Fully human antibodies targeting BAFF-R and their application in CAR-T therapy

Abstract

The present invention provides a fully human antibody targeting BAFF-R and its application in CAR-T therapy. The present invention obtains a fully human antibody or an antigen-binding fragment targeting BAFF-R by screening a fully human phage library, which specifically binds to BAFF-R with high affinity. Based on the screened antibody sequence, the present invention provides a fully human chimeric antigen receptor (CAR) targeting BAFF-R and a host cell expressing the CAR, such as a CAR-T cell, and provides the application of these CARs and CAR-T cells in the treatment of B cell tumors.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a fully human antibody targeting BAFF-R and its application in CAR-T therapy. Background Art

[0002] Lymphoma is a malignant tumor originating from lymph nodes and extranodal lymphoid tissues, accounting for 3%-4% of all malignant tumors in my country and the largest malignant tumor in the blood system. The two most common subtypes of B-cell lymphoma are diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive B-NHL, accounting for approximately 30% of newly diagnosed NHL cases. Currently, 10% of newly treated DLBCL are refractory cases after the standard first-line therapy R-CHOP, and 40% of those who are initially effective relapse after 2-5 years1. Autologous hematopoietic stem cell transplantation (ASCT) after achieving CR or PR after chemotherapy is currently the best treatment for relapsed and refractory DLBCL, but the 2-year disease-free survival rate after transplantation is still less than 15%. Follicular lymphoma (FL) is the most common indolent B-NHL, accounting for approximately 22% of newly diagnosed NHL cases. Although the disease progresses slowly, it is generally considered incurable, with an expected median survival of approximately 10 years, and approximately 15-28% of FL will transform into aggressive lymphoma within 10 years.

[0003] At present, CAR-T therapy for B-cell lymphoma is mainly based on the CD19 target. There are already 4 CD19-CAR-Ts on the market. The main indications of the four CD19-CAR-Ts are B-cell lymphoma (DLBCL) and follicular lymphoma (FL), with an ORR of approximately 70%-80% and a CR of 50-60%. Although CD19 CAR-T has achieved a higher remission rate than standard therapy for these two major B-NHLs, follow-up studies have shown that some patients relapse due to antigen escape, that is, mutations or deletions of CD19 protein expressed on the surface of tumor cells will render CAR-T cell therapy ineffective, accounting for about 10%-20%2. Therefore, for patients with relapse and refractory disease, it is urgent to seek new targets for CAR-T cell immunotherapy.

[0004] BAFF-R (B-cell activating factor receptor) is a member of the tumor necrosis factor (TNF) receptor superfamily. BAFF-R is expressed on the surface of peripheral B cells except plasma cells and centroblasts located in the dark zone of the germinal center. The researchers tested 116 B-cell lymphoma clinical samples and found that BAFF-R was highly expressed on a variety of B lymphoma cells, including chronic lymphocytic leukemia (CLL) (21 / 21, 100%), mantle cell lymphoma (MCL) (7 / 7, 100%), FL (13 / 16, 81%), and DLBCL (14 / 18, 78%)3. This shows that BAFF-R can be used as a new target for the treatment of B cell tumors in addition to CD19.

[0005] Currently, there are few reported clinical data on BAFF-R CAR-T. Only PMB-101 developed by City of Hope reported the efficacy data of the first patient in Phase I clinical trial at the end of 2022. The patient achieved CR after one month of treatment and no related neurotoxicity was observed. Therefore, the market is in urgent need of developing more BAFF-R-CAR products with better efficacy. Summary of the invention

[0006] The purpose of the present invention is to provide a fully human antibody targeting BAFF-R and its application in CAR-T therapy.

[0007] The object of the present invention is to provide an antibody targeting BAFF-R, a CAR and a host cell expressing a CAR targeting BAFF-R, as well as their preparation methods and uses.

[0008] In a first aspect of the present invention, an antibody targeting B cell activating factor receptor (BAFF-R) is provided, wherein the heavy chain variable region of the antibody comprises the following complementary determining regions CDR: CDR1 shown in SEQ ID NO.27, CDR2 shown in SEQ ID NO.28, and CDR3 shown in SEQ ID NO.29; or

[0009] The light chain variable region of the antibody comprises the following complementary determining regions CDR: CDR1 shown in SEQ ID NO.36, CDR2 shown in AAS, and CDR3 shown in SEQ ID NO.37, and the heavy chain variable region of the antibody comprises the following complementary determining regions CDR: CDR1 shown in SEQ ID NO.38, CDR2 shown in SEQ ID NO.39, and CDR3 shown in SEQ ID NO.40;

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

[0011] In another preferred embodiment, the light chain variable region and / or heavy chain variable region further includes a framework region (FR).

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

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

[0014] In another preferred example, the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO.25, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the above sequence.

[0015] In another preferred example, the light chain variable region has an amino acid sequence as shown in SEQ ID NO.30, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the above sequence, and the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO.32, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the above sequence.

[0016] In another preferred embodiment, the antibody is a single domain antibody.

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

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

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

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

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

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

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

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

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

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

[0027] In a second aspect of the present invention, a chimeric antigen receptor is provided, wherein the antigen binding domain of the chimeric antigen receptor comprises an antigen binding fragment targeting BAFF-R, and the heavy chain variable region of the antigen binding fragment comprises the following complementary determining regions CDR: CDR1 shown in SEQ ID NO.27, CDR2 shown in SEQ ID NO.28, and CDR3 shown in SEQ ID NO.29;

[0028] Or the light chain variable region of the antigen-binding fragment comprises the following complementary determining regions CDR: CDR1 shown in SEQ ID NO.36, CDR2 shown in AAS, and CDR3 shown in SEQ ID NO.37, and the heavy chain variable region of the antibody comprises the following complementary determining regions CDR: CDR1 shown in SEQ ID NO.38, CDR2 shown in SEQ ID NO.39, and CDR3 shown in SEQ ID NO.40.

[0029] In another preferred embodiment, the antigen-binding fragment is the heavy chain variable region of a single-domain antibody.

[0030] In another preferred embodiment, the antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO. 25 or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the above sequence.

[0031] In another preferred embodiment, the antigen binding domain of the chimeric antigen receptor comprises one or more antigen binding fragments.

[0032] In another preferred embodiment, the antigen-binding fragment is a single-chain antibody.

[0033] In another preferred embodiment, the antigen-binding fragment comprises a light chain variable region, a connecting peptide and a heavy chain variable region in sequence from segment N to segment C.

[0034] In another preferred embodiment, the antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO.34 or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the above sequence.

[0035] In another preferred embodiment, the CAR further comprises a transmembrane region derived from CD8a and / or an intracellular domain derived from CD3ζ.

[0036] In another preferred embodiment, the CAR comprises an intracellular co-stimulatory region derived from CD28.

[0037] In another preferred embodiment, the CAR comprises a co-stimulatory molecule derived from 4-1BB.

[0038] In another preferred embodiment, the structure of the chimeric antigen receptor is shown in Formula I below:

[0039] LVH-TM-C-CD3ζ(I)

[0040] In the formula,

[0041] Each "-" is independently a connecting peptide or a peptide bond;

[0042] L is the signal peptide sequence;

[0043] V is the antigen binding domain;

[0044] H is the hinge region;

[0045] TM is the transmembrane domain;

[0046] C is a co-stimulatory signal molecule;

[0047] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

[0048] In another preferred embodiment, the L is a signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

[0049] In another preferred embodiment, the L is a signal peptide derived from CD8.

[0050] In another preferred example, the amino acid sequence of the CD8-derived signal peptide is shown as SEQ ID NO.2.

[0051] In another preferred embodiment, the anti-BAFF-R single domain antibody includes fully human single domain antibodies, humanized single domain antibodies and camel-derived single domain antibodies, preferably fully human single domain antibodies.

[0052] In another preferred embodiment, the V is a fully human single domain antibody.

[0053] In another preferred embodiment, the H is a hinge region derived from a protein selected from the group consisting of Fc, CD8, CD28, CD137, or a combination thereof.

[0054] In another preferred embodiment, the H is a hinge region derived from CD8, and the amino acid sequence is shown in SEQ ID NO.4.

[0055] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the following group: ICOS, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0056] In another preferred embodiment, the TM is a transmembrane region derived from CD8.

[0057] In another preferred example, the amino acid sequence of the transmembrane region derived from CD8 is shown as SEQ ID NO.6.

[0058] In another preferred embodiment, C is a co-stimulatory signal molecule of a protein selected from the following group: ICOS, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.

[0059] In another preferred embodiment, the C is a co-stimulatory signal molecule derived from ICOS and / or 4-1BB.

[0060] In another preferred embodiment, the amino acid sequence of the co-stimulatory signal molecule derived from 4-1BB is shown in SEQ ID NO.8.

[0061] In another preferred example, the amino acid sequence of CD3ζ is shown as SEQ ID NO.10.

[0062] In another preferred embodiment, the CAR (preferably the C-terminus) also includes a cell suicide element.

[0063] In another preferred embodiment, the CAR is connected to the cell suicide element via a self-cleavage element.

[0064] In another preferred embodiment, the cell suicide element is connected to the CD3ζ of the CAR via T2A or P2A.

[0065] In another preferred embodiment, the cell suicide element is selected from the following group: HSV-TK, iCasp9, ΔCD20, mTMPK, ΔCD19, RQR8, EGFRt, or a combination thereof.

[0066] In another preferred embodiment, the cell suicide element is tEGFR.

[0067] In another preferred example, the amino acid sequence of tEGFR is shown as SEQ ID NO.16.

[0068] In another preferred example, the amino acid sequence of T2A is shown as SEQ ID NO.18.

[0069] In another preferred example, the amino acid sequence of the CAR is shown in SEQ ID NO.20.

[0070] The third aspect of the present invention provides a nucleic acid molecule, wherein the nucleic acid molecule encodes the antibody described in the first aspect of the present invention; and / or the chimeric antigen receptor described in the second aspect of the present invention.

[0071] In another preferred embodiment, the nucleotide sequence of the nucleic acid molecule encoding the chimeric antigen receptor according to the second aspect of the present invention is shown as SEQ ID NO.26.

[0072] The fourth aspect of the present invention provides a vector, wherein the vector contains the nucleic acid molecule described in the third aspect of the present invention.

[0073] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, or a combination thereof.

[0074] In another preferred embodiment, the vector is a lentiviral vector.

[0075] The fifth aspect of the present invention provides a host cell, wherein the host cell contains the vector described in the fourth aspect of the present invention, or an exogenous nucleic acid molecule described in the third aspect of the present invention integrated into the chromosome.

[0076] The sixth aspect of the present invention provides an engineered immune cell, which contains the vector described in the fourth aspect of the present invention, or has the exogenous nucleic acid molecule described in the third aspect of the present invention integrated into the chromosome, or expresses the antibody described in the first aspect of the present invention or the chimeric antigen receptor described in the second aspect of the present invention.

[0077] In another preferred embodiment, the immune cells are T cells or NK cells.

[0078] In another preferred embodiment, the TCR gene expression of the T cells is silenced.

[0079] In another preferred embodiment, the immune cells express exogenous cell suicide elements.

[0080] In another preferred embodiment, the engineered immune cells are selected from the following group:

[0081] (i) chimeric antigen receptor T cells (CAR-T cells); or

[0082] (ii) Chimeric antigen receptor NK cells (CAR-NK cells).

[0083] In another preferred embodiment, CAR and cell suicide elements are co-expressed in the engineered immune cells.

[0084] The seventh aspect of the present invention provides a preparation, which contains the engineered immune cells described in the sixth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0085] In another preferred embodiment, the preparation is a liquid preparation.

[0086] In another preferred embodiment, the preparation is in the form of an injection.

[0087] The eighth aspect of the present invention provides a use of the antibody described in the first aspect of the present invention, the chimeric antigen receptor described in the second aspect of the present invention, or the engineered immune cell described in the sixth aspect of the present invention for preparing a drug or preparation for preventing and / or treating BAFF-R related diseases.

[0088] In another preferred embodiment, the BAFF-R related diseases include tumor occurrence, growth and / or metastasis, tumor resistance related diseases, autoimmune diseases, inflammation, and metabolism related diseases.

[0089] In another preferred embodiment, the BAFF-R related diseases include BAFF-R highly expressed tumors.

[0090] In another preferred embodiment, the tumor includes leukemia, lymphoma, myeloma, breast cancer (such as triple-negative breast cancer), lung cancer (such as non-small cell lung cancer), pancreatic cancer, malignant glioma, gastric cancer, liver cancer, esophageal cancer, kidney cancer, colorectal cancer, bladder cancer, prostate cancer, endometrial cancer, ovarian cancer, cervical cancer, and angiosarcoma.

[0091] In another preferred embodiment, the tumor is a blood tumor.

[0092] In another preferred embodiment, the tumor is a plasma cell malignancy or a B cell malignancy.

[0093] A ninth aspect of the present invention provides a method for preparing an engineered immune cell, wherein the engineered immune cell expresses the chimeric antigen receptor according to the second aspect of the present invention, comprising the following steps:

[0094] The nucleic acid molecule described in the third aspect of the present invention or the vector described in the fourth aspect of the present invention is transduced into immune cells to obtain the engineered immune cells.

[0095] In another preferred embodiment, the immune cells are T cells or NK cells.

[0096] The tenth aspect of the present invention provides a kit for preparing the engineered immune cells described in the sixth aspect of the present invention, and the kit contains a container, and the nucleic acid molecule described in the third aspect of the present invention or the vector described in the fourth aspect of the present invention located in the container.

[0097] The eleventh aspect of the present invention provides a use of the engineered immune cells described in the sixth aspect of the present invention for preventing and / or treating BAFF-R related diseases.

[0098] The twelfth aspect of the present invention provides a method for treating a disease, comprising administering an appropriate amount of the engineered immune cells described in the sixth aspect of the present invention, or the preparation described in the seventh aspect of the present invention to a subject in need of treatment.

[0099] In another preferred embodiment, the disease is cancer or tumor.

[0100] In another preferred embodiment, the cancer or tumor is a blood tumor.

[0101] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] Figure 1The general process of the present invention for screening specific antibodies targeting BAFF-R from a phage antibody library is shown.

[0103] Figure 2 The results of enzyme-linked immunosorbent assay (ELISA) of some selected phage monoclones with target antigen and control antigen are shown.

[0104] Figure 3 Flow cytometric analysis results of binding of some phage monoclones to K562-BAFF-R and K562 cells are shown.

[0105] Figure 4A -B shows the flow cytometric analysis results (peak graph) of the binding of the screened phage monoclonals to various BAFF-R positive and negative cell lines. Figure 4A The figure shows the flow cytometric peak patterns of Clone1, 5, and 14 binding to various cell lines. The Negative Control is the negative control phage antibody clone. Figure 4B The figure shows the flow cytometry peak morphology of Clone77 and 80 binding to various cell lines. VAY736 is the positive control phage antibody clone.

[0106] Figure 5 The results of ELISA analysis of the screened phage monoclones with BAFF-R antigen proteins and non-related antigens from various companies are shown. Negative Control is a negative control phage antibody clone. anti-M13 phage mouse Ab / anti-mouse HRP Ab is a negative control without phage, only with the first antibody (Anti-M13phagemouse Ab) and the second antibody (anti-mouse HRP Ab); anti-mouse HRP Ab is a negative control with only the second antibody (anti-mouse HRP Ab) added, VAY736 Ab / anti-human IgG HRP Ab is a positive antibody control for the target antigen (BAFF-R-his-Bio), and anti-human IgG HRP and anti-his HRP are positive antibody controls for detecting antigen tags. Among them, Figure 5 In the figure, the bar graphs corresponding to each test antibody and control group represent the test results of the reagents KACTUS-BAFF-R-his Bio, ACRO-BAFF-R-Fc, KACTUS-CD19-Fc, ACRO-CD70-his, and SA from left to right.

[0107] Figure 6The schematic diagram of the structure of BAFF-R CAR vector construction is shown. CAR includes an extracellular signal peptide (SP), a ScFv (VL-linker-VH) or a single domain (V HH ), hinge region and transmembrane region between the cell membrane and the extracellular binding region (CD8a hinge+TM), 4-1BB co-stimulatory molecule, CD3ζ intracellular domain, T2A self-cleaving peptide, truncated tEGFR.

[0108] Figure 7 The expression of tEGFR in CAR-T cells of different clones is shown.

[0109] Figure 8 The killing results of CAR-T cells of different clones on various target cells JEKO1-luc (Figure A), MEC1-luc (Figure B), RS4-luc (Figure C), NALM6-luc (Figure D), and MOLT4-luc (Figure E) are shown.

[0110] Fig. 9 The results of CD107a degranulation of tumor cells from different tissues on CAR-T cells of different clones are shown.

[0111] Fig.10 The anti-tumor efficacy of CAR-T cells corresponding to different clones in JEKO1-luc tumor-bearing NCG mice is shown. Figure A shows the change of photon number over time after CAR-T cell infusion, and Figure B shows the survival rate of mice infused with CAR-T cells. Figure C shows the change of T cell-lymphocyte ratio in mice after CAR-T cell infusion.

[0112] Fig.11 Shown are IVIS photos of CAR-T cells corresponding to different clones at different time points after transfusion into JEKO1-luc tumor-bearing NCG mice. DETAILED DESCRIPTION

[0113] After extensive and in-depth research, the inventors used a large-capacity phage antibody library to screen fully human BAFF-R specific antibodies, and evaluated the specificity of these antibodies at the phage level through ELISA and FACS experiments. Finally, several fully human antibody clones with good specificity were obtained. Using fully human antibodies with lower immunogenicity, antibody drugs (including monoclonal antibodies, bispecific antibodies, ADCs, etc.), cell therapy drugs (including CAR-T, CAR-NK, etc.), etc. were developed, and the present invention was completed on this basis.

[0114] Specifically, using different antibody libraries, after panning with recombinant BAFF-R protein, a total of 182 monoclonal clones were selected for primary screening by enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS) detection, of which 39 clones specifically bound to BAFF-R-his-Bio antigen and BAFF-R expression-positive cells K562-BAFF-R, but not to control protein SA and BAFF-R expression-negative cells K562. After sequencing, 5 different monoclonal sequences were obtained. Subsequently, these five antibodies were identified by flow cytometry analysis (FACS) with a variety of BAFF-R positive (K562-BAFF-R, NALM6) and negative cell lines (K562, Jurkat), and by enzyme-linked immunosorbent assay (ELISA) with BAFF-R proteins from different companies (KACTUSBAFF-R-his bio, ACRO-BAFF-R-Fc, SB-BAFF-R-Fc) and non-related proteins (KACTUS-CD19-Fc, ACRO-CD70-his, SA). Four of the clones showed good binding and specificity on multiple cell lines and multiple protein antigens. The overall project process is as follows: Figure 1 shown.

[0115] The function of the candidate clones on CAR-T cells was further verified, among which clone 5 had the best in vivo antitumor efficacy and good mouse safety, and was a better candidate clone for later clinical development. The heavy chain variable region of clone 5 contained the following complementary determining region CDR: CDR1 shown in SEQ ID NO.27, CDR2 shown in SEQ ID NO.28, and CDR3 shown in SEQ ID NO.29. More specifically, the heavy chain variable region of clone 5 had an amino acid sequence as shown in SEQ ID NO.25. The in vivo antitumor efficacy of clone 1 was also relatively good, and it could be used as a candidate clone. The light chain variable region of clone 1 contained the following complementary determining region CDR: CDR1 shown in SEQ ID NO.36, CDR2 shown in AAS, and CDR3 shown in SEQ ID NO.37, and the heavy chain variable region of the antibody contained the following complementary determining region CDR: CDR1 shown in SEQ ID NO.38, CDR2 shown in SEQ ID NO.39, and CDR3 shown in SEQ ID NO.40.

[0116] the term

[0117] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0118] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0119] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0120] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0121] "Antibodies" refer to immunoglobulins secreted by plasma cells (effector B cells) and used by the body's immune system to neutralize foreign substances (peptides, viruses, bacteria, etc.). The foreign substances are correspondingly called antigens. The basic structure of an antibody molecule is a tetramer consisting of two identical heavy chains and two identical light chains. Based on the conservative differences in the amino acid sequences, the heavy chains and light chains are divided into a variable region (V) located at the amino terminus and a constant region (C) located at the carboxyl terminus. The variable region of a heavy chain (HCVR, also known as VH) and the variable region of a light chain (LCVR, also known as VL) interact to form an antigen binding site (Fv). In the variable region, the composition and arrangement order of amino acid residues in certain regions are more variable than other regions in the variable region (framework region, FR), which are called hypervariable regions (HVR). Hypervariable regions are actually the key sites for antibody binding to antigens. Since these hypervariable region sequences are complementary to the antigenic determinants, they are also called complementarity-determining regions (CDRs). Both the heavy chain and the light chain have three complementary determining regions, which are called HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3. The heavy chain constant region contains three constant domains CH1, CH2 and CH3. Each light chain contains a light chain variable region (abbreviated as VL herein) and a light chain constant region. The light chain constant region contains a constant domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementary determining regions (CDRs), which are interspersed with more conservative regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

[0122] "Single chain antibody" (single chain fragment variable, scFv) is composed of the variable region of the heavy chain and the variable region of the light chain of the antibody connected by a short peptide into a peptide chain. Through correct folding, the variable regions from the heavy chain and the light chain interact through non-covalent bonds to form the Fv segment, so scFv can better retain its affinity activity for the antigen.

[0123] "Single domain antibody (sdAb, or VHH)" and "nanobody" have the same meaning, which refers to cloning the variable region of the antibody heavy chain to construct a nanobody consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete functions. Usually, antibodies that naturally lack the light chain and heavy chain constant region 1 (CH1) are obtained first, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.

[0124] "Mouse antibodies" are antibodies produced by mice against specific antigens, usually antibodies produced by mouse B lymphocytes. In most cases, the mouse antibodies are monoclonal antibodies produced by hybridoma cells. The fully human antibodies of the present invention are obtained by screening human phage antibody libraries, which have lower immunogenicity than mouse antibodies and are more conducive to therapeutic use in humans.

[0125] As used herein, "fully human antibodies or single-chain antibodies or fragments thereof" generally refers to any form of antigen-binding molecules that can bind to a target antigen, for example, the antigen-binding molecule may be a protein or polypeptide, including, for example, antibodies and antigen-binding fragments thereof, single-chain scFv antibodies, single-domain antibodies, various fusions and conjugates constructed based on scFv, such as scFv-Fc antibodies, immunoconjugates, antibody-drug conjugates (ADCs), multi / bispecific antibodies, and chimeric antigen receptors (CARs).

[0126] "BAFF-R" or "BAFF-R" is the major mediating receptor for BAFF regulation of B-cell differentiation. The BAFF / BAFF-R signaling pathway plays an important role in the proliferation of B-cell tumor cells. Upregulated expression of BAFF-R is associated with disease progression in B-cell lymphomas and pre-B-ALL. BAFF-R has a high specificity, is mainly expressed in B cells, and only binds to BAFF, not other members of the TNF family. Therefore, BAFF-R is an effective therapeutic target for many cancers, autoimmune diseases, and various other diseases characterized by BAFF-R expression.

[0127] As used herein, when referring to amino acid or nucleotide sequences, the term "sequence identity" (also referred to as "sequence identity") refers to the amount of consistency between two amino acid or nucleotide sequences (e.g., a query sequence and a reference sequence), generally expressed as a percentage. Typically, before calculating the percentage of consistency between two amino acid or nucleotide sequences, the sequences are aligned and gaps (if any) are introduced. If the amino acid residues or bases in the two sequences are the same at a certain alignment position, the two sequences are considered to be consistent or matched at that position; if the amino acid residues or bases in the two sequences are different, they are considered to be inconsistent or mismatched at that position. In some algorithms, the number of matching positions is divided by the total number of positions in the alignment window to obtain sequence consistency. In other algorithms, the number of gaps and / or the length of the gaps are also taken into account. For the purposes of the present invention, the publicly available alignment software BLAST (available on the webpage ncbi.nlm.nih.gov) can be used to obtain the best sequence alignment and calculate the sequence consistency between two amino acid or nucleotide sequences using the default settings. In some embodiments, "at least 90% sequence identity" of the present invention includes, but is not limited to, at least 95%, at least 98%, at least 99% or even 100% sequence identity.

[0128] In some embodiments, the fully human antibodies provided by the present invention also include an amino acid sequence that has at least 90% sequence identity (e.g., at least 95%, at least 98%, at least 99% or even 100% sequence identity) to the sequence shown in SEQ ID NO:25.

[0129] It will be understood by those skilled in the art that, based on the specific sequences provided herein, a small number of amino acids can be replaced, deleted, added, and the binding ability or biological activity of the resulting products with the corresponding antigen BAFF-R can be verified or screened, thereby obtaining the corresponding variants of the BAFF-R targeting antibodies provided herein, and these variants should also be included in the scope of the present invention. For example, the fully human antibody or its single-chain antibody or antigen-binding fragment of the present invention may have at least 1 and no more than 10, or no more than 5, 4, 3, 2 or 1 amino acid changes in the full length or CDR sequence.

[0130] Those skilled in the art can also understand that, based on the specific heavy chain variable region sequences provided herein, the antibody light chain library (such as a human phage light chain library) can be screened using BAFF-R as an antigen to obtain a light chain variable region that matches the heavy chain variable region and maintains BAFF-R binding ability. Anti-BAFF-R antibody molecules obtainable in this manner are also included within the scope of the present invention.

[0131] In some embodiments, the antigen binding molecules of the present invention may further comprise post-translational modifications. Examples of post-translational protein modifications include phosphorylation, acetylation, methylation, ADP-ribosylation, ubiquitination, glycosylation, carbonylation, ubiquitination, biotinylation, or the addition of polypeptide side chains or hydrophobic groups. Therefore, the modified soluble polypeptide may comprise non-amino acid components, such as lipids, polysaccharides or monosaccharides, and phosphates. A preferred form of glycosylation is sialylation modification, in which one or more sialic acid groups are bound to the polypeptide. Sialic acid groups improve the solubility and serum half-life of proteins, while also reducing the possible immunogenetic properties of proteins. See Raju et al. Biochemistry. 2001 31; 40(30): 8868-76.

[0132] When referring to pharmaceutical compositions, the term "pharmaceutically acceptable carrier" refers to solid or liquid diluents, fillers, antioxidants, stabilizers and the like that can be safely administered, are suitable for administration to humans and / or animals without excessive adverse side effects, and are suitable for maintaining the activity of the drugs or active agents contained therein.

[0133] "Therapeutically effective amount" refers to the amount of active compound sufficient to cause the biological or medical response desired by the clinician in the subject. The "therapeutically effective amount" of the antibody of the present invention can be determined by those skilled in the art based on factors such as the route of administration, the subject's weight, age, and condition. For example, a typical daily dose range can be 0.01 mg to 100 mg of active ingredient per kg body weight. The administration of the antibody of the present invention includes, but is not limited to, injection, such as intravenous, intramuscular, intraarterial, subcutaneous, intraperitoneal, etc.

[0134] "Epitope" refers to the portion of a molecule that is bound by an antigen binding protein (e.g., an antibody). An epitope may comprise a non-contiguous portion of the molecule (e.g., amino acid residues in a polypeptide that are not adjacent in the primary sequence of the polypeptide but are close enough to each other in the trivalent and tetravalent structures of the polypeptide to be bound by an antigen binding protein).

[0135] The present invention provides a kit comprising one or more containers containing a large number of gene constructs encoding the polypeptide of the present invention and a pharmaceutically acceptable excipient. The kit may also include instructions for use. The kit may also have a notice in a form prescribed by a government agency that manages the production, use or sale of drugs or biological products, indicating that it has been approved by the production, use or sale agency for human drugs.

[0136] Chimeric Antigen Receptor (CAR)

[0137] The chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen binding domain). The intracellular domain includes a costimulatory signaling region and a ζ chain portion. The costimulatory signaling region refers to a portion of the intracellular domain including a costimulatory molecule. Costimulatory molecules are cell surface molecules required for the effective response of lymphocytes to antigens, rather than antigen receptors or their ligands.

[0138] Between the extracellular domain and the transmembrane domain of CAR, or between the cytoplasmic domain and the transmembrane domain of CAR, a joint may be incorporated. As used herein, the term "joint" generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane domain to the extracellular domain or cytoplasmic domain of a polypeptide chain. The joint may include 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

[0139] In a preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention includes an antigen binding domain targeting BAFF-R. When the CAR of the present invention is expressed in T cells, it is possible to perform antigen recognition based on antigen binding specificity. When it binds to its associated antigen, it affects tumor cells, causing tumor cells not to grow, to be caused to die or otherwise affected, and causes the patient's tumor load to be reduced or eliminated. The antigen binding domain is preferably fused with one or more intracellular domains from a costimulatory molecule and a ζ chain. Preferably, the antigen binding domain is fused with an intracellular domain of a combination of a 4-1BB signaling domain and a CD3ζ signaling domain.

[0140] As used herein, "antigen-binding domain" refers to a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or a single Fv fragment having antigen-binding activity.

[0141] In the present invention, fully human phages including a single domain library are used for antibody screening to obtain fully human single domain antibodies with lower immunogenicity than humanized mouse antibodies, and a CAR vector is constructed in which the antigen binding domain comprises one or more single domain antibodies that specifically recognize BAFF-R, thereby preparing CAR-T cells.

[0142] Carrier

[0143] The present invention includes a DNA construct comprising a CAR sequence, wherein the sequence includes a nucleic acid sequence of an antigen binding domain operably linked to a nucleic acid sequence of a signaling domain.

[0144] The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening a library from a cell expressing the gene, by obtaining the gene from a known vector comprising the gene, or by directly isolating from cells and tissues comprising the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically.

[0145] The present invention also provides a vector into which the DNA of the present invention is inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of the transgene and its propagation in daughter cells.

[0146] In brief summary, the expression of natural or synthetic nucleic acid encoding CAR is usually achieved by operably connecting a nucleic acid encoding a CAR polypeptide or a portion thereof to a promoter, and incorporating the construct into an expression vector. The vector is suitable for replication and integration of eukaryotic cells. A typical cloning vector comprises a transcription and translation terminator, an initial sequence, and a promoter that can be used to regulate the expression of the desired nucleic acid sequence.

[0147] The nucleic acid can be cloned into many types of vectors. For example, the nucleic acid can be cloned into such vectors, which include but are not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors.

[0148] Further, the expression vector can be provided to the cell in the form of a viral vector. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Typically, a suitable vector comprises a replication origin that functions in at least one organism, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers.

[0149] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to a subject's cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0150] Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. Typically, these are located in the 30-110 bp region upstream of the start site, although recently it has been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that when an element is inverted or moved relative to another, the promoter function is maintained. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it is shown that individual elements can work cooperatively or independently to start transcription.

[0151] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive any polynucleotide sequence operably connected thereto to express at a high level. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr (Epstein-Barr) virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the present invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered to be part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0152] In order to evaluate the expression of CAR polypeptides or parts thereof, the expression vector introduced into the cell may also include any one or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a cell population seeking to be transfected or infected by a viral vector. In other aspects, selectable markers may be carried on a single DNA segment and used for co-transfection procedures. Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences so that they can be expressed in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

[0153] Reporter gene is used to identify the cells of potential transfection and to evaluate the functionality of regulatory sequences. Generally, reporter gene is the following gene: it is not present in or expressed by a receptor organism or tissue, and it encodes a polypeptide whose expression is clearly indicated by some easily detectable properties such as enzymatic activity. After DNA has been introduced into the receptor cell, the expression of the reporter gene is measured at the appropriate time. Suitable reporter gene can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein gene (for example, Ui-Tei et al., 2000FEBS Letters479:79-82). Suitable expression systems are well known and can be prepared using known techniques or commercially available. Generally, the construct with minimum 5 flanking regions showing the highest level of reporter gene expression is identified as a promoter. Such a promoter region can be connected to a reporter gene and used to evaluate the ability of a reagent to regulate promoter-driven transcription.

[0154] Methods for introducing genes into cells and expressing genes into cells are known in the art. In the context of expression vectors, vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical or biological means.

[0155] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0156] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used methods for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others.

[0157] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0158] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. Consider using lipid preparations to introduce nucleic acid into a host cell (in vitro, ex vivo or in vivo). On the other hand, the nucleic acid can be associated with a lipid. Nucleic acids associated with lipids can be encapsulated in the aqueous interior of the liposome, dispersed in the lipid bilayer of the liposome, attached to the liposome through a connecting molecule associated with both the liposome and the oligonucleotide, trapped in the liposome, compounded with the liposome, dispersed in a solution comprising lipids, mixed with lipids, combined with lipids, included in lipids as a suspension, included in micelles or compounded with micelles, or otherwise associated with lipids. The lipids, lipid / DNA or lipid / expression vectors associated with the composition are not limited to any specific structure in the solution. For example, they can be present in a bilayer structure, as micelles or have a "collapsed (collapsed)" structure. They can also be simply dispersed in a solution, possibly forming aggregates of size or shape inhomogeneity. Lipid is a fatty substance, which can be a naturally occurring or synthetic lipid. For example, lipids include fat droplets that occur naturally in the cytoplasm as well as compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0159] In a preferred embodiment of the present invention, the vector is a lentiviral vector. The inventors have confirmed that the use of the lentiviral vector to construct the CAR of the present invention has a high transfection efficiency for T cells and is highly reproducible.

[0160] In a preferred embodiment of the present invention, the vector further comprises a signal peptide sequence. Preferably, the signal peptide sequence is connected upstream of the antigen tuberculosis domain nucleic acid sequence.

[0161] The main advantages of the present invention include

[0162] (1) The present invention uses fully human phages for antibody screening and directly obtains a series of fully human monoclonal antibodies with excellent performance. Compared with traditional hybridoma technology, the difficult step of humanizing mouse antibodies is omitted, and fully human antibodies have lower immunogenicity than humanized mouse antibodies, and have great application potential in the development of antibody drugs (including monoclonal antibodies, bispecific antibodies, ADCs, etc.) and cell therapy drugs (including CAR-T, CAR-NK, etc.).

[0163] (2) The high-affinity specific antibodies provided by the present invention can also be used in the development of detection reagents.

[0164] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify detailed conditions are usually based on conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0165] Example 1. Enrichment of specific antibody clones targeting BAFF-R protein from phage antibody library by affinity panning

[0166] Appropriate negative and positive panning strategies were used to enrich the specific antibody clones we needed from the phage antibody library.

[0167] Construction of phage antibody library

[0168] The phage antibody libraries we constructed include natural libraries, semi-synthetic libraries and single-domain libraries. The semi-synthetic phage antibody library, used together with the natural library, solves the problem that the natural library may lack BAFF-R high-affinity antibody clones. The single-domain phage antibody library is an antibody library composed only of amino acids in the variable region of heavy chain antibodies. Its molecular weight is only 12-15kDa, but it has similar or higher specificity and affinity than traditional antibodies. In addition, single-domain antibodies have attracted much attention due to their stable physicochemical properties, high affinity, easy recombinant expression and preparation, and easy combination with other target or epitope antibodies.

[0169] BAFF-R protein panning

[0170] Using BAFF-R-his-Bio as the positive panning protein, multiple rounds of panning were performed to obtain a phage pool enriched with the target antibody clone. The experimental steps are briefly described as follows:

[0171] 1) Block the SA magnetic beads with blocking solution for 2 hours, and then combine the target antigen (BAFF-R-his-Bio) with the blocked SA magnetic beads;

[0172] 2) Add phage library (containing 5x10 12 phage particles) and a clean SA magnetic bead were incubated together to remove the phage antibody clones that non-specifically bound to the SA magnetic beads;

[0173] 3) After incubation, the supernatant is transferred to SA magnetic beads bound to the target antigen and incubated further to allow the phage to bind to the target antigen;

[0174] 4) Wash the magnetic beads with washing solution to remove unbound phages;

[0175] 5) Elute the positive phage from the target antigen with an elution solution and add a neutralization solution to neutralize it;

[0176] 6) Re-infect the host bacteria XL1-blue with the eluted phages to amplify the recovered phages. Keep a small amount of sample for gradient dilution, infect the host bacteria, apply Amp resistance plates, and calculate the number of recovered phages;

[0177] 7) Repeat steps 1) to 6), usually 3 to 4 rounds of panning are required, until a significant increase in the phage recovery rate (number of eluted phages / number of input phages) is observed.

[0178] The enriched phage pool can be used for subsequent single clone selection and ELISA / FACS screening.

[0179] Main materials and reagents:

[0180] Fully human phage antibody library, including natural library, semi-synthetic library and single domain library;

[0181] Helper phage KO7, Thermo / Invitrogen, 18311019;

[0182] Recombinant biotinylated Human BAFF-R Protein,KACTUS,BAFF-R-HM401;

[0183] BeaverBeads TM Streptavidin, Beaver Bio, 22307-10;

[0184] High binding ELSIA plate,Costar,#3590

[0185] Blocking solution: PBS + 3% BSA

[0186] Rinse solution: PBS + 0.1% Tween20

[0187] Eluent: 0.2M Glycine, pH 2.2

[0188] Neutralization solution: 1M Tris, pH 9.1

[0189] Experimental results:

[0190] Using different antibody libraries, four rounds of protein panning were performed, and a significant increase in the recovery rate was observed in each panning (Table 1), demonstrating that the antibody clones were effectively enriched.

[0191] Table 1 Protein panning experimental results

[0192]

[0193] It can be seen that after 4 rounds of panning, different antibody libraries have been enriched (the recovery rate in the 4th round was significantly higher than that in the previous round).

[0194] Example 2. Enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS) from enriched phage Pool screening for specific clones

[0195] Purpose and Principle: The phage pool enriched by affinity panning contains phage antibodies of various properties: specific clones, non-specific clones, and negative clones. In order to obtain specific clones, we need to isolate monoclonal clones, package them into monoclonal phages, and screen a large number of monoclonal clones by enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS), and select monoclonal clones that specifically bind to BAFF-R protein and BAFF-R positive cell line K562-BAFF-R at the same time. The specific monoclonal clones are further sequenced by DNA to determine the unique antibody sequence contained therein.

[0196] In the ELISA primary screening, the biotinylated target antigen (BAFF-R-his-Bio) is closer to the antigen conformation of the natural state in the reaction solution through the binding of streptavidin and biotin. The clones that only bind to BAFF-R-his-Bio but not to the control protein SA are identified as specific clones. The FACS primary screening is carried out using the positive cell line K562-BAFF-R with high expression of BAFF-R and the BAFF-R negative cell line K562. The clones that only bind to the cells K562-BAFF-R but not to the K562 cells are identified as specific clones. Through the two primary screenings of ELISA and FACS, we can obtain candidate antibodies that can bind to the recombinantly expressed BAFF-R protein and recognize the natural state BAFF-R molecules on the cell surface for further screening.

[0197] Brief steps of ELISA experiment:

[0198] 1) Cultivate and package monoclonal phage in deep-well 96-well plates;

[0199] 2) Dilute Strepavidin to 2 μg / mL with PBS, add 100 μL / well to the high binding ELISA plate, and bind for 2 h at room temperature;

[0200] 3) Discard the coating solution, add 250 μL of blocking solution to each well, and block overnight at 4°C;

[0201] 4) Wash the plate twice with 250 μL of rinse solution;

[0202] 5) Dilute the biotin-tagged target protein and control protein to 2 μg / mL with PBS, add 100 μg / well to the ELISA plate pre-coated with Strepavidin, and bind at room temperature for 1 hour;

[0203] 6) Wash the plate twice with 250 μL of rinse solution;

[0204] 7) Add 100 μL of the cultured phage supernatant in step 1) to the wells coated with the target antigen and allow to bind for 2 h at room temperature;

[0205] 8) Wash the plate 4 times with 250 μL of rinse solution;

[0206] 9) Add 1:2000 diluted mouse anti M13 primary antibody, 100 μL / well, and incubate at room temperature for 45 min;

[0207] 10) Wash the plate 4 times with 250 μL of rinse solution;

[0208] 11) Add 1:2000 diluted HRP Donkey anti-mouse IgG, 100 μL / well, and incubate at room temperature for 45 min;

[0209] 12) Wash the plate 6 times with 250 μL of rinse solution;

[0210] 13) Add 100 μL TMB colorimetric substrate and allow color to develop for 5 to 10 min;

[0211] 14) Add 100 μL 2M H2SO4 to terminate the reaction and read the results on a microplate reader.

[0212] Brief steps of FACS screening experiment:

[0213] 1) Cultivate and package monoclonal phage in deep-well 96-well plates;

[0214] 2) Wash Raji and Jurkat cells twice with PBS and resuspend in PBS to 1x10 7 / mL concentration, and dispensed into 96-well deep-well plates at 50 μL;

[0215] 3) Add 50 μL of packaged monoclonal phage to each well, mix well, and allow to bind at 4°C for 2 h;

[0216] 4) Wash twice with 200 μL PBS;

[0217] 5) Add 1:2000 diluted mouse anti M13 primary antibody, 100 μL / well, pipette to mix, and incubate at room temperature for 45 min;

[0218] 6) Wash twice with 200 μL PBS;

[0219] 7) Add 1:300 diluted FITC horse anti mouse-IgG (H+L), 100 μL / well, pipette to mix, and incubate at room temperature for 45 min;

[0220] 8) Wash twice with 200 μL PBS; finally resuspend the cells with 200 μL PBS;

[0221] 9) Detect the fluorescence intensity of the sample FITC channel on a flow cytometer and analyze the results.

[0222] Main materials and reagents:

[0223] Helper phage KO7, Thermo / Invitrogen, 18311019

[0224] Streptavidin,Pierce,21125

[0225] Recombinant biotinylated Human BAFF-R Protein,KACTUS,BAFF-R-HM401;

[0226] High binding ELSIA plate,Costar,#3590

[0227] Corning 96 Well Clear Round Bottom TC-Treated Microplate,Costar,#3799

[0228] Blocking solution: PBS + 3% BSA

[0229] Rinse solution: PBS + 0.1% Tween20

[0230] Soluble one-component TMB substrate solution, Tiangen, PA-107-02

[0231] Anti-M13 Bacteriophage Coat Protein g8p antibody,abcam,ab9225

[0232] HRP Goat anti-mouse IgG(minimal x-reactivity)Antibody,Biolegend,405306

[0233] FITC horse anti mouse-IgG(H+L),Vector,FI2000

[0234] Experimental results:

[0235] Monoclonal clones were randomly selected from the enriched phage antibody pool, packaged into phage, and then the binding of monoclonal phage to BAFF-R-his-Bio protein and control protein SA was detected by phage ELISA to find BAFF-R specific phage antibody clones. The ELISA results of some clones are shown in Figure 2. Figure 2 As shown in the figure, clones H1-H2, H4-H7 bind well to the target antigen BAFF-R (BAFF-R-Bio-his), and do not bind to the control antigen SA, with good specificity. Clone H3 does not bind to either the target antigen BAFF-R-his-Bio or the control protein SA, and is a negative clone.

[0236] The initial FACS screening results of some clones are as follows Figure 3 Negative Control is a negative control phage antibody clone. Clone H1~H2, H4~H7 do not bind to K562, but bind to K562-BAFF-R cells, and are specific clones. Clone H3 does not bind to either K562-BAFF-R or K562, and is a negative clone.

[0237] Through ELISA detection and FACS initial screening, we obtained a total of 39 specific clones.

[0238] Example 3. Identification of monoclonal specificity by FACS using multiple cell lines

[0239] Purpose and principle of the experiment: Antibodies used for treatment must have very good target specificity, only bind to the target antigen, and not bind to any unrelated antigens; on the other hand, the amino acid sequence of the same antigen on different cell lines may be different (isomers or mutants) or the bound ligands may be different, and it is also necessary to examine whether our antibodies can bind to cells that are positive for various target proteins. In order to further analyze the specificity and universality of these monoclonal clones and find the best candidate clones, we further evaluated the specificity of the initial screening clones by flow cytometry. In this experiment, we used a variety of BAFF-R-positive cell lines and a variety of BAFF-R-negative cell lines to react with these monoclonal phage antibodies to analyze whether these clones can bind to BAFF-R antigens on different cell lines, and whether there is any non-specific binding with other cell lines that do not express BAFF-R. Through this experiment, we obtained several clones with excellent specificity.

[0240] Experimental method: Same as FACS initial screening;

[0241] Main samples and reagents:

[0242] K562-BAFF-R cell line, BAFF-R positive cell line;

[0243] K562 cell line, BAFF-R negative cell line;

[0244] NALM6 cell line, BAFF-R positive cell line;

[0245] Jurkat cell line, BAFF-R negative cell line;

[0246] The remaining reagents were the same as those for the FACS primary screening.

[0247] Experimental results:

[0248] Antibodies used for treatment must have very good target specificity. In order to further analyze the specificity of these monoclonal antibodies, we used enzyme-linked immunosorbent assay and flow cytometry to identify the only clone obtained in Example 2 on more antigens and cell lines. The results are shown in Figure 4A -B, Figure 4A The figure shows the flow cytometric peak patterns of Clone1, 5, and 14 binding to various cell lines. The Negative Control is the negative control phage antibody clone. Figure 4B The figure shows the flow cytometry peaks of Clone77 and 80 binding to various cell lines, and VAY736 is the positive control phage antibody clone. VAY736 antibody binds to all BAFF-R positive cell lines, but not to BAFF-R negative cell lines, indicating that the expression levels of BAFF-R antigen in the four selected cell lines are normal. Clone 5, 77, and 80 bind to all BAFF-R positive cell lines, while Clone 1 and 14 only bind to the BAFF-R overexpressing cell line K562-BAFF-R, but not to NALM6. They do not bind to BAFF-R negative cell lines, indicating good specificity.

[0249] Example 4. Identification of monoclonal specificity by ELISA using antigens from different companies

[0250] Purpose and principle of the experiment: Antibodies used for treatment must have very good target specificity, only binding to the target antigen and not binding to any unrelated antigens; on the other hand, the amino acid sequences of the same antigen produced by different companies will be different (isomers or mutants), and it is also necessary to examine whether our antibodies can bind to various target proteins. In order to further analyze the specificity and universality of these monoclonal clones and find the best candidate clones, we further evaluated the specificity of the initial screening clones by enzyme-linked immunosorbent assay (ELISA). In this experiment, we used BAFF-R antigens purchased from different companies and a variety of BAFF-R-unrelated antigens to react with these monoclonal phage antibodies to analyze whether these clones can bind to different BAFF-R antigens and whether there is any non-specific binding with other BAFF-R-unrelated antigens. Through this experiment, we obtained several clones with excellent specificity.

[0251] Experimental method: Same as ELISA initial screening;

[0252] Main samples and reagents:

[0253] Table 2

[0254]

[0255]

[0256] The remaining reagents are the same as those for the ELISA primary screening.

[0257] Experimental results:

[0258] Antibodies used for treatment must have very good target specificity. In order to further analyze the specificity of these monoclonal antibodies, we used enzyme-linked immunosorbent assay (ELISA) to identify multiple clones obtained in Example 2 on multiple antigens. The results are shown in Figure 5In the figure, Negative control is the negative control phage antibody clone. anti-M13phagemouse Ab / anti-mouse HRP Ab is a negative control without adding phage, only adding the first antibody (Anti-M13 phage mouse Ab) and the second antibody (anti-mouse HRP Ab); anti-mouse HRP Ab is a negative control with only adding the second antibody (anti-mouse HRP Ab); VAY736 Ab / anti-human IgG HRP Ab is a positive control with adding BAFF-R detection antibody; anti-human IgG HRP Ab / anti-his HRP Ab is a positive control for detecting antigen tag antibody. Clone 1, 5, 77, 80 binds to both BAFF-R antigens, but not to the non-related antigens KACTUS-CD19-Fc, Acro-CD70-his and SA, indicating that these four clones can bind to BAFF-R antigens of different conformations with good specificity. Clone14 weakly binds to the unrelated antigen Acro-CD70-his, and its specificity needs further confirmation.

[0259] Example 5. Detection of EGFRt expression on the surface of CAR-T cells

[0260] In order to verify the function of candidate clones on CAR-T cells, we constructed the following Figure 6 The CAR vector with the structure shown in the figure contains CD8α signal peptide, V HH Or scFv, CD8α hinge region, CD8α transmembrane region, 4-1BB co-stimulatory molecule and CD3ζ, and connected with T2A to a truncated EFGR molecule (EGFRt) can be used as a safety switch in clinical transformation. Then the CAR vector was packaged with lentivirus, and the expression of EGFRt was detected 5 to 7 days after T cells were transfected with the CAR lentivirus ( Figure 7 ). Since EGFRt is co-expressed with CAR molecules, it can be used as an indirect detection indicator for the distribution of CAR molecules on the surface of T cells without affecting the structure and function of CAR.

[0261] The brief experimental steps for CAR-T / EGFRt expression detection are as follows:

[0262] 1) Take 1×10 6 Each well of CAR-T / T cells was washed once with PBS, centrifuged at 300 g for 5 minutes, and the supernatant was discarded.

[0263] 2) Resuspend the cell pellet with 100 μL PBS, add 5 μL APC-CD5 antibody and 5 μL APC-EGFR antibody respectively, and incubate at 4°C in the dark for 15 minutes.

[0264] 3) Wash twice with PBS and centrifuge at 300g for 5 minutes.

[0265] 4) Resuspend in 200 μL PBS and detect on flow cytometry.

[0266] Main materials and reagents:

[0267] APC anti-human EGFR Antibody,Clone AY13,BioLegend,Cat.No.352906;

[0268] Fetal bovine serum (FBS), Gibco, Cat. No. 10099141;

[0269] Experimental results:

[0270] like Figure 7 As shown, on the seventh day, the expression of EGFR in T cells of each group was ≥30%.

[0271] Example 7. CD107a degranulation assay

[0272] Experimental principle and purpose:

[0273] CD107a is a marker of intracellular microvesicles. When microvesicles loaded with granzymes fuse with the cell membrane, CD107a on the cell membrane will increase. When monesin (purchased from BioLegend) is used to block its recovery, the intensity of microvesicle release can be quantitatively reflected. When CAR-T is stimulated by target antigens on target cells, granzymes will be released, and the activation of T cells can be determined by flow cytometry to detect the increase in CD107a.

[0274] Brief experimental steps of CD107a degranulation:

[0275] 1) The CAR-T cells and target cells to be tested were centrifuged at 300g for 5 min at room temperature, the supernatant was discarded, and the cells were resuspended in 1640 medium + 10% FBS to a concentration of 2x10 6 cells / mL;

[0276] 2) Add 100 μL of the CAR-T cells to be tested and 100 μL of the target cells to a 96-well plate and mix well;

[0277] 3) Add 1.5 μL PE / Cy7 anti-human CD107a antibody and 0.2 μL monensin to each well of cells, and then place in a cell culture incubator at 37°C, 5% CO2 for 4 h;

[0278] 4) After incubation, centrifuge at 300 g for 5 min at 4°C, discard the supernatant, and wash the cells twice with 200 μL PBS;

[0279] 5) Resuspend the cells with 100 μL PBS, add 1.5 μL BV421 anti-human CD8 and 1.5 μL APC anti-human EGFR antibodies respectively, mix well and incubate on ice in the dark for 20 min;

[0280] 6) After incubation, wash the cells three times with 200 μL PBS; resuspend with 100 μL PBS and detect using flow cytometry.

[0281] Main samples and reagents:

[0282] Target cellsNALM6-luc(BAFF-R+),RS4-luc(BAFF-R+),MEC1-luc(BAFF-R+)

[0283] JEKO1-luc(BAFF-R+), THP1-luc(BAFF-R-), Molm13-luc(BAFF-R-), CCRF-luc(BAFF-R-), MOLT4-luc(BAFF-R-);

[0284] Fetal bovine serum (FBS), Gibco, Cat. No. 10099141;

[0285] Monensin,BioLegend,Cat.No.420701;

[0286] PE / Cy7 mouse anti-human CD107a, BD, Cat.No.561348;

[0287] BV421 mouse anti-human CD8,BD,Cat.No.301036;

[0288] APC anti-human EGFR Antibody, Clone AY13, BioLegend, Cat. No. 35290.

[0289] Experimental results:

[0290] CAR-T cells were obtained by lentiviral transduction and cultured in vitro for 9-12 days before CD107a degranulation experiment. The CAR-T cells to be tested were incubated with target cells, monensin and CD107a antibody for 4 hours. The cell density of CAR-T cells and target cells was 2×10 5 cells / mL. Then, the samples were labeled with CD8 antibody and EGFR antibody, and flow cytometry was performed. In the Flowjo software, the live cell gate (P1) was selected in the scatter plot to remove cell debris; the cells in the P1 gate were analyzed and the single scattered cell gate (P2) was selected; then, CD8-positive cells (P3) were further selected in the P2 gate; finally, in the P3 gate, the proportion of CD107a-positive cells in cells stained positive for EGFR antibody (i.e., CAR-positive cells) was analyzed. The analysis results are shown in Table 3. The degranulation level of BAFF-R CAR-T was significantly increased only after co-incubation with BAFF-R-positive cells, while there was no significant increase in the degranulation level after co-incubation with negative cell lines. Among them, the degranulation level of BAFF-R-positive cells of clone numbers 1, 5, 14, 77, and 80 was high, and all higher than the control Vay736 CAR (CAR scFV sequence derived from Ianalumab).

[0291] Table 3 Results of CD107a degranulation effects of different target cells on CAR-T cells in each group.

[0292]

[0293] Example 8. In vitro cell killing assay

[0294] Experimental purpose and principle:

[0295] In vitro cell killing experiments used human acute B lymphoblastic leukemia cells NALM6, human acute lymphoblastic leukemia cells RS4, human mucoepidermoid carcinoma cells MEC1, and human mantle cell lymphoma cells JEKO1 as BAFF-R positive target cells, and MOLT4 cells as BAFF-R negative target cells to evaluate the antigen-specific killing ability of BAFF-RCAR-T cells. Among them, the above target cells were transduced by lentivirus to obtain target cells that stably expressed firefly luciferase, so the activity of luciferase in the sample can reflect the number of target cells. CAR-T cells and target cells were co-incubated and cultured. When the target cells are killed by CAR-T cells, luciferase will be released and quickly inactivated (the half-life of firefly luciferase is about 0.5h). If the target cells are not killed or inhibited by CAR-T cells, more luciferase will be produced as the target cells proliferate and the luciferase continues to be expressed. Therefore, the activity of luciferase can be used to detect the killing of target cells by CAR-T.

[0296] Brief experimental steps of in vitro cell killing:

[0297] 1) The above target cells were centrifuged at 500g for 5 min at room temperature, the supernatant was discarded, and the cells were resuspended in 1640+10% FBS medium to a concentration of 1x10 5 cells / mL; 100 μL of target cells were added to each well of a 96-well plate;

[0298] 2) According to the CAR positivity rate and effector-target ratio of the CAR-T sample to be tested, 100 μL of CAR-T cells were added to each well of the 96-well plate and mixed with the target cells; then placed in a carbon dioxide incubator for incubation for 24 hours;

[0299] 3) Use a luciferase detection kit to detect the luciferase activity in each well of the sample.

[0300] Main samples and reagents:

[0301] BAFF-R positive target cells: NALM6-luc, RS4-luc, MEC1-luc, JEKO1-luc

[0302] BAFF-R negative target cells Raji-luc, CCRF-luc;

[0303] Steady-Glo Luciferase Assay System, Promega, Cat. No. E2520.

[0304] Experimental results:

[0305] CAR-T cell samples and a fixed number of target cells (1x10 4 After mixing at different effector-target ratios (E:T), the samples were incubated for 24 hours, and then the luciferase activity (RLU) in the samples was detected. Since the luciferase activity can reflect the number of target cells in the sample, the killing / inhibition ability of CAR-T cells on target cells can be obtained by the change in luciferase activity in the sample. The lower the luciferase activity reading (RLU), the more target cells are killed.

[0306] like Figure 8 As shown in the figure, all CAR-T cell samples can effectively kill BAFF-R positive target cells, and the killing ability is similar to that of the control CAR Vay736. Among them, clone 14 has a certain degree of killing against negative cells MOLT4. Clones 1, 5, 77, and 80 have no non-specific killing against negative cells.

[0307] Through CD107a degranulation experiments and in vitro cell killing experiments, it was found that clones 1, 5, 77, and 80 were similar to the control CAR Vay736 in in vitro function. The CAR specificity and in vivo efficacy of these three clones were further evaluated below.

[0308] Example 9. Determination of the performance of organ-derived tumor cells to stimulate CAR cell degranulation

[0309] CD107a is a marker of intracellular microvesicles. When microvesicles loaded with granzymes fuse with the cell membrane, CD107a on the cell membrane will increase. When monesin (purchased from BioLegend) is used to block its recovery, the intensity of microvesicle release can be quantitatively reflected. When CAR-T is stimulated by target antigens on target cells, granzymes will be released, and the activation of T cells by target cells can be judged by flow cytometry by detecting the increase in CD107a.

[0310] Brief experimental steps of CD107a degranulation:

[0311] 1) The CAR-T cells and target cells to be tested were centrifuged at 300g for 5 min at room temperature, the supernatant was discarded, and the cells were resuspended in 1640 medium + 10% FBS to a concentration of 2x10 6 cells / mL;

[0312] 2) Add 100 μL of the CAR-T cells to be tested and 100 μL of the target cells to a 96-well plate and mix well;

[0313] 3) Add 1.5 μL PE / Cy7 anti-human CD107a antibody and 0.2 μL monensin to each well of cells, and then place in a cell culture incubator (37°C, 5% CO2, incubate for 4 hours);

[0314] 4) After incubation, centrifuge at 300 g for 5 min at 4°C, discard the supernatant, and wash the cells twice with 200 μL PBS;

[0315] 5) Resuspend the cells with 100 μL PBS, add 1.5 μL BV421 anti-human CD8 and 1.5 μL APC anti-human EGFR antibodies respectively, mix well and incubate on ice in the dark for 20 min;

[0316] 6) After incubation, wash the cells three times with 200 μL PBS; resuspend with 100 μL PBS and detect using flow cytometry.

[0317] Main samples and reagents:

[0318] Target cellsNALM6, 293T, A375, HepG2, NCHI460, OVCAR-3, Raji, HCT116, Panc-1, MDA-MB-468, KATOIII

[0319] Fetal bovine serum (FBS), Gibco, Cat. No. 10099141;

[0320] Monensin,BioLegend,Cat.No.420701;

[0321] PE / Cy7 mouse anti-human CD107a, BD, Cat.No.561348;

[0322] BV421 mouse anti-human CD8,BD,Cat.No.301036;

[0323] APC anti-human EGFR Antibody, Clone AY13, BioLegend, Cat. No. 35290.

[0324] Experimental results:

[0325] CAR-T cells were obtained by lentiviral transduction and cultured in vitro for 9-12 days before CD107a degranulation experiment. The CAR-T cells to be tested were incubated with target cells, monensin and CD107a antibody for 4 hours. The cell density of CAR-T cells and target cells was 2×10 6 cells / mL. The samples were then labeled with CD8 antibodies and EGFR antibodies and flow cytometry was performed. In the Flowjo software, the live cell gate (P1) was selected in the scatter plot to remove cell debris; the cells in the P1 gate were analyzed and the single scattered cell gate (P2) was selected; then, CD8-positive cells (P3) were further selected in the P2 gate; finally, in the P3 gate, the proportion of CD107a-positive cells in cells stained positive for EGFR antibodies (i.e., CAR-positive cells) was analyzed. The analysis results are as follows: Fig. 9 As shown, the degranulation level of BAFF-R CAR-T clones 1, 5, 77, and 80 was significantly increased only after co-incubation with BAFF-R positive cells, but there was no significant increase in degranulation level after co-incubation with organ-derived tumor cell lines. This shows that BAFF-R CAR-T cells are not easily activated by normal tissue and organ cells and have high safety.

[0326] The above in vitro experiments showed that the CAR-T cells of candidate clones 1, 5, 77, and 80 can effectively recognize BAFF-R antigens on tumor cells and produce effective killing. In vitro negative cell verification showed no nonspecific activation and killing activity, and the clinical safety expectations were good. Repeated stimulation experiments showed that the four candidate clones can be efficiently amplified under in vitro target antigen stimulation and have the potential to maintain long-term effective killing of tumor cells.

[0327] Example 10. Antitumor efficacy experiment on NCG tumor-bearing mice

[0328] In order to further verify the anti-tumor efficacy of the four candidate clones in mice, we identified the tumor cell line JEKO1-Luc, which expresses firefly luciferase (ffLuc), and identified that it can normally express firefly luciferase, and JEKO1 itself is a natural cell line with high expression of BAFF-R. JEKO1-luc cells were inoculated into immunodeficient NCG mice through the tail vein. After the mice formed tumors, the candidate clone CAR-T cells were inoculated to verify their anti-tumor effects.

[0329] Brief test process :

[0330] a) Establishing a firefly luciferase (ffLuc) overexpressing cell line JEKO1-luc and verifying that it can normally express firefly luciferase;

[0331] b) After the cells are cultured to the logarithmic growth phase, the cells are collected;

[0332] c) 48 NCG mice were purchased, raised for 1 week, and JEKO1-luc cells were injected into the tail vein at 5×10 6 cells / 200 μL PBS, and the general condition of the mice was observed every day after injection;

[0333] d) On day 10, mice were divided into groups and injected into the tail vein with CAR-T cells (3E+06CAR+ cells / mouse), control untransfected Mock-T cells (2E+07 cells / mouse) and PBS buffer.

[0334] e) Regular imaging observations, weighing of body weight, observation and recording of the general condition of the mice, and regular determination of the proliferation of T cells in the mouse blood.

[0335] Experimental results:

[0336] Fig.10 , Fig.11 The candidate clones showed anti-tumor efficacy in mice. Fig.10 A and Fig.11As shown in the figure, the tumor burden of PBS, mockT, 1500 (Vay CAR), 1499 (city of hope CAR), clone 77, 80 CAR-T cells continued to increase after reinfusion. The tumor burden of clone 1 CAR-T cells after reinfusion was slightly lower than that of PBS and mockT, but the inhibitory effect on tumor growth was weaker. The tumor burden of clone 5 could be significantly inhibited. Fig.10 As shown in B, the 35-day survival rate of clone 5 mice was greater than 80%. Fig.10 C shows the proliferation of T cells in mouse blood. The proliferation of BS, mockT, 1500 (Vay CAR), 1499 (city of hope CAR), and clone77 CAR in vivo was not obvious. The proliferation of clone 80 T cells only decreased rapidly in the first two weeks, which may be the reason for its poor efficacy. Clone 1 and 5 can maintain continuous and effective proliferation in vivo, which is significantly better than other clones, and the start time of clone 5 expansion is earlier than clone 1.

[0337] In summary, Clone 5 has the best in vivo antitumor efficacy and good safety, and is a better candidate clone for later clinical development. Clone 1 also has a certain antitumor effect and good safety, and can be used as a candidate clone.

[0338] References

[0339] 1.Wang S,Wang L,Hu J,Qian W,Zhang X,Hu Y,Zhu Q,Chen B,Wu D,Chang CHet al:Outcomes in refractory diffuse large B-cell lymphoma:results from amulticenter real-world study in China.Cancer Commun(Lond)2021,41(3):229-239.

[0340] 2.Spiegel JY,Patel S,Muffly L,Hossain NM,Oak J,Baird JH,Frank MJ,Shiraz P,Sahaf B,Craig J et al:CAR T cells with dual targeting of CD19 andCD22 in adult patients with recurrent or

[0341] refractory B cell malignancies:a phase 1trial. Nat Med 2021,27(8):1419-1431.

[0342] 3. Rodig SJ, Shahsafaei A, Li B, Mackay CR, Dorfman DM: BAFF-R, the major Bcell-activating factor receptor, is expressed on most mature B cells and B-cell lymphoproliferative disorders. Hum Pathol 2005, 36(10): 1113-1119.

[0343] Sequences of the present invention

[0344] CD8α signal peptide nucleic acid sequence SEQ ID NO.1

[0345] ATGGCCCTGCCTGTGACAGCTCTGCTCCTCCCTCTGGCCCTGCTGCTCCATGCCGCCAGACCC

[0346] CD8α signal peptide protein sequence SEQ ID NO.2

[0347] MALPVTALLLPLALLLHAARP

[0348] CD8α hinge region nucleic acid sequence SEQ ID NO.3

[0349] TTCGTGCCCGTGTTCCTGCCCGCCAAACCTACTACTACCCCTGCACCTAGGCCTCCCACCCAGCCCCAACAATCGCCAGCCAGCCTCTGTCTCTGCGGCCCGAAGCCTGTAGACCTGCTGCCGGCGGAGCCGTGCACACCAGAGGCCTGGACTTCGCCTGCGAC

[0350] CD8α hinge region protein sequence SEQ ID NO.4

[0351] FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0352] CD8α transmembrane region nucleic acid sequence SEQ ID NO.5

[0353] ATCTACATCTGGGCCCCTCTGGCCGGCACCTGTGGCGTGCTGCTGCTGAGCCTGGTGATCACCCTGTACTGCAACCACCGGAAC

[0354] CD8α transmembrane protein sequence SEQ ID NO.6

[0355] IYIWAPLAGTCGVLLLSLVITLYCNHRN

[0356] 4-1BB intracellular domain nucleic acid sequence SEQ ID NO.7

[0357] AAACGGGGCAGAAAGAAACTCCTGTATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG

[0358] 4-1BB intracellular domain protein sequence SEQ ID NO.8

[0359] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0360] CD3z intracellular signaling domain nucleic acid sequence SEQ ID NO.9

[0361] AGAGTGAAGTTCAGCAGATCCGCCGACGCCCCTGCCTACCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAACCTGGGCAGACGGGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCCGGGACCCCGAGATGGGCGGAAAGCCCAGACGGAAGAACCCCCAGGAA GGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGCGGCAAGGGCCACGATGGCCTGTACCAGGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCCAGA

[0362] CD3z intracellular signaling domain protein sequence SEQ ID NO.10

[0363] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0364] Cleavage peptide T2A nucleic acid sequence SEQ ID NO.11

[0365] GAGGGAAGGGGCAGCTTATTAACATGTGGCGATGTGGAAGAGAACCCCGGTCCC

[0366] Cleavage peptide T2A protein sequence SEQ ID NO.12

[0367] EGRGSLLTCGDVEENPGP

[0368] CSF2RA signal nucleic acid sequence SEQ ID NO.13

[0369] ATGCTGCTGCTCGTGACCTCTTTACTGTTATGTGAGCTGCCCCACCCCGCTTTTTTACTGATCCCT

[0370] CSF2RA signal protein sequence SEQ ID NO.14 MLLLVTSLLLCELPHPAFLLIP

[0371] EGFRt nucleic acid sequence SEQ ID NO.15

[0372]

[0373] EGFRt protein sequence SEQ ID NO.16

[0374] RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM

[0375] Nucleic acid sequence of P2A SEQ ID NO.17

[0376] gctactaacttcagcctgctgaagcaggctggtgacgtcgaggagaatcctggcccc

[0377] Protein sequence of P2A SEQ ID NO.18

[0378] ATNFSLLKQAGDVEENPGP

[0379] BAFF-R-5 CAR VHH nucleic acid sequence SEQ ID NO.19

[0380] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCACCTGTGCAGCCTCTGGATTCACGTTCAGTACCTCTGGCATGTACTGGATTCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTAGTAGTAGTACCATATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTATTGTGTGACTCAGTCGTGTGGTGCTGGTAAATGCTACCCGGGGGTCTGGGGCCAAGGGACCACGGTCACTGTCTCCTCA

[0381] BAFF-R-5 CARVHH amino acid sequence SEQ ID NO.20

[0382] EVQLVESGGGVVQPGRSLRLTCAASGFTFSTSGMYWIRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTQSCGAGKCYPGVWGQGTTVTVSS

[0383] Vay736 nucleic acid sequence SEQ ID NO.21

[0384] CAGGTGCAGCTGCAGCAGAGCGGCCCCGGCCTGGTGAAGCCCAGCCAGACCCTGAGCCTGACCTGCGCCATCAGCGGCGACAGCGTGAGCAGCAACAGCGCCGCCTGGGGCTGGATCCGCCAGAGCCCCGGCCGCGGCCTGGAGTGGCTGGGCCGCATCTACTACCGCAGCAAGTGGTACAACAGCTACGCCGTGAGCGTGAAGAGCCGCATCACCATCAACCCCGACACCAGCAAGAACCAGTTCAGCCTGCAGCTGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGCTACCAGTGGGTGCCCAAGATCGGCGTGTTCGACAGCTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGAGGAGGCGGCTCCGGCGGCGGCGGCAGCGGCGGAGGAGGCTCTGACATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGTTCATCCTGCCCGAGTACCTGAGCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGGCAGCAGCAGCCGCGCCACCGGCGTGCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTTCTACAGCAGCCCCCTGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG

[0385] The amino acid sequence of Vay736 SEQ ID NO.22

[0386] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWGWIRQSPGRGLEWLGRIYYRSKWYNSYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARYQWVPKIGVFDSWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPATLSLSPGERATLSCRASQFILPEYLSWYQQKPGQAPRLLIYGSSSRATGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFYSSPLTFGQGTKVEIK

[0387] City of Hope CAR scFV nucleic acid sequence SEQ ID NO.23

[0388] GTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCCAGACCCTGAGCCTTACTTGCACGGTGAGCGGTGACAGCATCACCAGCGGCTACTGGAACTGGATCCGCCAGCACCCCGGCAAGGGCCTGGAGTACATCGGCTACATCAGCTACAGCGGCAGCACCTACTACAACCCCAGCCTGAAGAGCCGCGTGACCATCAGCCGCGACACCAGCAAGAACCAGTACAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCAGCCCCAACTACCCCTTCTACGCCATGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCGAGAGCGTGGACAACTACGGCATCAGCTTCATGAACTGGTTCCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGCCGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAGCAAGGAGGTGCCCTGGACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGCGC

[0389] City of Hope CAR scFV Amino Acid Sequence SEQ ID NO.24

[0390] VQLQESGPGLVKPSQTLSLTCTVSGDSITSGYWNWIRQHPGKGLEYIGYISYSGSTYYNPSLKSRVTISRDTSKNQYSLKLSSVTAADTAVYYCASPNYPFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPATLSLSPGERATLSCRASESVDNYGISFMNWFQQKPGQAPRLLIYAASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSKEVPWTFGGGTKVEIKR

[0391] 05VH amino acid sequence SEQ ID NO.25

[0392] EVQLVESGGGVVQPGRSLRLTCAASGFTFSTSGMYWIRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVTQSCGAGKCYPGVWGQGTTVTVSS

[0393] 05VH nucleotide sequence SEQ ID NO.26

[0394] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCACCTGTGCAGCCTCTGGATTCACGTTCAGTACCTCTGGCATGTACTGGATTCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTAGTAGTAGTACCATATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTATTGTGTGACTCAGTCGTGTGGTGCTGGTAAATGCTACCCGGGGGTCTGGGGCCAAGGGACCACGGTCACTGTCTCCTCA

[0395] 05VH CDR1 SEQ ID NO.27 GFTFSTSG

[0396] 05VH CDR2 SEQ ID NO.28 ISSSSSTI

[0397] 05VH CDR3 SEQ ID NO.29 VTQSCGAGKCYPGV

[0398] 01VL Amino Acid Sequence SEQ ID NO.30

[0399] DIQMTQSPSSLSASVGDRVTITCRASQKISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGADFTLTISSLQPEDFATYYCQQSYSPPTTFGGGTKVDIKR

[0400] 01VL Nucleotide Sequence SEQ ID NO.31

[0401] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTTGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAAAATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGGCAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAGCAGAGTTACAGTCCCCCGACCACTTTCGGCGGAGGGACCAAAGTGGATATCAAACGT

[0402] 01VH Amino Acid Sequence SEQ ID NO.32

[0403] MAEVQLVESGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARNTYYYDSSGYPNDAFDIWGQGTTVTVSS

[0404] 01VH Nucleotide Sequence SEQ ID NO.33

[0405] ATGGCCGAGGTGCAGCTGGTGGAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAG AAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAAATACGTATTACTATGATAGTAGTGGTTACCCAAATGATGCTTTTGATATCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

[0406] The amino acid sequence of the scFv (VL-VH) contained in the CAR constructed by clone 1 (01) is SEQ ID NO.34

[0407] DIQMTQSPSSSLSASVGDRVTITCRASQKISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGADFTLTISSLQPEDFATYYCQQSYSPPTTFGGGTKVDIKRSRGGGGSGGGGSGGGGSLE MAEVQLVESGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSSTAYMELRSLRSDDTAVYYCARNTYYYDSSGYPNDAFDIWGQGTTVTVSS

[0408] The nucleotide sequence of the scFv (VL-VH) contained in the CAR constructed by clone 1 (01) is SEQ ID NO.35

[0409] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTTGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAAAATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGGCAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAGCAGAGTTACAGTCCCCCGACCACTTTCGGCGGAGGGACCAAAGTGGATATCAAACGTTCTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCCTCGAGATGGCCGAGGTGCAGCTGGTGGAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAAATACGTATTACTATGATAGTAGTGGTTACCCAAATGATGCTTTTGATATCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

[0410] 01VL CDR1 SEQ ID NO.36 QKISSY

[0411] 01VL CDR2 AAS

[0412] 01VL CDR3 SEQ ID NO.37 QQSYSPPTT

[0413] 01VH CDR1 SEQ ID NO.38 GYTFTSYG

[0414] 01VH CDR2 SEQ ID NO.39 ISAYNGNT

[0415] 01VH CDR3 SEQ ID NO.40 ARNTYYYDSSGYPNDAFDI

[0416] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. An antibody targeting B cell activating factor receptor (BAFF-R), characterized in that The heavy chain variable region of the antibody comprises the following complementary determining regions CDR: CDR1 shown in SEQ ID NO.27, CDR2 shown in SEQ ID NO.28 and CDR3 shown in SEQ ID NO.29; or The light chain variable region of the antibody comprises the following complementary determining regions CDR: CDR1 shown in SEQ ID NO.36, CDR2 shown in AAS, and CDR3 shown in SEQ ID NO.37, and the heavy chain variable region of the antibody comprises the following complementary determining regions CDR: CDR1 shown in SEQ ID NO.38, CDR2 shown in SEQ ID NO.39, and CDR3 shown in SEQ ID NO.40; Any of the above amino acid sequences also includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and is able to retain BAFF-R binding affinity.

2. The antibody according to claim 1, characterized in that The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO.25 or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the above sequence.

3. The antibody according to claim 1, characterized in that The light chain variable region has an amino acid sequence as shown in SEQ ID NO.30, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the above sequence; the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO.32, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the above sequence.

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

40.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody of claim 1; and / or the chimeric antigen receptor of claim 4.

6. A carrier, characterized in that The vector contains the nucleic acid molecule according to claim 5.

7. A host cell, characterized in that The host cell contains the vector according to claim 6, or the exogenous nucleic acid molecule according to claim 5 is integrated into the chromosome.

8. An engineered immune cell, characterized in that: The immune cell contains the vector according to claim 6, or has the exogenous nucleic acid molecule according to claim 5 integrated into its chromosome, or expresses the antibody according to claim 1 or the chimeric antigen receptor according to claim 4.

9. A preparation, characterized in that The preparation contains the engineered immune cells according to claim 8, and a pharmaceutically acceptable carrier, diluent or excipient.

10. A use of the antibody of claim 1, the chimeric antigen receptor of claim 4 or the engineered immune cell of claim 8, characterized in that: Used for preparing drugs or preparations for preventing and / or treating BAFF-R related diseases.

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