilled in the art should know that imbalances in the BAFF-BAFF-R signaling pathway can cause a variety of diseases, including autoimmune diseases, graft-versus-host diseases, and tumors. The tumors include all B-cell malignancies except plasma cell lesions (multiple myeloma), such as mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zone lymphoma, Burkitt lymphoma, lymphoblastic leukemia, chronic lymphocytic leukemia, and hairy cell leukemia. The autoimmune diseases include, for example, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, glomerulonephritis, Sjögren's syndrome, or autoimmune hemolytic anemia.

[0072] As used herein, the terms “effective dose” or “therapeutic dose” mean an amount of a pharmaceutical composition comprising one or more peptides, proteins, nucleic acids, or their variants, variants, analogs, or derivatives disclosed herein, in which a patient or examinee receiving such “effective dose” or “therapeutic dose” of the pharmaceutical composition can obtain a reasonable benefit / risk ratio of medical treatment, thereby alleviating or preventing at least one symptom of a disease or condition and achieving the desired therapeutic or preventive effect.

[0073] As used herein, the term “about” refers to the normal range of error for each value, which is readily apparent to those skilled in the art. References to “about” a value or parameter herein include (and are described) embodiments relating to that value or parameter itself. Where used herein, when the term “about” precedes a number, it means a range of plus or minus 10% of that number. For example, “about 100” covers both 90 and 110.

[0074] As used herein, the singular forms "a," "an," and "the" refer to multiple subjects unless otherwise specified.

[0075] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains.

[0076] Chimeric antigen receptor This application provides a receptor that specifically recognizes BAFF-R. In some embodiments, the receptor that specifically recognizes BAFF-R is a chimeric antigen receptor (CAR). When the receptor that specifically recognizes BAFF-R is a chimeric antigen receptor (CAR), it comprises a specific recognition domain, an activation-stimulating domain, and a transmembrane domain located between the specific recognition domain and the activation-stimulating domain, where the specific recognition domain refers to the portion of the CAR that specifically binds to the target antigen on the target cell. In some embodiments, the specific recognition domain may include an antibody or its functional equivalent, or a fragment or derivative thereof, such as a full-length heavy chain, a Fab fragment, a single-chain Fv(scFv) fragment, a bivalent single-chain antibody, or a diabody, each specific to the target antigen. However, many alternatives exist, such as cytokines (which recognize cells with cytokine receptors), aphibodies, ligand-binding domains derived from native receptors, and soluble protein / peptide ligands of the receptor (e.g., on tumor cells), each of which can be used in various embodiments of the present invention. Those skilled in the art should understand that virtually any molecule or domain that binds to the target antigen with high affinity can be used as the specific recognition domain. In some embodiments, the specific recognition domain includes a T cell receptor (TCR) or its antigen-recognizing portion, such as a single-chain TCR (scTCR). Methods for preparing such TCRs are known in the art. For example, an scTCR can be manipulated to include Vαl and Vβ genes from a T cell clone linked by a linker (e.g., a flexible peptide).

[0077] In some embodiments, the specific recognition domain includes an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 12, or an amino acid sequence having about 70% or more sequence identity with them, for example, an amino acid sequence having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity with them. In some embodiments, the specific recognition domain is A heavy chain variable region containing an amino acid sequence selected from SEQ ID NOs. 6, SEQ ID NOs. 7, and SEQ ID NOs. 8, or an amino acid sequence having approximately 70% or more sequence identity with them, for example, an amino acid sequence having approximately 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity with them; and The light chain variable region includes an amino acid sequence selected from SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, or an amino acid sequence having approximately 70% or more sequence identity with them, for example, an amino acid sequence having approximately 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity with them.

[0078] In some embodiments, the heavy chain variable region comprises three complementarity-determining regions CDR H1, CDR H2, and CDR H3, where CDR H1, CDR H2, and CDR H3 each comprise the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, or amino acid sequences having approximately 70% or more sequence identity with them, for example, amino acid sequences having approximately 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity with them, and the light chain variable region also comprises three complementarity-determining regions CDR L1, CDR L2, and CDR L3, where CDR L1, CDR L2, and CDR Each L3 contains the amino acid sequences of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, or amino acid sequences having approximately 70% or more sequence identity with them, for example, amino acid sequences having approximately 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity with them.

[0079] In some embodiments, the heavy chain variable region and the light chain variable region further include a framework region, preferably a human antibody framework region.

[0080] In some embodiments, the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having about 70% or more sequence identity thereto, for example, an amino acid sequence having about 75%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity thereto, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having about 70% or more sequence identity thereto, for example, an amino acid sequence having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity thereto.

[0081] In some embodiments, the heavy chain variable region and the light chain variable region are linked via a linker to form an scFv, the scFv comprising the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence having about 70% or more sequence identity thereto, for example, an amino acid sequence having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity thereto.

[0082] CARs typically consist of a specific recognition domain, an activation / stimulus domain, and a transmembrane domain between them. There are three conventional CAR structures, the main difference of which lies in the activation / stimulus domain, including the following: First-generation CARs have an intracellular signaling domain in their activating / stimulating domain, but lack a co-stimulatory domain; Second-generation CARs include an intracellular signaling domain and one co-stimulatory domain in their activating / stimulating domain; Third-generation CARs include an intracellular signaling domain and two co-stimulatory domains in their activation-stimulating domain.

[0083] Therefore, in some embodiments of this application, the CAR is a first-generation CAR in which the signaling domain may be located in the cytoplasm and can transmit effector functional signals and instruct the cell to perform its specific function. Examples of the signaling domain include, but are not limited to, the ζ chain or any homolog of the T cell receptor (e.g., η chain, FcεR1γ, β chain, MB1(Igα) chain, B29(Igβ) chain, etc.), CD3 polypeptides (γ, δ, and ε), syk family tyrosine kinases (e.g., Syk, ZAP70), src family tyrosine kinases (e.g., Lck, Fyn, Lyn), and other molecules involved in T cell transduction, such as CD2, CD5, and CD28. Specifically, the intracellular signaling domain may be the human CD3ζ chain, FcγRIII, FcεRI, the cytoplasmic tail region of the Fc receptor, a cytoplasmic receptor having an immunoreceptor tyrosine activation motif (ITAM), or a combination thereof. Other intracellular signaling domains are apparent to those skilled in the art and can be used in conjunction with alternative embodiments of the present invention. In some embodiments, the signaling domain included in the activation-stimulating domain is a signaling domain comprising an immune receptor tyrosine activation motif. In some embodiments, the intracellular signaling domain comprises one or more signaling domains selected from the intracellular regions of the CD3ζ, CD3γ, CD3δ, CD3ε, FcεRIγ, FcεR1β, CD79α, CD79β, FcγRIIa, DAP10, and DAP12 molecules, or variants thereof that retain the same function. In some preferred embodiments, the intracellular signaling domain comprises the intracellular region of CD3ζ or FcεRIγ, or variants thereof that retain the same function. In some preferred embodiments, the intracellular signaling domain is derived from the intracellular region of CD3ζ. In some embodiments, the intracellular signaling domain includes the amino acid sequence shown in SEQ ID NO: 20, or an amino acid sequence having approximately 70% or more sequence identity thereto, for example, an amino acid sequence having approximately 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity thereto.

[0084] In some embodiments of the present application, the CAR is a second-generation CAR, the signal transduction domain is the above-mentioned signal transduction domain, and the co-stimulatory domains further included are CD27, CD28, 4-1BB, OX40, CD30, CD40, CD2, LFA-1, LIGHT, NKG2C, B7-H3, PD-1, ICOS, CDS, ICAM-1, GITR, BAFFR, LIGHTR, SLAMF7, CD7, NKp80(KLRF1), CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, These are ligands that specifically bind to CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, CD229, CD160, PSGL1, CD100, CD69, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and CD83, or the intracellular domain of any one of these molecules, or variants that retain the same function.

[0085] In some embodiments of the present application, the CAR is a third-generation CAR, the signal transduction domain is the above-mentioned signal transduction domain, and the co-stimulatory domains further included are CD27, CD28, 4-1BB, OX40, CD30, CD40, CD2, LFA-1, LIGHT, NKG2C, B7-H3, PD-1, ICOS, CDS, ICAM-1, GITR, BAFFR, LIGHTR, SLAMF7, CD7, NKp80 (KLRF1), CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103 These are ligands that specifically bind to ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, CD229, CD160, PSGL1, CD100, CD69, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and CD83, or the intracellular domains of any two, three, or more molecules among their variants that retain the same function.

[0086] In some preferred embodiments, the co-stimulatory domain is preferably a 4-1BB intracellular domain, a CD28 intracellular domain, an OX40 intracellular domain, or a variant having the same function as those thereafter. In some specific embodiments, the co-stimulatory domain includes an intracellular region derived from 4-1BB. In some specific embodiments, the co-stimulatory domain includes the amino acid sequence shown in SEQ ID NO: 18, or an amino acid sequence having approximately 70% or more sequence identity thereto, for example, approximately 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity thereto.

[0087] Those skilled in the art will understand that by appropriately arranging and combining the signal transduction domain and the costimulatory domain, effector cells containing the CAR can obtain an appropriate activation signal when the CAR binds to a ligand.

[0088] In some embodiments, the transmembrane domain between the specific recognition domain and the activation stimulus domain may be derived from the transmembrane sequence of any protein having a transmembrane domain (including any type I, type II, or type III transmembrane proteins). Furthermore, the transmembrane domain of the CAR of the present invention may also include an artificial hydrophobic sequence. In some embodiments, the transmembrane domain may be derived from TCRα, TCRβ, TCRγ, CD3ζ, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1, ICOS (CD278), 4-1BB, CTLA-4, GITR, CD40, BAFFR, LIGHTR, SLAMF7, NK p80, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49α, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD10 3, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, CD226, SLAMF4, CD84, CD96, CEACAM1, CRT The molecule comprises a transmembrane domain of a molecule selected from the group consisting of AM, CD229, CD160, PSGL1, CDIOO, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, or NKG2C, preferably a transmembrane domain of CD4, CD8α, CD28, CD3ζ, PD1, or 4-1BB, or a variant thereof that retains the same function.

[0089] In some embodiments, the transmembrane domain may include one or more other amino acids adjacent to the transmembrane domain, for example, one or more amino acids that associate with the extracellular region of the protein from which the transmembrane domain originates (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10-15 of the extracellular region) and / or one or more other amino acids that associate with the intracellular region of the protein from which the transmembrane protein originates (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10-15 of the intracellular region). In one embodiment, the transmembrane domain originates from the same protein from which the signaling domain, co-stimulatory domain, or hinge domain originates. In another embodiment, the transmembrane domain does not originate from the same protein from which the other domains of CAR originate. In some preferred embodiments of the present application, the transmembrane domain includes the transmembrane domain of CD8α. In some embodiments, the transmembrane domain includes the amino acid sequence shown in SEQ ID NO: 16, or an amino acid sequence having about 70% or more sequence identity thereto, for example, an amino acid sequence having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity thereto.

[0090] In some embodiments, the transmembrane domain is directly linked to the specific recognition domain. In some embodiments, the transmembrane domain and the specific recognition domain are linked via a hinge region. In some embodiments, the hinge region includes an amino acid sequence selected from the group consisting of human CD8α or an antibody Fc fragment or its functional equivalent, fragment or derivative, human CD8α or an antibody hinge region or its functional equivalent, fragment or derivative, an antibody CH2 region, an antibody CH3 region, an artificial spacer sequence, and combinations thereof, preferably an IgG, IgD, CD8α, or CD28 hinge region amino acid sequence. In some exemplary embodiments, the hinge region includes any one of the following: (i) the hinge region, CH2 region, and CH3 region of IgG4; (ii) the hinge region of IgG4; (iii) the hinge region and CH2 region of IgG4; (iv) the hinge region of CD8α; (v) the hinge region, CH2 region, and CH3 region of IgG1; (vi) the hinge region of IgG1; (vi) the hinge region and CH2 region of IgG1; or (vii) one or more combinations thereof. In some specific embodiments, the hinge region is the hinge region of human CD8α. In some specific embodiments, the hinge region includes the amino acid sequence shown in Sequence ID No. 14, or an amino acid sequence having about 70% or more sequence identity thereto, for example, an amino acid sequence having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity thereto.

[0091] In some embodiments, the CAR further comprises a signal peptide. Typically, signal peptides (SPs) are located at the N-terminus of a protein, carry information about the protein's secretion, and are involved in determining the protein's secretory pathway and distribution. Those skilled in the art will know that proteins localized in the nucleus, mitochondria, and cytoplasm, as well as free proteins in the cytosol and proteins that are secreted or ultimately anchored to the cell membrane, typically have different signal peptides. Those skilled in the art can select a signal peptide sequence suitable for the function of a particular protein through software prediction and signal peptide databases. Thus, in some embodiments of this application, the signal peptide is selected from any signal peptide sequence of a secreted protein or cell membrane protein, or a variant thereof that retains the same function. In some embodiments, the signal peptide sequence is the signal peptide of CD8α. In some embodiments, the signal peptide sequence includes the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having about 70% or more sequence identity thereto, for example, an amino acid sequence having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity thereto.

[0092] Manipulated T cell receptor On the other hand, this application also provides an engineered T cell receptor (TCR) that specifically recognizes BAFF-R. Specific receptors on the surface of T cells are responsible for recognizing antigens presented by the major histocompatibility complex (MHC). T cell receptors are heterodimers composed of two distinct subunits. Typically, 95% of T cell receptors consist of α and β subunits, and the remaining 5% consist of γ and δ subunits. Each subunit contains two extracellular domains: a variable region and a constant region. The variable region of each subunit (e.g., Vα of the α subunit, Vβ of the β subunit, Vγ of the γ subunit, and Vδ of the δ subunit) contains at least three highly variable complementarity-determining regions, namely CDR1, CDR2, and CDR3. CDR3 plays a role in directly binding to polypeptides presented by MHC. The α and β subunits of CDR1 act on the N-terminus and C-terminus of the polypeptide, respectively, while CDR2 is involved in MHC recognition.

[0093] In some embodiments, the manipulated T cell receptor is Vα containing an amino acid sequence selected from SEQ ID NOs. 6, SEQ ID NOs. 7, and SEQ ID NOs. 8, or an amino acid sequence having approximately 70% or more sequence identity with them; and Vβ containing an amino acid sequence selected from SEQ ID NOs. 10, SEQ ID NOs. 11, and SEQ ID NOs. 12, or an amino acid sequence having approximately 70% or more sequence identity with them; or Vβ contains an amino acid sequence selected from SEQ ID NOs. 6, SEQ ID NOs. 7, and SEQ ID NOs. 8, or an amino acid sequence having approximately 70% or more sequence identity with them; and Vα contains an amino acid sequence selected from SEQ ID NOs. 10, SEQ ID NOs. 11, and SEQ ID NOs. 12, or an amino acid sequence having approximately 70% or more sequence identity with them.

[0094] In other embodiments, the manipulated T cell receptor is Vγ containing an amino acid sequence selected from SEQ ID NOs. 6, SEQ ID NOs. 7, and SEQ ID NOs. 8, or an amino acid sequence having approximately 70% or more sequence identity with them; and Vδ containing an amino acid sequence selected from SEQ ID NOs. 10, SEQ ID NOs. 11, and SEQ ID NOs. 12, or an amino acid sequence having approximately 70% or more sequence identity with them; or Vδ contains an amino acid sequence selected from SEQ ID NOs. 6, SEQ ID NOs. 7, and SEQ ID NOs. 8, or an amino acid sequence having approximately 70% or more sequence identity with them; and Vγ contains an amino acid sequence selected from SEQ ID NOs. 10, SEQ ID NOs. 11, and SEQ ID NOs. 12, or an amino acid sequence having approximately 70% or more sequence identity with them.

[0095] The manipulated TCR specifically recognizes the BAFF-R presented by the MHC and, together with the signaling-carrying CD3δ / ε dimer, CD3γ / ε dimer, and CD247ζ / ζ or ζ / η dimer, and in some embodiments together with co-receptors, activates downstream signaling pathways.

[0096] nucleic acid molecule This application also provides nucleic acid molecules encoding a receptor or fragment thereof that specifically recognizes BAFF-R. In some embodiments, the receptor that specifically recognizes BAFF-R is the chimeric antigen receptor described above. In some embodiments, the receptor that specifically recognizes BAFF-R is the engineered T cell receptor described above. The nucleic acid molecule comprises a polynucleotide sequence encoding the chimeric antigen receptor or engineered T cell receptor or fragment thereof. The nucleic acid molecule may be DNA or RNA. In some embodiments, the acid molecule is linear, and in some embodiments, the nucleic acid molecule is cyclic. In some embodiments, the nucleic acid molecule is double-stranded. In some embodiments, the nucleic acid molecule is single-stranded. In some embodiments, the nucleic acid molecule is chemically synthesized. In some embodiments, the nucleic acid molecule includes chemical modifications to make it more stable in cells or animals. In some embodiments, the nucleic acid molecule is synthesized by bacterial cells, fungal cells, or animal cells. In some embodiments, the nucleic acid molecule is a plasmid, a viral vector, or an oligonucleotide.

[0097] In some embodiments, the polynucleotide sequence includes a sequence encoding a specific recognition domain of a receptor that specifically recognizes the BAFF-R. In some embodiments, the specific recognition domain is scFv. In some embodiments, the polynucleotide sequence includes the DNA sequence shown in Sequence ID No. 3 and / or a DNA sequence complementary thereto, or a corresponding or complementary RNA sequence, or a polynucleotide sequence having about 70% or more sequence identity with the DNA or RNA sequence described in this paragraph, for example, a polynucleotide sequence having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity. As used herein, “complementarity” of nucleic acids means that two polynucleotides can hybridize under stringent conditions, and stringent hybridization means that when two polynucleotides hybridize, each base or each nucleotide follows the Watson-Crick base pairing rules, i.e., A matches T or U, C matches G or I. As used herein, RNA "corresponding" to a DNA sequence means a ribonucleotide (RNA) sequence that matches the base sequence of the DNA sequence and in which all T bases are replaced with U bases.

[0098] In some embodiments, the nucleic acid molecule includes a coding sequence for a membrane-localized signal peptide molecule. In some embodiments, the membrane-localized signal peptide is a CD8α membrane-localized signal peptide. In some embodiments, the coding sequence for the membrane-localized signal peptide molecule includes the DNA sequence shown in Sequence ID No. 1 and / or a DNA sequence complementary thereto, or a corresponding or complementary RNA sequence, or a polynucleotide sequence having about 70% or more sequence identity with the DNA or RNA sequence described in this paragraph, for example, a polynucleotide sequence having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity.

[0099] In some embodiments, the nucleic acid molecule includes a sequence encoding a hinge region. In some embodiments, the encoded hinge region is the hinge region of CD8α. In some embodiments, the sequence encoding the hinge region includes the DNA sequence shown in Sequence ID No. 13 and / or a DNA sequence complementary thereto, or a corresponding or complementary RNA sequence, or a polynucleotide sequence having about 70% or more sequence identity with the DNA or RNA sequence described in this paragraph, for example, a polynucleotide sequence having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity.

[0100] In some embodiments, the nucleic acid molecule includes a sequence encoding a signaling domain. In some embodiments, the signaling domain is the CD3ζ signaling domain. In some embodiments, the sequence encoding the signaling domain includes the DNA sequence shown in Sequence ID No. 19 and / or a DNA sequence complementary thereto, or a corresponding or complementary RNA sequence, or a polynucleotide sequence having about 70% or more sequence identity with the DNA or RNA sequence described in this paragraph, for example, a polynucleotide sequence having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity.

[0101] In some embodiments, the nucleic acid molecule includes a sequence encoding a co-stimulatory domain. In some embodiments, the co-stimulatory domain is a signaling domain at 4-1BB. In some embodiments, the sequence encoding the signaling domain includes the DNA sequence shown in Sequence ID No. 17 and / or a DNA sequence complementary thereto, or a corresponding or complementary RNA sequence, or a polynucleotide sequence having about 70% or more sequence identity with the DNA or RNA sequence described in this paragraph, for example, a polynucleotide sequence having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity.

[0102] In some embodiments, the nucleic acid molecule includes a sequence encoding the costimulatory domain, a sequence encoding the signaling domain, a sequence encoding the hinge region, a sequence encoding the membrane-localized signal peptide molecule, and a sequence encoding the specific recognition domain. After the nucleic acid molecule is introduced into cells, it will be apparent to those skilled in the art how the sequences contained in the nucleic acid molecule are arranged and what relevant components may be added to modulate expression in order to successfully synthesize a complete receptor that specifically recognizes the BAFF-R. In some specific embodiments, the nucleic acid molecule includes, sequentially from the 5' end to the 3' end, the polynucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19, or polynucleotide sequences having about 70% or more sequence identity thereto, for example, polynucleotide sequences having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity thereto, where other sequences may be included between the polynucleotide sequences.

[0103] This application also provides a mixture comprising multiple nucleic acid molecules. Each of the multiple nucleic acid molecules comprises one or more sequences selected from the following: a sequence encoding the above-mentioned co-stimulatory domain, a sequence encoding the above-mentioned signal transduction domain, a sequence encoding the above-mentioned hinge region, a sequence encoding the above-mentioned membrane-localized signal peptide molecule, and a sequence encoding the above-mentioned specific recognition domain. By simultaneously introducing this mixture into cells, a complete receptor that specifically recognizes the BAFF-R can be synthesized.

[0104] Manipulated immune effector cells Engineered immune effector cells comprising a receptor that specifically recognizes the BAFF-R or the nucleic acid molecule.

[0105] In some embodiments, the receptor that specifically recognizes BAFF-R is the chimeric antigen receptor described above, and the manipulated immune effector cells are CAR-T cells or CAR-NK cells.

[0106] In some embodiments, the receptor that specifically recognizes BAFF-R is the above-described engineered T cell receptor, and the engineered immune effector cell is a TCR-T cell. In some embodiments, the receptor that specifically recognizes BAFF-R is the above-described engineered T cell receptor, and the engineered immune effector cell is a TCR-NK cell.

[0107] Virus particles, liposomes, lipid nanoparticles, pharmaceutical compositions The application also provides a viral plasmid, wherein the packaged viral vector comprises the above-mentioned chimeric antigen receptor or engineered T cell receptor, and / or a polynucleotide sequence encoding the said chimeric antigen receptor or engineered T cell receptor. The application also provides liposomes or lipid nanoparticles comprising a receptor or fragment thereof that specifically recognizes the said BAFF-R, or a nucleic acid molecule encoding the receptor or fragment thereof.

[0108] This application further provides a pharmaceutical composition comprising a therapeutically effective amount of one or more selected from the above-mentioned chimeric antigen receptors, T cell receptors, nucleic acid molecules, engineered immune effector cells, viral particles, liposomes, and lipid nanoparticles. In some embodiments, the pharmaceutical composition further comprises suitable excipients, carriers, and / or stabilizers. Such acceptable carriers, excipients, or stabilizers are nontoxic to the recipient at the dose and concentration used and include, for example, buffers such as phosphates, citrates, or acetates (typically pH 5.0–8.0, possibly 6.0–7.0); salts that achieve isotonicity, such as sodium chloride and potassium chloride; antioxidants; preservatives; low molecular weight polypeptides; proteins; hydrophilic polymers such as polysorbate 80; amino acids such as glycine; carbohydrates; chelating agents; sugars; and other standard components known to those skilled in the art (Remington: The Science and Practice of Pharmacy, 22nd edition, Loyd V. Allen et al ed., Pharmaceutical Press (2012)).

[0109] treatment This application also discloses a method for treating or preventing a disease or condition in a test subject requiring such treatment, comprising administering an effective amount of the above-mentioned pharmaceutical composition to the test subject, wherein the disease is selected from diseases caused by imbalances in the B-cell BAFF-BAFF-R signaling pathway. Optionally, the method further comprises administering to the test subject a second therapeutic agent, such as a monoclonal antibody capable of binding to the CD20 antigen, a monoclonal antibody capable of binding to CD19, a monoclonal antibody capable of binding to an immune checkpoint such as PD-1 / PD-L1, or engineered immune effector cells targeting CD20, CD19, and PD-1. In some embodiments, the disease is selected from any of autoimmune diseases, graft-versus-host diseases, or tumors.

[0110] In some embodiments, the disease is cancer. Optionally, the cancer is lymphoma, leukemia, or myeloma. Optionally, the cancer is lymphoma. Optionally, the lymphoma is mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zone lymphoma, or Burkitt lymphoma. Optionally, the cancer is leukemia. Optionally, the leukemia is lymphoblastic leukemia, chronic lymphocytic leukemia, or hairy cell leukemia. Optionally, the cancer is myeloma. Optionally, the myeloma is multiple myeloma.

[0111] In some embodiments, the disease is an autoimmune disease. Optionally, the autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, glomerulonephritis, Sjögren's syndrome, or autoimmune hemolytic anemia.

[0112] In another aspect, the application also provides a method for inhibiting cell proliferation. This method comprises contacting the cells with a pharmaceutical composition provided in the application to form contact cells. The anti-BAFF-R receptor or a functional fragment thereof binds to and contacts the BAFF-R protein on the cells, thereby inhibiting cell proliferation. Optionally, the cells are lymphocytes. Optionally, the cells are B cells or cancer cells. Optionally, the cells are lymphoma cells.

[0113] As used herein, treating or preventing a disease or condition means a method of obtaining a beneficial or desired outcome, including a clinical outcome. A beneficial or desired clinical outcome may include, but is not limited to, the alleviation or improvement of one or more symptoms or conditions; reduction of the severity of a condition, symptoms, or disease; stabilization of the condition, symptoms, or disease; prevention of the onset of a condition, symptoms, or disease; prevention of the spread of a condition, symptoms, or disease; delaying or slowing the progression of a condition, symptoms, or disease; delaying or slowing the onset of a condition, symptoms, or disease; improvement or mitigation of the condition, symptoms, or disease; and partial or overall improvement. Treatment may also mean extending the survival of the subject beyond what would have been expected without treatment. Treatment may also mean inhibiting the progression of a condition, symptoms, or disease, temporarily slowing the progression of a condition, symptoms, or disease, but in some cases including permanently stopping the progression of a condition, symptoms, or disease.

[0114] Although preferred embodiments of the present invention have been described in detail above, the present invention is not limited thereto. Within the scope of the technical idea of ​​the present invention, various simple modifications can be made to the proposed technical ideas of the present invention, including various combinations of technical features by other appropriate methods. These simple modifications and combinations should also be considered within the scope of the disclosure of the present invention and fall within the scope of protection of the present invention.

[0115] It should be understood that the above description and the following examples are for illustrative purposes only and do not limit the scope of the present invention. Other aspects, advantages, and modifications within the scope of the present invention will be apparent to those skilled in the art to which the present invention pertains. [Examples]

[0116] Example 1: Design of a chimeric antigen receptor targeting BAFF-R The present invention constructs an anti-BAFF-R chimeric antigen receptor (H90-11 CAR, comprising the heavy chain variable region and light chain variable region of an H90-11 mAb monoclonal antibody as shown in U.S. Patent Application Publication No. 2021 / 0261676A1, the contents of which are incorporated herein by reference). As shown in the schematic structural diagram of Figure 1, the chimeric antigen receptor comprises a CD8α signal peptide sequence (leader), a single-chain antibody sequence (scFv) that specifically binds to the BAFF-R antigen, a human CD8α hinge region (Hinge) and transmembrane domain sequence (Transmembrane), a 4-1BB costimulatory domain sequence and a CD3ζ signaling domain sequence, the specific order of each part is as follows; please refer to Table 1 for specific sequences. The polynucleotide sequence of the human CD8α molecular signal peptide (leading signal) is shown in Sequence ID No. 1, and the amino acid sequence is shown in Sequence ID No. 2. The polynucleotide sequence of the humanized BAFF-R single-chain antibody (scFv) is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4. Here, the heavy chain variable region (VH) of the scFv is SEQ ID NO: 5, and the amino acid sequences of the three complementarity-determining regions CDR H1, CDR H2, and CDR H3 contained in the VH are SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. The light chain variable region (VL) of the scFv is SEQ ID NO: 9, and the amino acid sequences of the three complementarity-determining regions CDR L1, CDR L2, and CDR L3 contained in the VL are SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively. The polynucleotide sequence of the human CD8α hinge region (CD8 Hinge) is shown in SEQ ID NO: 13, and the amino acid sequence is shown in SEQ ID NO: 14. The polynucleotide sequence of the human CD8α transmembrane domain (CD8™) is shown in SEQ ID NO: 15, and the amino acid sequence is shown in SEQ ID NO: 16. The polynucleotide sequence of the intracellular region (4-1BB) of the human 4-1BB molecule is shown in SEQ ID NO: 17, and the amino acid sequence is shown in SEQ ID NO: 18. The nucleotide sequence of the intracellular region (CD3ζ) of the human CD3ζ molecule is shown in SEQ ID NO: 19, and the amino acid sequence is shown in SEQ ID NO: 20.

[0117] [Table 1-1]

[0118] [Table 1-2]

[0119] Example 2: Construction of a chimeric antigen receptor expression vector The anti-BAFF-R H90-11 CAR lentivirus expression vector pCDH-EF1a-H90-11CAR-4-1BB-EGFRt and the control EGFRt CAR lentivirus expression vector pCDH-EF1a-EGFRt-AT-Free(delete T2A) were both synthesized and constructed using Nanjing GenScript. The specific experimental procedure is as follows. The anti-BAFF-R scFv gene sequence was synthesized by Nanjing GenScript. Following the manufacturer's protocol for the In-Fusion Snap assembly seamless cloning reagent, the synthesized scFv sequence was inserted into the pCDH-EF1a-4-1BB-EGFRt vector (Nanjing GenScript, order number: C046EEE310) to obtain the anti-BAFF-R chimeric antigen receptor lentiviral expression vector pCDH-EF1a-H90-11CAR-4-1BB-EGFRt (Nanjing GenScript, order number: C9401FG210). The vector map is shown in Figure 2. The pCDH-EF1a-T2A-EGFRt (provided by iCarTab) lentiviral expression vector has an ATG start codon in the T2A linker, preventing EGFRt from being properly expressed due to a frameshift mutation. Following the protocol of the manufacturer of the in-fusion snap assembly seamless cloning reagent, the T2A in the pCDH-EF1a-T2A-EGFRt vector was deleted to obtain the control EGFRt CAR lentiviral expression vector pCDH-EF1a-EGFRt-AT-Free(delete T2A) (constructed by Nanjing GenScript). The vector map is shown in Figure 3.

[0120] Example 3: Packaging and titer measurement of lentiviruses Lentivirus packaging 1 × 10⁶ 293T cells in a 15cm culture dish 7Inoculate and add complete medium of DMEM (Gibco, catalog number: 41965-062) containing 10% FBS (Gibco, catalog number: 10099-141), place at 37 °C, and culture overnight in a 5% CO2 incubator. Put 2 mL of PBS (Gibco, catalog number: 14190-250) into one well of a 6-well plate, add 20 μg of lentiviral expression plasmid (pCDH-EF1a-H90-11 CAR-4-1BB-EGFRt or pCDH-EF1a-EGFRt-AT-Free (delete T2A)) and 30 μL of lentiviral packaging plasmid Mix (iCarTab, catalog number: LVP2MIX) respectively, pipette up and down to mix well, add 150 μL of LVTransm transfection reagent (iCarTab, catalog number: LVTran100), immediately pipette up and down to mix uniformly, and let stand at room temperature for 10 minutes. Dropwise add the above DNA / PEI complex one drop at a time to a 15 cm culture dish (petri dish), gently shake the culture dish, and mix well. Place the culture dish in a 37 °C, 5% CO2 incubator, culture for 6 - 8 hours, then discard the medium containing the transfection reagent and replace it with fresh complete medium. After continuing the culture for 48 hours, collect the virus-containing culture supernatant in the culture dish, filter it through a 0.45 μm filter membrane, transfer it to a centrifuge tube, and centrifuge at 20,000 g at 4 °C for 2 hours. After centrifugation, in a biosafety cabinet, discard the liquid in the centrifuge tube, add 500 μL of PBS buffer to resuspend the virus pellet, aliquot, and then store the virus in a -80 °C refrigerator. Measurement of virus titer Resuscitate 293T cells and adjust the cell state to the logarithmic growth phase. Prepare a new 6-well plate and seed 293T cells at 8×10 per well 5Cells were seeded, and the culture medium was replenished until the final volume reached 2 mL. The 6-well plates were placed in a 37°C, 5% CO2 incubator and incubated overnight. 50 μL of concentrated lentivirus was added to the above 6-well plates, and simultaneously, polyblen (Sigma, catalog number: 107689-10G) at a final concentration of 6 μg / mL was added. The 6-well plates were returned to the 37°C, 5% CO2 incubator, and incubation was continued for 48 hours. After culturing, the cells in each well were washed with PBS, and genomic DNA was extracted using the MiniBEST Universal Genomic DNA Extraction Kit (Takara, catalog number: 9765). The concentration of the extracted genomic DNA was measured using NanoDrop2000. A fluorescence quantitative PCR reaction master mix was prepared according to the PCR reaction system shown in Table 2 below.

[0121] [Table 2]

[0122] The sequence and fluorescent groups of the primers used are shown in Table 3 below.

[0123] [Table 3]

[0124] The pUC-LTR (purchased from iCarTab) and pUC-ALB plasmid (purchased from iCarTab) were removed from the refrigerator and diluted 10-fold each to a total of 6 samples to prepare calibration curve samples necessary for fluorescence quantitative PCR. A new 96-well PCR reaction plate was prepared, 5 μL of the genome sample or calibration curve sample was added to each well, then 15 μL of the PCR reaction master mix prepared above was added to each well, the plate was sealed with sealing film, and gently centrifuged for 1 minute. The PCR reaction was performed according to the PCR procedure in Table 4 below.

[0125] [Table 4]

[0126] After the PCR reaction was complete, the Ct values ​​of the calibration curve and the sample were obtained using the Roche 480 support software. A calibration curve was created using the Ct value on the y-axis and the logarithm of the copy number on the x-axis, and the copy number of the sample was calculated based on the calibration curve. Then, the viral titer was calculated according to the following formula.

[0127]

number

[0128] [Table 5]

[0129] As can be seen from Table 5, high-titer lentiviruses are packaged and can then be used to infect T cells and prepare CAR-T cells.

[0130] Example 4: Preparation of CAR-T cells Isolation of T cells from peripheral blood PBMCs A 10 mL anticoagulated peripheral blood sample was transferred to a 15 mL sterile centrifuge tube and centrifuged at 800 g for 20 minutes. The pale yellow serum layer at the top was removed, and an equal volume of PBS was added to the peripheral blood cell layer at the bottom. The tube was gently inverted to mix thoroughly. Lymphocyte separatory solution (Hao Yang Biotechnology, catalog number: 1077) was removed and mixed thoroughly by inverting the tube several times. 5 mL of lymphocyte separatory solution was added to another 15 mL centrifuge tube. To avoid mixing the separatory solution with the blood sample, the diluted blood sample was slowly added along the tube wall to the upper layer of lymphocyte separatory solution, and the tube was centrifuged at 800 g for 20 minutes. After centrifugation, the centrifuge tube was gently removed, the central white mononuclear cell layer was aspirated into a new sterile centrifuge tube, an equal volume of physiological saline was added and mixed gently, and the tube was centrifuged at 800 g for 5 minutes. After centrifugation, the supernatant was removed, the PBMCs were washed again, and the cell density was set to 5 × 10⁶. 7The cells were adjusted to the desired concentration per mL and transferred to 2 mL cell cryopreservation tubes at a rate of 1 mL per tube. Dynabeads® Human T-Expander CD3 / CD28 (Thermo, catalog number: 11141D) was washed twice with PBS. An appropriate amount of Dynabeads® Human T-Expander CD3 / CD28 was added to the PBMCs in a 2 mL cell cryopreservation tube, gently mixed until uniform, and incubated at room temperature for 20 minutes. The 2 mL cell cryopreservation tube was inserted into the magnetic pole, left at room temperature for 1 minute, and then gently inverted to drain the liquid from the tube. Remove the cell cryopreservation tubes from the magnetic poles, add an appropriate amount of X-Vivo 15 medium (containing 200 IU / mL IL-2 (Beijing Yuance Pharmaceutical Co., Ltd., catalog number: 20150713B)), 10 ng / mL IL-7 (PrimeGene, catalog number: 101-07), and 5 ng / mL IL-15 (PrimeGene, catalog number: 101-15), resuspend the cells, count them, and determine a cell density of 0.5 to 1 × 10⁶. 6 The cells were adjusted to the desired concentration (cells / mL), transferred to a 6-well plate, and incubated in a 37°C, 5% CO2 incubator for 48 hours of continuous incubation. T-cell lentiviral infection The density of T cells after culture is 1 × 10⁻⁶ 6 The lentivirus was adjusted to cells / mL, removed from a -80°C ultracold refrigerator, rapidly thawed in a 37°C water bath, and added to T cells prepared with polyblen (Sigma, catalog number: 107689-10G) to a final concentration of 6 μg / mL. An appropriate amount of virus (infection multiplicity MOI=25) was added, and the mixture was homogeneously mixed by gently pipetting. The culture vessel was sealed with Parafilm and centrifuged at 800g at room temperature for 1 hour. After centrifugation, incubation was continued for 24 hours, the medium was changed, and incubation was continued in a 37°C, 5% CO2 incubator. Massive proliferation of T cells The Dynabeads® cell pellet in the culture system was gently pipettered and counted daily until completely isolated. The cell density was 1 × 10⁶. 6If the cell density exceeds 10⁻¹⁴ cells / mL, add X-Vivo 15 medium (containing 200 IU / mL IL-2, 10 ng / mL IL-7, and 5 ng / mL IL-15) to increase the cell density to 0.5 × 10⁻¹⁴. 5 The cells were adjusted to the desired cell / mL concentration, and culture was continued. Cells were collected on day 10 of growth and culture and stored in a cell cryopreservation solution. Detection of CAR-T positive rate When H90-11 CAR-T cells (T cells infected with pCDH-EFIa-H90-11CAR-4-1BB-EGFRt lentivirus) and EGFRt CAR-T cells (T cells infected with pCDH-EF1a-EGFRt-AT-Free(delete T2A) lentivirus) were incubated with FITC-Anti-EGFR antibody (iCARTAB, catalog number: IAB006A) (final concentration 10 μg / mL), respectively, and incubated in the dark at room temperature for 30 minutes, flow cytometry was used to detect EGFRt expression, which reflects the CAR-T cell positivity rate. As shown in Figure 4, the positivity rate for H90-11 CAR-T cells was 36.1%, and the positivity rate for EGFRt CAR-T cells was 78.1%. Simultaneously, H90-11 CAR-T cells and EGFRt CAR-T cells were incubated with BAFF-R Liama ​​IgG2b Fc Tag recombinant protein (Acro, catalog number: BAR-H5258) (final concentration 10 μg / mL), respectively, for 30 minutes at room temperature. After centrifugation at 500 g for 5 minutes, the supernatant was discarded, and FITC-anti Liama ​​IgG (H+L) (Novus, catalog number: NBP1-47627) was diluted 1:1000 in 0.5% BSA PBS. The cell pellet was resuspended in 100 μL / sample and incubated in the dark at room temperature for 30 minutes. The positivity of CAR-T cells was then detected by FACS, and as shown in Figure 5, the positivity of H90-11 CAR-T cells was 30.2%, and the positivity of EGFRt CAR-T cells was 0.65%. H90-11 CAR-T cells expressed both EGFRt protein and anti-BAFF-R CAR molecule, and their positivity rates were essentially the same. However, EGFRt CAR-T cells expressed only EGFRt protein and not the anti-BAFF-R CAR molecule. This indicates that both H90-11 CAR-T cells and EGFRt CAR-T cells were successfully prepared.

[0131] Example 5: Evaluation of in vitro tumor cell killing by BAFF-R-targeting chimeric antigen receptor T cells. CAR-T cell lysis of target cells Nalm6-luciferase cells are acute lymphoblastic leukemia cells that highly express human BAFF-R. They are luciferase-overexpressing cells obtained by infecting Nalm6 cells (purchased from Genio Biotech) with a lentivirus. The cytotoxicity of H90-11 CAR-T cells against Nalm6-luciferase target cells was detected using a luciferase detection kit (Promega, catalog number: E2610), and EGFRt CAR-T cells were used as a control. The specific steps are as follows. Nalm6-luciferase target cells were resuspended in complete medium (RPMI1640 + 10% FBS), and 2 × 10⁶ target cells were placed in the medium. 4 Cells were inoculated into 96-well plates at a rate of one cell / well and incubated overnight in a 37°C, 5% CO2 incubator. Prepared CAR-T cells were harvested by centrifugation and resuspended in 1640 medium containing 10% FBS. The 96-well plates were removed from the incubator, the medium in the wells was completely aspirated, the cells were gently washed once with sterile PBS, and CAR-T cells were added in effect-to-target ratios of 0.5:1, 1:1, 2.5:1, 5:1, and 10:1. Double wells were set up, and the final volume was refilled to 200 μL / well. Equal numbers of target cells were inoculated into the Maxi lysis wells and Mini lysis wells, but no CAR-T cells were added. The 96-well plates were incubated in a 37°C, 5% CO2 incubator for 18 hours. After culturing, luciferase activity, which reflects the ability of recombinant CAR-T cells to lyse target cells, was detected according to the instructions for the luciferase detection kit (Promega, catalog number: E2610). Formula for calculating target cell lysis rate: Lysis% = (1 - [RLU]_Sample) / ([RLU]_Max) × 100% The results are shown in Figure 6. The killing effect of H90-11 CAR-T cells against Nalm6-luciferase cells expressing BAFF-R was much higher than that of the EGFRt CAR-T group. When the effect-to-target ratio was 0.5:1, 1:1, 2.5:1, 5:1, and 10:1, the killing rates of H90-11 CAR-T cells against target cells were 62.7%, 83.1%, 95.4%, 98.9%, and 99.7%, respectively, while the killing rates of the EGFRt CAR-T group against target cells were 2.1%, 10.5%, 10.5%, 18.1%, and 24.7%, respectively. This indicates that chimeric antigen receptor T cells targeting BAFF-R prepared by the method of the present invention have strong tumor-killing activity. Detection of CAR-T cytokine secretion levels The expression levels of IL-2 and IFN-γ in the supernatant of co-cultures of target cells (Nalm6-luciferase) and prepared CAR-T cells were detected by ELISA. Nalm6-luciferase target cells were resuspended in complete medium, and 2 × 10⁶ target cells were placed in a 96-well plate. 4 Cells were seeded per well and cultured overnight in a 37°C, 5% CO2 incubator. Recombinant H90-11 CAR-T cells and EGFRt CAR-T cells were used as effector cells, respectively, to establish co-culture systems of effector cells and target cells. Double wells were set up with effect-to-target ratios of 0.5:1, 1:1, 2.5:1, 5:1, and 10:1. Well plates were cultured for 18 hours in a 37°C, 5% CO2 incubator. After the culture was complete, the well plate was removed from the incubator, and the 96-well plate was centrifuged at 1200g at room temperature for 5 minutes. The well plate was then gently removed, and the supernatant was transferred to the ELISA detection kit (Fcmacs Biotech Co., Ltd., catalog number: FMS-ELH003) to detect the expression levels of IL-2 and IFN-γ (Fcmacs Biotech Co., Ltd., catalog number: FMS-ELH035) (for detailed procedures, please refer to the protocol of the ELISA detection kit). The results are shown in Figures 7A (IL-2) and 7B (IFN-γ). IL-2 and IFN-γ cytokine levels in the co-culture supernatant of BAFF-R-expressing Nalm6-luciferase cells and H90-11 CAR-T cells were significantly higher and increased in a gradient compared to the EGFRt CAR-T group. This result indicates that H90-11 CAR-T cells can secrete Th1-related cytokines when stimulated by target cells expressing BAFF-R.

[0132] Example 6: Evaluation of tumor cell killing by BAFF-R-targeting chimeric antigen receptor T cells in mice Acute lymphoblastic leukemia Nalm6-luciferase cell lines were xenotransplanted into an NSG mouse model, and the in vivo activity of H90-11 CAR-T was evaluated. Twenty 6-8 week old NSG mice (purchased from Beijing Biocytogen) (body weight 18-22g) were taken and cultured for one week, after which Nalm6-luciferase was inoculated into the tail vein. 1 × 10⁶ mice were given each. 6 Numerous tumor cells were inoculated. Five days after inoculation of the tumor cells into the tail vein, the first in vivo imaging was performed, but the imaging effect was not ideal. Eight days after inoculation, mice that failed to model were excluded from the subsequent experimental group, and the mice were divided into groups according to the in vivo imaging results. The mice were randomly divided into two groups, with 8 mice in each group, resulting in 2.25 × 10⁶ mice. 6 H90-11 CAR-T cells or EGFRt CAR-T cells were injected into the tail vein of mice, and D-luciferin (Sinochrome / Cytogenetics, catalog number: BC219-10) was administered intraperitoneally every 7 days while the mice were anesthetized using a gas anesthesia machine. In vivo imaging data of the tumors were collected, and changes in the mice's body weight were recorded to create survival curves. In vivo imaging observations were performed on small animal in vivo imaging devices on days 8, 15, and 22 after modeling. The imaging results are shown in Figure 8A, and the absolute luminescence curve is shown in Figure 8B. According to the imaging results, H90-11 CAR-T showed a significant killing effect on tumor cells compared to the EGFRt CAR-T control group; after CAR-T cell injection therapy on day 8, the increase in tumor load (absolute luminescence) in mice injected with H90-11 CAR-T cells was slower from injection to day 15 compared to the EGFRt CAR-T control group, and the tumor load decreased sharply after day 15. The graph of the drawn mouse body weight change curve is shown in Figure 8C. The body weight of mice injected with EGFRt CAR-T control cells was equivalent to that of mice injected with H90-11 CAR-T cells. During the experiment, the body weight of the mice increased gradually, which indicates that CAR-T cell injection does not cause serious toxic reactions.

Claims

1. A chimeric antigen receptor molecule that specifically recognizes BAFF-R, comprising a specific recognition domain, an activation-stimulating domain, and a transmembrane domain located between the specific recognition domain and the activation-stimulating domain, wherein the specific recognition domain comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises three complementarity-determining regions CDR H1, CDR H2, and CDR H3, wherein CDR H1 comprises the amino acid sequence shown in SEQ ID NO: 6, CDR H2 comprises the amino acid sequence shown in SEQ ID NO: 7, and CDR H3 comprises the amino acid sequence shown in SEQ ID NO:

8. The light chain variable region includes CDR L1, CDR L2, and CDR L3, wherein CDR L1 includes the amino acid sequence shown in SEQ ID NO: 10, CDR L2 includes the amino acid sequence shown in SEQ ID NO: 11, and CDR L3 includes the amino acid sequence shown in SEQ ID NO:

12. A chimeric antigen receptor molecule that specifically recognizes BAFF-R, wherein the heavy chain variable region is linked to the light chain variable region to form an scFv, and the scFv contains the amino acid sequence shown in SEQ ID NO:

4.

2. A chimeric antigen receptor molecule that specifically recognizes BAFF-R according to claim 1, wherein the heavy chain variable region and the light chain variable region further include a human antibody framework region.

3. A chimeric antigen receptor molecule that specifically recognizes BAFF-R according to claim 1, wherein the activation stimulating domain comprises a signal transduction domain, and the signal transduction domain comprises an immune receptor tyrosine activation motif.

4. A chimeric antigen receptor molecule that specifically recognizes BAFF-R according to claim 3, wherein the signal transduction domain includes the signal transduction domain of the intracellular region of the CD3ζ, CD3γ, CD3δ, CD3ε, FcεRIγ, FcεR1β, CD79α, CD79β, FcγRIIa, DAP10, or DAP12 molecule.

5. The activation stimulating domain further comprises one, two, three or more co-stimulating domains, wherein the co-stimulating domains are CD27, CD28, 4-1BB, OX40, CD30, CD40, CD2, LFA-1, LIGHT, NKG2C, B7-H3, PD-1, ICOS, ICAM-1, GITR, BAFFR, LIGHTR, SLAMF7, CD7, NKp80 (KLRF1), CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, V LA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, C A chimeric antigen receptor molecule that specifically recognizes BAFF-R according to claim 3, derived from the intracellular domain of one or more molecules of ligands that specifically bind to D11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, CD229, CD160, PSGL1, CD100, CD69, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and CD83.

6. A chimeric antigen receptor molecule that specifically recognizes BAFF-R according to claim 5, wherein the co-stimulatory domain includes the amino acid sequence shown in SEQ ID NO:

18.

7. A chimeric antigen receptor molecule that specifically recognizes BAFF-R according to any one of claims 1 to 3, wherein the transmembrane domain comprises a transmembrane domain selected from the group consisting of CD4, CD8α, CD28, or CD3ζ transmembrane domains.

8. The transmembrane domain and the specific recognition domain are linked via a hinge region, the hinge region being selected from IgG, IgD, or CD8α / CD28 hinge regions. A chimeric antigen receptor molecule that specifically recognizes BAFF-R according to any one of claims 1 to 3.

9. A nucleic acid molecule encoding a chimeric antigen receptor molecule that specifically recognizes BAFF-R as described in any one of claims 1 to 3.

10. The nucleic acid molecule according to claim 9, comprising the polynucleotide sequence shown in Sequence ID No.

3.

11. The nucleic acid molecule according to claim 9, further comprising a coding sequence for a membrane-localized signal peptide molecule.

12. The nucleic acid molecule according to claim 11, comprising the polynucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19 in this order from the 5' end to the 3' end.

13. Engineered immune effector cells comprising a chimeric antigen receptor molecule that specifically recognizes BAFF-R as described in any one of claims 1 to 3.

14. A pharmaceutical composition comprising a therapeutically effective amount of the manipulated immune effector cells described in claim 13.

15. A pharmaceutical composition according to claim 14, used for treating or preventing a disease, The aforementioned disease is selected from diseases caused by an imbalance in the B-cell BAFF-BAFF-R signaling pathway. Pharmaceutical composition.

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