CN116396389B

CN116396389B - Single-domain antibody targeting BCMA, chimeric antigen receptor and application thereof

Info

Publication number: CN116396389B
Application number: CN202310496243.8A
Authority: CN
Inventors: 周丹; 袁肖; 何雨辰; 孙海; 邹强
Original assignee: Chengdu Yousainuo Biotechnology Co ltd
Current assignee: Chengdu Yousainuo Biotechnology Co ltd
Priority date: 2023-05-05
Filing date: 2023-05-05
Publication date: 2024-01-23
Anticipated expiration: 2043-05-05
Also published as: CN116396389A

Abstract

The invention relates to the technical field of immunotherapy, and provides a single-domain antibody targeting BCMA, a Chimeric Antigen Receptor (CAR) targeting BCMA constructed by using the single-domain antibody and an engineered immune effector cell. The invention also provides application of the single domain antibody targeting BCMA and the engineered immune effector cell containing the CAR in preparing medicines for treating BCMA related diseases.

Description

Single-domain antibody targeting BCMA, chimeric antigen receptor and application thereof

Technical Field

The invention belongs to the technical field of immunotherapy, and in particular relates to a single-domain antibody targeting BCMA, a chimeric antigen receptor of the targeting BCMA constructed by the single-domain antibody and an engineered immune effector cell. The invention also relates to a method of treating a disease or disorder in a subject, in particular to T cell immunotherapy based on chimeric antigen receptors.

Background

Multiple Myeloma (MM) is a currently incurable invasive plasma malignancy, classified as a B-cell related malignancy, and proliferates in the bone marrow in an uncontrolled manner, thereby interfering with the normal metabolism of blood cells and causing a painful bone lesion. Multiple myeloma can clinically manifest hypercalcemia, renal insufficiency, anemia, bone lesions, bacterial infections, hyperviscosity, and amyloidosis. The number of cases of myeloma is expected to rise year by year due to aging population. As with many cancers, the cause of multiple myeloma is still unclear and currently incurable. Traditional treatments for multiple myeloma, such as those including chemotherapy, radiotherapy, stem cell transplantation, bone marrow transplantation, etc., although current therapies generally result in remission of the disease, almost all patients eventually relapse. Therefore, these patients still have urgent demands for more therapeutic treatment means for multiple myeloma.

B cell maturation antigen (B-cell maturation antigen, BCMA), also known as CD269, consists of 184 amino acid residues, the intracellular region of which contains 80 amino acid residues, the extracellular region sequence is very short, and there is only one B cell surface molecule of the carbohydrate recognition domain. BCMA belongs to a class i transmembrane signaling protein lacking a signaling peptide, a member of the tumor necrosis factor receptor family (TNFR), and binds to both B cell activating factor (BAFF) and proliferation-inducing ligand (APRIL), respectively. In normal tissues, BCMA is expressed on the surfaces of mature B cells and plasma cells, and researches show that the immune system of a BCMA gene knockout mouse is normal, has a normal spleen structure, and B lymphocytes are normal in development, but the number of the plasma cells is obviously reduced, so that the BCMA plays an important role in maintaining the survival of the plasma cells, and the mechanism mainly comprises that the BCMA is combined with BAFF proteins and up-regulates anti-apoptosis genes Bcl-2, mcl-1, bclw and the like so as to maintain the cell growth. Similarly, this mechanism plays an important role in myeloma cells and plays an important role in promoting malignant proliferation of myeloma cells. The results of previous studies have shown that BCMA is ubiquitously expressed in multiple myeloma cell lines and that consistent results are also detected in multiple myeloma patients. Based on the prior reports by Kochenderfer et al, the expression profile of BCMA molecules was studied extensively using Q-PCR, flow Cytometry and immunohistochemistry, confirming that BCMA was not expressed in normal human tissues outside mature B cells and plasma cells, and also in CD34+ hematopoietic cells (see J.N.Kochenderfer et al B-cell maturation antigen is a promising target for adoptive T-cell therapy of multiple myela. Clin Cancer Res.2013Apr 15;19 (8): 2048-60). These studies suggest that BCMA can be one of the very potential therapeutic targets for B cell-related malignancies (particularly multiple myeloma) or autoimmune diseases, applied to various cellular immunotherapy.

Single-domain antibodies (sdabs) differ from traditional 4-chain antibodies by having a single variable domain of a monomeric antibody. For example, camelids and sharks produce antibodies that naturally lack light chains, which are referred to as heavy chain-only antibodies, or simply heavy chain antibodies (hcabs). The antigen binding fragment in each arm of a camelid heavy chain antibody has a single heavy chain variable domain, known as a heavy chain single domain antibody (VHH), which can have high affinity for antigens without the aid of light chains. The camelid-derived VHH antibodies are referred to as the smallest functional antigen binding fragments and have a molecular weight of only about 15kD and are thus also referred to as Nanobodies (Nbs). VHH antibodies have the natural advantages of good solubility, high stability, strong penetration and wide binding epitopes, and since they were discovered, they have been increasingly focused by researchers in the field of immunotherapy, and various studies on them have become mature. In application, aiming at hematological malignant diseases, autoimmune diseases, virus infection and the like, the composition gradually enters a clinical research stage, and has great advantages in the aspects of treating malignant tumors, autoimmune diseases, anti-infection and the like.

Chimeric antigen receptor (Chimeric Antigen Receptor, CAR) modified T cells are receiving wide attention and application as an immunotherapeutic strategy in tumor therapy, especially in hematological malignancies. The principle is that through genetic engineering modification, T cells express a receptor structure (for example, a single-chain antibody) capable of specifically recognizing tumor cell surface antigens, and after the receptor is specifically combined with the tumor cell surface antigens, the downstream immune co-stimulatory factors and the T cells are activated, so that the activated T cells secrete relevant cytokines, the tumor cells are specifically killed, and the restriction of a main histocompatibility complex (Major Histocompatibility Complex, MHC) with specificity to target tumor antigens is avoided. The representative structure of a CAR consists of four parts, an extracellular antigen binding domain (typically a single chain antibody with antigen recognition function), a hinge region, a transmembrane domain, and an intracellular signaling domain. Classical CAR structures are currently classified into the first generation (without costimulatory molecules), the second generation (comprising one costimulatory molecule) and the third generation (comprising two costimulatory molecules) depending on whether or not costimulatory molecules are added to the intracellular signaling domain, the second generation CAR structure being the most used by the current market products and clinical research stage.

Monoclonal antibody-based therapies are expected to overcome the problem of chemotherapy resistance and potential toxicity associated with the treatment, one of the most promising is chimeric antigen receptor modified T cell (CAR-T cell) therapies, which can break through MHC restriction, directly recognize tumor antigens, and specifically kill tumor cells. Currently CAR-T cells have been widely used in hematological malignancies, in particular CAR-T cell immunotherapy with CD19 as target antigen has been a breakthrough development and demonstrated surprising efficacy, but the indications are mainly limited to acute B-lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma (DLBCL) and Mantle Cell Lymphoma (MCL), and there is still a need for more CAR-T cell immunotherapy against other target antigens. Because CAR-T cell manufacturing techniques require the use of single chain antibodies that have good binding activity and high binding epitope efficiency, one of the key technical parts of CAR-T cell therapies is the screening of high affinity antibodies that have good specificity, strong binding capacity, and efficient binding epitope. However, conventional BCMA antibodies are limited by the problems of large molecular weight, weak binding force with antigen, low affinity, difficult transformation, poor stability, and the like, and further single-chain transformation is required, so that flexible construction of CAR-T cells is difficult to realize by using conventional BCMA antibodies (monoclonal antibodies, etc.).

Thus, there remains a broad need to screen and develop improved single domain antibodies targeting BCMA, their constructed chimeric antigen receptors targeting BCMA, and engineered immune effector cells. In particular, stable and small BCMA-targeting single domain antibodies suitable for more effective and more efficient CAR-T cell therapies were developed.

Disclosure of Invention

One of the purposes of the present invention is to provide a single domain antibody targeting BCMA, which has a natural single chain structure, has the advantages of small molecular weight, high solubility, high stability, low immunogenicity, high tissue permeability and no need of additional folding and assembling steps or linker optimization modification, and is a promising alternative to scFv single chain antibodies with larger molecular weight, and has very remarkable tumor cell killing ability after being constructed into CAR-T cells.

The single domain antibody for targeting BCMA provided by the invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 comprises the amino acid sequences shown in SEQ ID NOS.1-5, wherein CDR2 comprises the amino acid sequences shown in SEQ ID NOS.6-16, and wherein CDR3 comprises the amino acid sequences shown in SEQ ID NOS.17-33.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 1, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 6, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 17.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 2, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 7, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 18.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 2, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 8, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 19.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 3, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 9, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 20.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 2, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 10, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 21.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 4, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 11, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 22.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 2, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 12, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 23.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 2, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 13, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 24.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 2, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 12, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 25.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 2, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 12, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 26.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 1, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 13, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 27.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 2, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 10, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 28.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 1, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 12, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 29.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 2, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 14, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 30.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 1, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 15, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 31.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 1, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 12, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 32.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 5, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 16, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 33.

The single domain antibody for targeting BCMA provided by the invention comprises CDR1, CDR2 and CDR3 regions; wherein the determination of CDR1, CDR2 and CDR3 is according to the IMGT numbering scheme, the Kabat numbering scheme, the AbM numbering scheme, the Chothia numbering scheme or the Contact numbering scheme.

The technical scheme disclosed by the invention, wherein the specific amino acid sequence information shown in SEQ ID NO 1-33 is shown in Table 1.

TABLE 1

The single domain antibody targeting BCMA provided by the invention comprises an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO. 34-50.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID No. 34.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID No. 35.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID No. 36.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID No. 37.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID No. 38.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID No. 39.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID No. 40.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID No. 41.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID No. 42.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID No. 43.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID No. 44.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID No. 45.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID No. 46.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID No. 47.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID No. 48.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID No. 49.

In some embodiments, the present invention provides a single domain antibody targeting BCMA comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID No. 50.

The single domain antibody for targeting BCMA provided by the invention comprises an amino acid sequence shown in a table 2 or shown in SEQ ID NO. 34-50.

In some embodiments, the single domain antibodies targeting BCMA provided herein comprise the amino acid sequence shown as SEQ ID NO 34.

In some embodiments, the single domain antibodies targeting BCMA provided herein comprise an amino acid sequence as set forth in SEQ ID NO. 35.

In some embodiments, the single domain antibodies targeting BCMA provided herein comprise the amino acid sequence shown as SEQ ID NO. 36.

In some embodiments, the single domain antibodies targeting BCMA provided herein comprise the amino acid sequence shown as SEQ ID NO. 37.

In some embodiments, the single domain antibodies targeting BCMA provided herein comprise the amino acid sequence shown as SEQ ID NO. 38.

In some embodiments, the single domain antibodies targeting BCMA provided herein comprise the amino acid sequence shown as SEQ ID NO 39.

In some embodiments, the single domain antibodies targeting BCMA provided herein comprise the amino acid sequence shown as SEQ ID NO. 40.

In some embodiments, the single domain antibodies targeting BCMA provided herein comprise the amino acid sequence shown as SEQ ID NO. 41.

In some embodiments, the single domain antibodies targeting BCMA provided herein comprise an amino acid sequence as set forth in SEQ ID NO. 42.

In some embodiments, the single domain antibodies targeting BCMA provided herein comprise the amino acid sequence shown as SEQ ID NO. 43.

In some embodiments, the single domain antibodies targeting BCMA provided herein comprise the amino acid sequence shown as SEQ ID NO. 44.

In some embodiments, the single domain antibodies targeting BCMA provided herein comprise the amino acid sequence shown as SEQ ID NO. 45.

In some embodiments, the single domain antibodies targeting BCMA provided herein comprise the amino acid sequence shown as SEQ ID NO. 46.

In some embodiments, the single domain antibodies targeting BCMA provided herein comprise an amino acid sequence as set forth in SEQ ID NO. 47.

In some embodiments, the single domain antibodies targeting BCMA provided herein comprise the amino acid sequence shown as SEQ ID NO. 48.

In some embodiments, the single domain antibodies targeting BCMA provided herein comprise the amino acid sequence shown as SEQ ID NO. 49.

In some embodiments, the single domain antibodies targeting BCMA provided herein comprise the amino acid sequence shown as SEQ ID NO. 50.

The technical scheme disclosed by the invention, wherein the specific amino acid sequence information shown in SEQ ID NO 34-50 is shown in Table 2.

TABLE 2

One of the purposes of the present invention is to provide a chimeric antigen receptor, which has a very remarkable capability of killing tumor cells after transfection to prepare CAR-T cells.

The present invention provides chimeric antigen receptors comprising

(a) An extracellular antigen-binding domain,

(b) A transmembrane domain, and

(c) An intracellular signaling domain;

wherein the extracellular antigen-binding domain comprises a single domain antibody that targets BCMA as described previously.

In some embodiments, the present invention provides chimeric antigen receptors wherein the transmembrane domain is derived from CD8 a, CD28, cd3ζ, cd3γ, cd3δ, cd3ε, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, tcra, tcrβ, tcrγ, tcrδ, tcrζ, OX40, ICOS, LAG-3, 2B4, BTLA, CTLA-4, PD-1, or any combination thereof.

In some embodiments, the present invention provides chimeric antigen receptors wherein the transmembrane domain is derived from CD8 a.

In some embodiments, the present invention provides chimeric antigen receptors in which the transmembrane domain comprises the amino acid sequence shown as SEQ ID NO. 53.

In some embodiments, the present invention provides chimeric antigen receptors in which the intracellular signaling domain is derived from cd3ζ, cd3γ, cd3δ, cd3ε, CD22, CD66d, CD79a, CD79b, fcrβ, fcrγ, or any combination thereof.

In some embodiments, the present invention provides chimeric antigen receptors wherein the intracellular signaling domain is derived from cd3ζ.

In some embodiments, the present invention provides chimeric antigen receptors in which the intracellular signal transduction domain comprises the amino acid sequence set forth in SEQ ID NO. 55.

In some embodiments, the invention provides chimeric antigen receptors in which the intracellular signaling domain further comprises a costimulatory signaling domain.

In some embodiments, the present invention provides chimeric antigen receptors in which the costimulatory signaling domain is derived from 4-1BB (CD 137), CD28, OX40, ICOS, ICAM, LFA-1, TLR1-10, CARD11, CD2, CD3, CD7, CD8 alpha, CD27, CD30, CD40, CD83, HVEM, BTLA, B7-H3, GITR, DAP10, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, ZAP70, CD83 ligand, or any combination thereof.

In some embodiments, the present invention provides chimeric antigen receptors wherein the costimulatory signaling domain is derived from 4-1BB (CD 137).

In some embodiments, the present invention provides chimeric antigen receptors in which the costimulatory signaling domain comprises the amino acid sequence depicted as SEQ ID NO. 54.

In some embodiments, the chimeric antigen receptor provided herein further comprises a hinge region located between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain.

In some embodiments, the present invention provides chimeric antigen receptors wherein the hinge region is derived from CD8 a, CD28, CD137, igG1, igG4, or any combination thereof.

In some embodiments, the invention provides chimeric antigen receptors in which the hinge region is derived from CD8 a.

In some embodiments, the present invention provides chimeric antigen receptors in which the hinge region comprises the amino acid sequence set forth in SEQ ID NO. 52.

In some embodiments, the chimeric antigen receptor provided by the invention further comprises a signal peptide located at the N-terminus of the chimeric antigen receptor polypeptide.

In some embodiments, the present invention provides chimeric antigen receptors wherein the signal peptide is derived from HLA-A, CD8 a, CD4, CD33, CD137, GM-CSFR a, igG1, ig kappa, IL-2, or any combination thereof.

In some embodiments, the present invention provides chimeric antigen receptors wherein the signal peptide is derived from CD8 a.

In some embodiments, the present invention provides chimeric antigen receptors wherein the signal peptide is derived from HLA-A.

In some embodiments, the present invention provides chimeric antigen receptors wherein the signal peptide comprises the amino acid sequence set forth in SEQ ID NO. 51.

The technical scheme disclosed by the invention, wherein the specific amino acid sequence information shown in SEQ ID NO 51-55 is shown in Table 3.

TABLE 3 Table 3

The chimeric antigen receptor provided by the invention comprises an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO. 56-72.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 56.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 57.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 58.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 59.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 60.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 61.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 62.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 63.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 64.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 65.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 66.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 67.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 68.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 69.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 70.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 71.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 72.

The chimeric antigen receptor provided by the invention comprises an amino acid sequence shown in table 4 or SEQ ID NO. 56-72.

In some embodiments, the chimeric antigen receptor provided by the invention comprises the amino acid sequence shown as SEQ ID NO. 56.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 57.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 58.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as shown in SEQ ID NO. 59.

In some embodiments, the chimeric antigen receptor provided by the invention comprises the amino acid sequence as shown in SEQ ID NO. 60.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as shown in SEQ ID NO. 61.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as shown in SEQ ID NO. 62.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as shown in SEQ ID NO. 63.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 64.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 65.

In some embodiments, the chimeric antigen receptor provided by the invention comprises the amino acid sequence as set forth in SEQ ID NO. 66.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 67.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 68.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as shown in SEQ ID NO. 69.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 70.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as shown in SEQ ID NO. 71.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as shown in SEQ ID NO. 72.

The technical scheme disclosed by the invention, wherein the specific amino acid sequence information shown in SEQ ID NO 56-72 is shown in Table 4.

TABLE 4 Table 4

It is an object of the present invention to provide an isolated nucleic acid comprising a nucleic acid sequence encoding a chimeric antigen receptor as described above. In some embodiments, the invention provides isolated nucleic acids comprising a nucleic acid sequence as set forth in Table 5 or as set forth in SEQ ID NOS: 73-89.

The technical scheme disclosed by the invention, wherein the specific nucleic acid sequence information shown in SEQ ID NO 73-89 is shown in Table 5.

TABLE 5

It is an object of the present invention to provide a vector comprising a nucleic acid encoding a nucleic acid sequence of a chimeric antigen receptor as described above.

It is an object of the present invention to provide an engineered immune effector cell comprising a chimeric antigen receptor, an isolated nucleic acid, or a vector as described above.

In some embodiments, the engineered immune effector cells provided herein are selected from T cells, B cells, NK cells, NKT cells, DNT cells, macrophages, dendritic cells, induced pluripotent stem cells (ipscs), or any combination thereof.

It is an object of the present invention to provide a pharmaceutical composition comprising a single domain antibody or engineered immune effector cell targeting BCMA as described above and a pharmaceutically acceptable carrier or excipient.

It is an object of the present invention to provide a method of treating a disease or disorder in a subject comprising administering to the subject a therapeutically effective amount of a single domain antibody, engineered immune effector cell or pharmaceutical composition as described previously that targets BCMA.

In some embodiments, the invention provides a method of treating a disease or disorder in a subject, wherein the disease or disorder is a B-cell related disease or disorder and/or a BCMA related disease or disorder.

In some embodiments, the invention provides a method of treating a disease or disorder in a subject, wherein the disease or disorder is cancer or tumor.

In some embodiments, the invention provides a method of treating a disease or disorder in a subject, wherein the disease or disorder is a B cell-related malignancy. For example, the B cell-related malignancy is Multiple Myeloma (MM). More specifically, wherein the disease or disorder is selected from marginal zone lymphoma (e.g., splenic marginal zone lymphoma), diffuse large B-cell lymphoma (DLBCL), mantle Cell Lymphoma (MCL), primary Central Nervous System (CNS) lymphoma, primary mediastinum large B-cell lymphoma (PMLBCL), small Lymphocytic Lymphoma (SLL), B-cell prolymphocytic leukemia (B-PLL), follicular Lymphoma (FL), burkitt's lymphoma, primary intraocular lymphoma, chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), hairy Cell Leukemia (HCL), precursor B-lymphocytic leukemia, non-hodgkin's lymphoma (NHL), high grade B-cell lymphoma (HGBL), and/or Multiple Myeloma (MM).

In some embodiments, the invention provides a method of treating a disease or disorder in a subject, wherein the disease or disorder is a B-cell related autoimmune and/or inflammatory disease. More specifically, wherein the B cell-related autoimmune and/or inflammatory disease is associated with inappropriate or enhanced B cell numbers and/or activation.

It is an object of the present invention to provide a method for treating a B cell-related malignancy, a B cell-related autoimmune disease, and/or an inflammatory disease, comprising administering to a subject in need thereof a single domain antibody, an engineered immune effector cell, or a pharmaceutical composition as described above.

Interpretation of the terms

The term "antibody" as used herein includes monoclonal antibodies (including full length antibodies having an immunoglobulin Fc region), antibody compositions having multi-epitope specificity, multi-specific antibodies (e.g., bispecific antibodies), diabodies and single chain molecules, and antibody fragments, particularly antigen binding fragments such as Fab, F (ab') 2 and Fv. In some embodiments of the invention, the terms "immunoglobulin (Ig)" and "antibody" are used interchangeably.

"variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domains of the heavy and light chains may be referred to as "VH" and "VL", respectively. These domains are typically the most variable parts of an antibody (relative to other antibodies of the same type) and contain antigen binding sites.

The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). IgM antibodies consist of 5 basic heterotetramer units and a further polypeptide called a J chain, comprising 10 antigen binding sites; whereas IgA antibodies comprise 2-5 basic 4-chain units, which can polymerize with J-chains to form multivalent assemblies. In the case of IgG, the 4-chain unit is typically about 150,000 daltons. Each light chain is linked to the heavy chain by one covalent disulfide bond, while the two heavy chains are linked to each other by one or more disulfide bonds, the number of disulfide bonds being dependent on the isotype of the heavy chain. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at the N-terminus, followed by three (CH 1, CH2 and CH3 for each alpha and gamma chain) and four (CH 1, CH2, CH3 and CH 4) constant domains (CH) for the mu and epsilon isoforms and a Hinge region (Hinge) between the CH1 domain and the CH2 domain. Each light chain has a variable domain (VL) at the N-terminus followed by a constant domain (CL) at its other end. VL and VH are aligned together, while CL and the first constant domain of the heavy chain (CH 1) are aligned together. Specific amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The paired VH and VL together form an antigen binding site. For the structure and properties of different classes of antibodies, see also Basic and Clinical immunology, weight edition, daniel p. Sties, abba i.terr and Tristram g. Parsol w. Appleton & Lange, norwalk, ct.1994, page 71and Chapter 6. Light chains from any vertebrate species can be classified, based on their constant domain amino acid sequences, into one of two distinct types called kappa and lambda. Immunoglobulins may be assigned to different classes or isotypes depending on their heavy chain constant domain (CH) amino acid sequence. There are five classes of immunoglobulins: igA, igD, igE, igG and IgM have heavy chains called α, δ, ε, γ and μ, respectively. Based on the relatively small differences in CH sequence and function, the gamma and alpha classes can be further divided into subclasses, e.g., humans express the following subclasses: igG1, igG2A, igG2B, igG3, igG4, igA 1and IgA2.

Heavy chain antibodies are antibodies derived from camelidae or cartilaginous fish organisms. In contrast to the 4-chain antibodies described above, the heavy chain antibody lacks the light and heavy chain constant regions 1 (CH 1), comprising only 2 heavy chains consisting of variable regions (VHH) and other constant regions, which are linked to the constant regions by hinge-like structures. Each heavy chain of a camelidae heavy chain antibody comprises 1 variable region (VHH) and 2 constant regions (CH 2 and CH 3), and each heavy chain of a cartilaginous fish heavy chain antibody comprises 1 variable region and 5 constant regions (CH 1-CH 5). Antigen binding fragments of heavy chain antibodies include VHH and single chain heavy chain antibodies. Heavy chain antibodies can have CH2 and CH3 of human IgG Fc by fusion to the constant region of human IgG Fc.

The terms "single domain antibody", "BCMA-targeting single domain antibody", "heavy chain variable region domain of heavy chain antibody", "VHH", "nanobody" as used herein are used interchangeably and refer to a single domain antibody that specifically recognizes and binds to BCMA. Single domain antibodies are the variable regions of heavy chain antibodies. In general, single domain antibodies contain three CDR regions (antigen complementarity determining regions) and four FR regions (framework regions). Single domain antibodies are the smallest functional antigen binding fragments. Typically, a single domain antibody consisting of only one heavy chain variable region is constructed by cloning the variable region of the heavy chain of the antibody after the heavy chain antibody is obtained with naturally deleted light and heavy chain constant regions 1 (CH 1).

One skilled in the art can alter the sequences of the invention by one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids to obtain variants of the antibody or functional fragment sequences thereof without substantially affecting the activity of the antibody. These variants include, but are not limited to: deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminal and/or N-terminal end. Conservative substitutions with amino acids of similar or similar properties generally do not alter the function of the protein in the art. Amino acids having similar properties are substituted, for example, in the FR and/or CDR of the variable region. Amino acid residues that may be conservatively substituted are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. For example, the addition of one or several amino acids at the C-terminal and/or N-terminal end will not normally alter the function of the protein. They are all considered to be included within the scope of the present invention.

In some embodiments, the sequences of the variants of the invention may have at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the sequence from which they were derived. Sequence identity as described herein can be measured using sequence analysis software. Such as computer programs BLAST, in particular BLASTP or TBLASTN, using default parameters. The invention also includes those molecules having antibody heavy chain variable regions with CDRs, provided that the CDRs are 90% or more (preferably 95% or more, most preferably 98% or more) homologous to the CDRs identified herein.

The single domain antibodies, nanobodies or VHH antibodies of the invention can be prepared using methods conventional in the art, such as phage display techniques well known in the art. Alternatively, the various antibodies of the invention may be expressed in other cell lines. Suitable mammalian host cells may be transformed with sequences encoding the various antibodies of the invention. Transformation may be performed using any known method, including, for example, packaging the polynucleotide in a virus (or viral vector) and transducing the host cell with the virus (or vector). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide in liposomes, and direct microinjection of DNA into the nucleus, etc. Host mammalian cell lines useful for expression are well known in the art, for example, a variety of immortalized cell lines available from the American Type Culture Collection (ATCC) including, but not limited to, chinese Hamster Ovary (CHO) cells, heLa cells, baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human hepatocellular carcinoma cells (e.g., hepG 2), and the like. Particularly preferred cell lines are selected by determining which cell lines have high expression levels and produce antibodies with substantial BCMA binding properties.

The term "Chimeric Antigen Receptor (CAR)" as used herein, comprises an extracellular antigen-binding domain comprising a single domain antibody (SdAb), e.g., VHH antibody, that binds BCMA as disclosed herein.

In some embodiments, the Chimeric Antigen Receptor (CAR) of the present disclosure comprises a polypeptide comprising: (a) An extracellular antigen-binding domain comprising a single domain antibody (SdAb) to BCMA disclosed herein; (b) a transmembrane domain; (c) an intracellular signaling domain. Each domain and additional regions will be described in more detail below.

The invention discloses CARs comprising an extracellular antigen-binding domain comprising one or more single domain antibodies. The sdabs may have the same or different sources, and have the same or different sizes. Exemplary sdabs include, but are not limited to, heavy chain variable domains (e.g., VHH) from heavy chain-only antibodies, binding molecules that are naturally devoid of light chains, single domains (e.g., VH or VL) derived from conventional 4-chain antibodies, humanized-only heavy chain antibodies, human single domain antibodies produced by transgenic mice or rats expressing human heavy chain fragments, and engineering domains and single domain scaffolds that are not derived from antibodies. Any SdAb known in the art or disclosed by the invention, including the single domain antibodies disclosed herein, can be used to construct a CAR described herein. The SdAb may be derived from any species, including but not limited to mouse, rat, human, camel, llama, lamprey, shark, goat, rabbit, and cow. The single domain antibodies contemplated by the present invention also include naturally occurring single domain antibody molecules from species other than camelidae and shark.

In some embodiments, the extracellular antigen-binding domains disclosed herein comprise at least one binding domain, and the at least one binding domain comprises a single domain antibody disclosed herein that binds BCMA, e.g., a VHH antibody.

In some embodiments, the anti-BCMA SdAb is camelid, chimeric, human, or humanized.

In some embodiments, a CAR of the present disclosure comprises a polypeptide comprising: (a) An extracellular antigen-binding domain comprising an anti-BCMA SdAb; (b) a transmembrane domain; (c) an intracellular signaling domain; wherein the anti-BCMA SdAb comprises a polypeptide sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to sequence SEQ ID NOs 34-50.

In some embodiments, a CAR of the present disclosure comprises a polypeptide comprising: (a) An extracellular antigen-binding domain comprising an anti-BCMA SdAb; (b) a transmembrane domain; (c) an intracellular signaling domain; wherein the anti-BCMA SdAb comprises a polypeptide sequence as set forth in SEQ ID NO. 34-50.

In addition to the antigen binding domains disclosed herein, the CARs disclosed herein may further comprise one or more of the following structures: a linker (e.g., a peptide linker), a signal peptide, a hinge region, a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain. These domains will be described in detail below.

In some embodiments, the intracellular signal transduction domain comprises a primary intracellular signal transduction domain of an immune effector cell (e.g., a T cell). In some embodiments, the primary intracellular signaling domain is derived from cd3ζ, cd3γ, cd3δ, cd3ε, BCMA, CD79a, CD79b, CD66d, fcrγ, fcrβ. In some embodiments, the primary intracellular signaling domain is derived from cd3ζ. In some embodiments, the intracellular signaling domain further comprises a costimulatory signaling domain. In some embodiments, the costimulatory signal domain is derived from costimulatory molecules derived from one or more of CD27, CD28, CD137 (4-1 BB), OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83 ligand. In some embodiments, the costimulatory signaling domain is derived from CD28. In some embodiments, the costimulatory signaling domain is derived from 4-1BB (CD 137).

In some embodiments, BCMACAR further comprises a hinge domain (e.g., a CD8 a hinge domain) located between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain.

In some embodiments, the BCMA CAR further comprises a signal peptide (e.g., HLA-A signal peptide or CD8 a signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus: HLA-A signal peptide or CD8 a signal peptide, extracellular antigen binding domain, CD8 a hinge region, CD8 a transmembrane domain, costimulatory signal domain derived from 4-1BB (CD 137), and intracellular signal transduction domain derived from cd3ζ.

In some embodiments, the different domains of the CAR can also be fused to each other by a peptide linker. Depending on the structural and/or functional characteristics of the single domain antibody and/or the various domains, each peptide linker in the CAR may have the same or different length and/or sequence. Each peptide linker can be independently selected and optimized by one skilled in the art. In some embodiments, the peptide linker comprises flexible residues (e.g., glycine and serine) such that adjacent domains can move freely relative to each other. Among these, glycine-serine duplex may be a suitable peptide linker.

The peptide linker may have any suitable length. In some embodiments, the peptide linker is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or more amino acids in length. In some embodiments, the peptide linker is no more than about 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or less amino acids in length. In some embodiments, the peptide linker is about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids long, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids in length.

The peptide linker may have a naturally occurring sequence or a non-naturally occurring sequence. For example, sequences derived from the hinge region of heavy chain-only antibodies may be used as linkers. See, for example, the linker disclosed in WO 1996/34103. In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include, but are not limited to, glycine polymer (G) n, glycine-serine polymer (e.g., (GS) n, (GSG) n, (GGGS) n, and (GGGGS) n, where n is an integer of at least 1), glycine-alanine polymer, alanine-serine polymer, and other flexible linkers known in the art.

Nucleic acids encoding the various antibodies or Chimeric Antigen Receptors (CARs) described above are also disclosed. The present invention provides polynucleotides encoding the heavy chain variable region, the light chain variable region, the heavy chain, the light chain, and the CDRs. The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

As is well known to those skilled in the art, due to the degeneracy of the genetic code, a very large number of nucleic acids can be made, all of which encode an antibody or chimeric antigen receptor of the invention. Thus, where a particular amino acid sequence has been identified, one of skill in the art can prepare any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not alter the amino acid sequence encoding the protein. Thus, the present invention also relates to polynucleotides which hybridize to the above polynucleotide sequences and which have at least 70%, preferably at least 80%, more preferably at least 90% identity between the two sequences. The invention relates in particular to polynucleotides which hybridize under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" means: (1) Hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturing agents such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42℃and the like during hybridization; or (3) hybridization only occurs when the identity between the two sequences is at least 90% or more, more preferably 95% or more. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

The full length nucleic acid sequences of the various antibodies or Chimeric Antigen Receptors (CARs) of the invention, or fragments thereof, are typically obtained by PCR amplification, recombinant methods, or synthetic methods. One possible approach is to synthesize the sequences of interest by synthetic means, in particular with short fragment lengths. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. In addition, the heavy chain coding sequence and the expression tag (e.g., 6 His) may be fused together to form a fusion protein. Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, polypeptides, etc.) to which the present invention relates include biomolecules that exist in isolated form. At present, it is entirely possible to obtain DNA sequences encoding the polypeptides of the invention (or fragments or derivatives thereof) by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can be introduced into the polypeptide sequences of the invention by chemical synthesis.

The invention also relates to nucleic acid constructs, such as expression vectors and recombinant vectors, comprising the appropriate DNA sequences as described above and appropriate promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the polypeptide or protein. Vectors typically contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. The sequences (collectively referred to as "flanking sequences" in certain embodiments) typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a leader sequence encoding for secretion of the polypeptide, a ribosome binding site, a polyadenylation sequence, a multiple linker region for inserting a nucleic acid encoding the polypeptide to be expressed, and selectable marker elements.

The host cell to which the present invention relates may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are bacterial cells such as E.coli, streptomyces, salmonella typhimurium, etc.; fungus cells such as yeast; insect cells such as Drosophila S2 or Sf 9; animal cells such as CHO, COS7 and 293 cells.

In some embodiments, the host cell may be a variety of functional cells well known in the art, such as a variety of killer cells, including, but not limited to, cytokine-induced killer Cells (CIK), dendritic cell-stimulated cytokine-induced killer cells (DC-CIK), cytotoxic T Lymphocytes (CTLs), γδ T cells, natural killer cells (NK), tumor Infiltrating Lymphocytes (TIL), lymphokine activated killer cells (LAK), CD3AK cells (anti-CD 3 mab killer cells). In certain embodiments, the killer cells are T cells or NK cells. Exemplary NK cells include, but are not limited to, primary NK cells, NK cell lines (e.g., NK 92), and NKT cells. In certain embodiments, the NK cells are primary NK cells. Exemplary T cells include, but are not limited to, T cells of mixed cell populations such as peripheral blood T lymphocytes, umbilical cord blood T lymphocytes, cytotoxic killer T Cells (CTLs), helper T cells, suppressor/regulatory T cells, γδ T cells, and cytokine-induced killer Cells (CIKs), tumor Infiltrating Lymphocytes (TILs), and the like. In certain embodiments, the T cells are peripheral blood T lymphocytes or umbilical cord blood T lymphocytes.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which can take up DNA, can be obtained after the exponential growth phase and then treated with CaCl ₂ The process steps used are well known in the art. Another approach is to use MgCl ₂ . Alternatively, transformation may be performed by electroporation. When the host is eukaryotic, DNA transfection methods such as calcium phosphate co-precipitation, microinjection, electroporation, liposome packaging, and the like may be used.

The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media, such as a serum-containing medium or a serum-free medium, depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.

The polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations of these methods.

The invention also discloses vectors for cloning and expressing any of the CARs of the invention. In some embodiments, the vector is suitable for replication and integration in eukaryotic cells, such as mammalian cells. In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentiviral vectors, vaccinia vectors, herpes simplex virus vectors, and derivatives thereof. Viral vector technology is well known in the art and is described in Sambrook et al (see Molecular Cloning: A Laboratory Manual, cold Spring Harbor Laboratory Press, cold Spring harbor (2001)) and other virology and molecular biology manuals.

Many virus-based systems have been developed in the art for transferring genes into mammalian cells. For example, retroviruses provide a convenient system for gene delivery. The heterologous nucleic acid can be inserted into the vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated in vitro or ex vivo and delivered to engineered mammalian cells. Many retroviral systems are known in the art. In some embodiments, an adenovirus vector is used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. In some embodiments, self-inactivating lentiviral vectors are used. For example, self-inactivating lentiviral vectors carrying an immunomodulatory agent (e.g., immune checkpoint inhibitor) coding sequence and/or self-inactivating lentiviral vectors carrying a chimeric antigen receptor may be prepared using techniques known in the art. The resulting lentiviral vector may be used to transduce mammalian cells (e.g., primary human T cells) using methods known in the art. Vectors derived from retroviruses (e.g., lentiviruses) are suitable tools for achieving long-term gene transfer, as they allow long-term, stable integration of transgenes and their propagation in offspring cells. Lentiviral vectors also have the advantage of low immunogenicity and can transduce non-proliferating cells.

In some embodiments, the vector comprises any nucleic acid encoding a CAR of the invention. The nucleic acid may be cloned into a vector using any molecular cloning method known in the art, including, for example, the use of restriction endonuclease sites and one or more selection markers. In some embodiments, the nucleic acid is operably linked to a promoter. A variety of promoters have been explored for gene expression in mammalian cells, and any promoter known in the art may be used in the present invention. Promoters can be further classified as constitutive promoters or regulated promoters (e.g., inducible promoters).

In some embodiments, the nucleic acid encoding the CAR is operably linked to a constitutive promoter. Constitutive promoters allow for constitutive expression of a heterologous gene (also referred to as a transgene) in a host cell. Exemplary constitutive promoters contemplated by the present invention include, but are not limited to, the Cytomegalovirus (CMV) promoter, the human elongation factor-1 alpha (hEF 1 alpha) promoter, the ubiquitin C (Ubic) promoter, the phosphoglycerate kinase (PGK) promoter, the Simian Virus 40 (SV 40) early promoter, and the chicken beta-actin coupled to CMV early enhancer (CAGG) promoter. The efficiency of such constitutive promoters in driving transgene expression has been widely compared in a number of studies. For example Michael C.Milone et al (see Molecular Therapy,17 (8): 1453-1464 (2009)) compared the efficiencies of CMV, hEF 1. Alpha., ubic and PGK in driving chimeric antigen receptor expression in human primary T cells and concluded that the hEF 1. Alpha. Promoter not only induced the highest levels of transgene expression, but also remained optimal in human CD4 and CD 8T cells. In some embodiments, the nucleic acid encoding the CAR is operably linked to the hef1α promoter.

In some embodiments, the nucleic acid encoding the CAR is operably linked to an inducible promoter. Inducible promoters are among the regulatory promoters. The inducible promoter may be induced by one or more conditions, such as physical conditions, microenvironment of the engineered immune effector cell or physiological state of the engineered immune effector cell, an inducer, etc. In some embodiments, the induction conditions do not induce expression of an endogenous gene in the engineered mammalian cell and/or in the subject receiving the pharmaceutical composition. In some embodiments, the induction conditions are selected from the group consisting of an inducer, radiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox status, tumor environment, and activation status of the engineered mammalian cell.

In some embodiments, the vector further comprises a selectable marker gene or reporter gene to select a CAR expressing cell from a population of host cells transfected with the lentiviral vector. The selectable marker and the reporter gene may be flanked by appropriate regulatory sequences for expression in the host cell. For example, the vector may contain transcription and translation terminators, initiation sequences, and promoters for regulating expression of the nucleic acid sequences.

The term "immune effector cell" as used herein is an immune cell that can perform an immune effector function. In some embodiments, the immune effector cells express at least fcyriii and perform ADCC effector function. Examples of immune effector cells that can mediate ADCC include, but are not limited to: t cells, B cells, NK cells, macrophages, dendritic cells, induced pluripotent stem cells (ipscs), and the like.

In some embodiments, the immune effector cell is a T cell. In some embodiments, the T cell is a CD4+/CD8-, CD4-/CD8+, CD4+/CD8+, CD4-/CD8-T cell or a combination thereof. In some embodiments, the T cells produce cytokines such as IL-2, ifnγ, and/or tnfα after expressing the CAR and binding to a target cell (e.g., bcma+ tumor cell). In some embodiments, the cd8+ T cells lyse antigen-specific target cells after expression of the CAR and binding to the target cells.

In some embodiments, the immune effector cell is an NK cell. In other embodiments, the immune effector cell may be an established cell line, such as NK-92 cells.

In some embodiments, immune effector cells may be differentiated from stem cells, such as hematopoietic stem cells, pluripotent stem cells, ipscs, or embryonic stem cells.

The engineered immune effector cells of the invention are prepared by introducing a CAR into an immune effector cell (e.g., T cell). In some embodiments, the CAR is introduced into the immune effector cell by transfection of any one of the isolated nucleic acids or any one of the vectors described above.

Methods for introducing vectors or isolated nucleic acids into mammalian cells are known in the art. The vectors described can be transferred into immune effector cells by physical, chemical or biological means.

Physical methods for introducing the vector into immune effector cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for preparing cells comprising vectors and/or exogenous nucleic acids are well known in the art (see Sambrook, J., fritsch, E.F. and Maniatis, T. (2001) molecular μLar Cloning: ALABORATION Manual. Cold Spring Harbor Laboratory Press, cold Spring harbor.). In some embodiments, the vector is introduced into the immune effector cell by electroporation.

Biological methods for introducing vectors into immune effector cells include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammals (e.g., human cells).

Chemical methods for introducing the carrier into immune effector cells include colloidal dispersion systems, including, for example, macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including, for example, oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro delivery vehicle is a liposome.

In some embodiments, an RNA molecule encoding any CAR described herein can be prepared by conventional methods (e.g., in vitro transcription), and then introduced into immune effector cells by known methods such as mRNA electroporation (see Peter M rabinovich. Human Gene Therapy,17:1027-1035 (2006)).

In some embodiments, the transduced or transfected immune effector cells proliferate ex vivo after introduction of the vector or isolated nucleic acid. In some embodiments, the transduced or transfected immune effector cells are cultured to proliferate for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 14 days. In some embodiments, the transduced or transfected immune effector cells can be further evaluated or screened to select for engineered immune effector cells.

Reporter genes can be used to identify potentially transfected cells and to evaluate the function of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by a recipient organism or tissue and which encodes a polypeptide that expresses some readily detectable property, such as enzymatic activity. The expression of the reporter gene is determined at a suitable time after introduction of the DNA into the recipient cell. Suitable reporter genes may include genes encoding luciferases, beta-galactosidases, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or green fluorescent protein (see Kumiko Ui-tei.febs Letters,479:79-82 (2000)). Suitable expression systems are known in the art and may be prepared or commercially available using known techniques. Other methods of confirming the presence of a nucleic acid encoding a CAR in an engineered immune effector cell include: molecular biological testing methods well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; biochemical assay methods, such as detecting the presence or absence of a particular peptide; immunological methods such as ELISA.

The pharmaceutical composition disclosed by the invention contains the single-domain antibody or the engineered immune effector cell targeting the BCMA, and a pharmaceutically acceptable carrier or excipient. Pharmaceutically acceptable carriers or excipients include, but are not limited to, diluents, solubilizers, emulsifiers, preservatives and/or adjuvants, also commonly referred to as adjuvants. The adjuvant is preferably non-toxic or substantially non-toxic to the recipient at the dosage and concentration employed. Such excipients include, but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, the pharmaceutical composition may contain substances for improving, maintaining or retaining, for example, pH, permeability, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or permeation of the composition. The optimal pharmaceutical composition can be determined depending on the intended route of administration, the mode of delivery and the dosage required.

Pharmaceutical compositions for in vivo administration are generally provided in the form of sterile formulations. Sterilization is achieved by filtration through sterile filtration membranes. In lyophilizing a composition, the method may be used to sterilize prior to or after lyophilization, reconstitution, dilution. The pharmaceutical compositions of the present invention may be selected for parenteral delivery. Compositions for parenteral delivery may be stored in lyophilized form or in solution. For example, by using physiological saline or an aqueous solution containing glucose and other auxiliary agents by conventional methods. Parenteral compositions are typically placed in a container having a sterile access port, such as an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. Alternatively, the composition may be selected for inhalation or delivery through the digestive tract (such as orally). The preparation of the pharmaceutically acceptable compositions is within the skill of the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations comprising antibodies or engineered immune effector cells in sustained or controlled release delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery means, such as liposome carriers, bioerodible particles or porous beads, and depot injections, are also well known to those skilled in the art.

Once formulated, the pharmaceutical compositions are typically stored in sterile vials in the form of solutions, suspensions, gels, emulsions, solids, crystals, or as lyophilized powders. The formulation may be stored in a ready-to-use form or in a form that is reconstituted prior to administration (e.g., lyophilized). The invention also provides kits for producing single dose administration units. Kits of the invention may each contain a first container having a dried protein and a second container having an aqueous formulation. In certain embodiments of the invention, kits are provided that contain single and multi-chamber prefilled syringes (e.g., liquid syringes and lyophilized syringes).

The invention also provides a method of treating a patient, particularly a patient suffering from a BCMA-related disease, by administering a single domain antibody targeting BCMA, an engineered immune effector cell, or a pharmaceutical composition thereof according to any one of the embodiments of the invention. The terms "patient", "subject", "individual", "subject" are used interchangeably herein to include any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, etc.), and most preferably a human. "treating" refers to a subject employing the methods of treatment described herein to achieve at least one positive therapeutic effect (e.g., reduced number of cancer cells, reduced tumor volume, reduced rate of infiltration of cancer cells into peripheral organs, reduced rate of tumor metastasis or tumor growth). The therapeutic methods effective to treat a patient may be adjusted according to a variety of factors, such as the disease state, age, weight, and ability of the patient to elicit an anti-cancer response in the subject.

The therapeutically effective amount of the pharmaceutical composition comprising the BCMA-targeted single domain antibody or engineered immune effector cell of the present invention to be employed will depend on, for example, the extent of treatment and the goal. Those skilled in the art will appreciate that the appropriate dosage level for treatment will vary depending in part on the molecule delivered, the indication, the route of administration, and the patient's condition (body weight, body surface or organ size) and/or condition (age and general health). In certain embodiments, the clinician may titrate the dose and alter the route of administration to obtain the optimal therapeutic effect.

The frequency of administration will depend on the pharmacokinetic parameters of the BCMA-targeted single domain antibody or engineered immune effector cells in the formulation used. The clinician typically administers the pharmaceutical composition until a dose is reached that achieves the desired effect. The pharmaceutical composition may thus be administered as a single dose, or over time as two or more doses (which may or may not contain the same amount of the desired molecule or cell), or as a continuous infusion through an implanted device or catheter.

The route of administration of the pharmaceutical composition is conventional in the art, e.g. oral, nasal, by intravenous, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, portal vein or intralesional route injection, and may also be administered by a sustained release system or by an implanted device.

Drawings

FIG. 1A shows the expression rate of BCMAAR molecules on each of the groups of CD4+ T cells prepared.

FIG. 1B shows the expression rate of BCMAAR molecules on each of the resulting CD8+ T cells.

FIG. 2 shows the results of the following procedures on various target cells: expression of BCMA antigen on the surface of mm.1s cells, RPMI 8226 cells, nalm6 BCMA positive cells.

Fig. 3A shows killing of mm.1s cells by each group of effector cells on the first day when the effective target ratio (E: T) =1:3.

Fig. 3B shows killing of mm.1s cells by each group of effector cells on the first day when the effective target ratio (E: T) =1:1.

Fig. 4A shows the killing rate of RPMI 8226 cells by each group of effector cells on the first day when the effective target ratio (E: T) =1:3.

Fig. 4B shows the killing rate of RPMI 8226 cells by each group of effector cells on the first day when the effective target ratio (E: T) =1:1.

Fig. 5A shows killing rates of Nalm6 cells by each group of effector cells on the first day when the effective target ratio (E: T) =1:9.

Fig. 5B shows the killing rate of Nalm6 cells by each group of effector cells on the first day when the effective target ratio (E: T) =1:3.

Fig. 6A shows killing rates of various groups of effector cells on the first day against Nalm6 BCMA positive cells at an effective target ratio (E: T) =1:9.

Fig. 6B shows killing of Nalm6 BCMA positive cells by each group of effector cells on the first day when the effective target ratio (E: T) =1:3.

In the above figures: unT is a negative control group, TN1 is a TN1-BCMA-CAR-T group, TN62 is a TN62-BCMA-CAR-T group, TN65 is a TN65-BCMA-CAR-T group, TN79 is a TN79-BCMA-CAR-T group, TN81 is a TN81-BCMA-CAR-T group, TN84 is a TN84-BCMA-CAR-T group, TN85 is a TN85-BCMA-CAR-T group, TN87 is a TN87-BCMA-CAR-T group, TN88 is a TN88-BCMA-CAR-T group, TN89 is a TN89-BCMA-CAR-T group, TN90 is a TN90-BCMA-CAR-T group, TN91 is a TN91-BCMA-CAR-T group, TN92 is a TN92-BCMA-CAR-T group, TN93 is a TN93-BCMA-CAR-T group, TN94 is a TN 94-BCMA-T group, TN107 is a TN 85-BCMA-T group, and TN109 is a TN 109-BCMA-T group.

The achievement of the objects, functional features and advantageous effects of the present invention will be further described with reference to the following embodiments and with reference to the accompanying drawings.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the invention thereto. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

Example 1 preparation of a single domain antibody (VHH) targeting BCMA

(1) Animal immunity and immune response test

Healthy alpaca was selected as the immunization subject. For the first immunization, complete Freund's adjuvant and BCMA antigen (0.8 mg) are mixed in a ratio of 1:1, emulsified and injected subcutaneously in multiple points, and the subsequent booster immunization is carried out by mixing incomplete Freund's adjuvant and BCMA antigen in a ratio of 1:1, wherein the immunization interval period is 2 weeks, and the total immunization is 5 times. After each immunization, 5mL of peripheral blood is collected, serum is separated, the immune response is monitored by ELISA method, and the animal serum antibody titer is determined to be 10 ⁵ After the level 50mL peripheral blood was collected.

(2) Construction of antibody phage libraries

1) Extraction and reverse transcription of RNA

After animal immunization is finished, lymphocytes in peripheral blood are separated, RNA is extracted, and the obtained total RNA is subjected to reverse transcription by using a Takara reverse transcription kit to obtain cDNA; dividing the RNA obtained in the last step into two parts for reverse transcription into cDNA according to the specification of a reverse transcription kit, respectively storing the cDNA into 2 centrifuge tubes, and respectively using Oligo dT primers and random m primers for reverse transcription primers.

2) PCR amplification

Amplifying a specific antibody fragment from the reverse transcribed cDNA, and performing PCR amplification using Taq DNA Polymerase Hot Start enzyme; carrying out 1% agarose gel electrophoresis on all PCR products, and cutting gel to recover a band with the size of a target fragment of about 600-700bp, namely the PCR amplification product of the first round;

And (3) performing a second PCR reaction by taking the first PCR amplification product as a template, performing 1% agarose gel electrophoresis after the reaction is finished, finally cutting glue to recover a single target band with the fragment size of about 400bp, and performing DNA purification on the PCR reaction liquid by using a universal DNA purification recovery kit.

3) Enzyme digestion and ligation

The target gene fragment amplified by the second round of PCR and the pComb3XSS phage plasmid vector are respectively digested by restriction enzymes SpeI and SacI, and the VHH target gene fragment is connected to the pComb3XSS phage plasmid vector by ligase after the digestion is completed, so as to construct a recombinant plasmid.

4) Bacterial library and phage library construction

a. Taking a 50 mu L TG1 competent cell, placing on ice for 5-10min to melt; 100ng of the ligation product was added and transferred to pre-chilled 1mm apart electric rotating cups, where the parameters were set as: 1800V, 1mm, click button conversion; and (3) immediately adding 1mL of preheated SOC culture solution at 37 ℃ after the electrotransformation is finished, uniformly mixing, and placing the mixture at 37 ℃ for shaking and resuscitating for 1h at 200 rpm.

b. More than 20 of 100ng of the ligation system was used to perform electrotransformation using competence as described above.

c. Taking 100 mu L from the resuscitated bacterial liquid, carrying out 10-time gradient dilution, then coating the solution on a plate, and culturing overnight at 37 ℃; calculating the number of transformed colonies obtained by all reactions according to the dilution times and the number of single colonies, namely the library capacity of the bacterial library; meanwhile, a plurality of monoclonal antibodies are randomly selected from the gradient dilution plate to carry out colony PCR, and the cloning positive rate of the bacterial library is verified.

d. All bacterial liquid is collected and evenly spread into 20 more than 15cm culture plates (2 XYT contains 100 mug/mL Amp,2% agarose), and the culture is carried out upside down at 37 ℃ for overnight; the overnight cultured plate colonies were scraped off using 2 XYT liquid medium, placed in 50mL centrifuge tubes, OD600 values were measured, and glycerol at a final concentration of 20% was added for preservation at-80 ℃.

e. Inoculating the bacterial library into 100mL 2 XYT liquid culture medium (containing 100 μg/mL Amp) to make the initial OD600 value 0.1, and culturing at 37deg.C and 250rpm until OD600 is 0.5-0.55; according to 1:20 (bacterial number: phage number), helper phage were added and incubated at 37℃and 250rpm for 30min; adding Kana with a final concentration of 50 μg/mL, and culturing at 30deg.C and 250rpm overnight; after the bacterial liquid cultured overnight is placed at 4 ℃ and 13000rpm for 5min, transferring the supernatant to a new centrifuge tube, adding 1/4 volume of precooled PEG/NaCl, incubating on ice for 30min, placing at 4 ℃ and 13000rpm for 10min, removing the supernatant, and adding 1mL of PBS buffer solution for dissolving the precipitate; after adding 1/4 volume of PEG/NaCl again and incubating on ice for 10min, centrifuging at 4℃for 10min at 12000g, removing the supernatant and dissolving the precipitate in 1mL of PBS to obtain a purified phage library.

(3) Phage selection

1) 50 μg of antigen was added to 2mL of PBS and to the immune tube, coated overnight at 4 ℃ while BSA was coated in parallel; the supernatant from the overnight coated immune tubes was discarded, the immune tubes were washed 3 times with PBS buffer at room temperature and spun 5 min/time; 2mL of a blocking solution (3% nonfat milk powder) was added, the supernatant was discarded after blocking for 2 hours at room temperature, and the immune tube was washed 3 times at room temperature with 2mL of PBST buffer, and rotated 5 min/time.

2) Removing the wash solution from the immune tube and adding about 10% of the phage library prepared ¹² pfu is used as a first round of screening an input phage library, PBS buffer is added to 2mL, and the phage library is rotated and incubated for 1h at room temperature; the supernatant was discarded, and 2mL of PBS buffer containing 0.1% Tween20 was added for 20 times at room temperature, each time rotated for 5min; removing the liquid in the immune tube, adding 1mL of 0.25mg/mL Trypsin solution, and performing rotary elution at room temperature for 30min; adding 10 μl of 10% AEBSF to stop elution, transferring the solution in the immune tube into a new 1.5mL centrifuge tube, and eluting the phage for the first round of screeningAnd (3) liquid.

3) Amplifying and purifying the eluted phage; repeating the experiment for 3 times, taking the phage of the first round as a second round of screening input phage library to obtain a phage eluent of the second round of screening; and then taking the phage of the second round as a third round of screening input phage library to obtain a third round of screening phage eluent.

(4) Monoclonal ELISA detection

1) Performing gradient dilution on phages obtained after three rounds of screening, adding 100 mu L of phages into TG1 bacterial liquid with OD600nm of 0.5, culturing at 37 ℃ for 30min, coating on a 2XYT culture plate containing Amp, and culturing at 37 ℃ overnight; 192 single colonies were randomly picked into 96-well cell culture plates containing ampicillin in 2XYT medium and incubated overnight at 37 ℃; 5. Mu.L of the overnight cultured bacterial liquid was added to a new 96-well plate (each well contains 200. Mu.L of 2XYT fresh culture liquid, 100. Mu.g/mL of Amp), helper phage was added to the culture well after 5 hours, and Kana was added to a final concentration of 50. Mu.g/mL after incubation at 37℃for 30 minutes, and the culture was allowed to stand at 30℃overnight.

2) Centrifuging the 96-well culture plate after overnight culture to obtain a supernatant containing phage; coating the screening antigen coated ELISA plate at 4 ℃ overnight, and simultaneously coating BSA with the same concentration in parallel as a control; the liquid in the ELISA plate is discarded, 200 mu L of PBS buffer containing 0.1% Tween20 is added into each hole to wash for 3 times, and each time is 10min; adding the phage supernatant obtained in the previous step after 3% BSA blocking, and incubating for 1h at room temperature; washing 3 times with PBS buffer containing 0.1% Tween20 for 10min each; m13 Bacteriophage Antibody (HRP) was added to each well, mouse Mab,1:30000 diluted in blocking solution, 100. Mu.L/well, incubated for 1h at room temperature; the ELISA plate was discarded and washed 6 times with PBS buffer containing 0.1% Tween20 for 5min each; 100 mu L of TMB single-component color development liquid is added into each hole, color development is carried out for 1-3min in a dark place, then 100 mu L of 1M HCl is added for termination, and an OD450 value is read by an enzyme-labeling instrument.

3) And (3) carrying out gene sequencing on the positive clone colony to obtain the gene sequence of the VHH single domain antibody.

EXAMPLE 2 construction of chimeric antigen receptor targeting BCMA and immunocyte expression

(1) Construction of BCMA-CAR

First, the coding nucleotide sequences for each set of BCMA-targeting CAR nucleotide sequences (SEQ ID NO: 73-89), each set of sequences comprising the HLA-A signal peptide (SEQ ID NO: 51), the extracellular antigen binding domain of BCMA-VHH (SEQ ID NO: 34-50), the CD8 a hinge region (SEQ ID NO: 52), the CD8 a transmembrane domain (SEQ ID NO: 53), the 4-1BB (CD 137) costimulatory signal domain (SEQ ID NO: 54) and the CD3 zeta intracellular signal transduction domain (SEQ ID NO: 55) were designed and artificially synthesized for expression of each experimental set of complete BCMA-CAR polypeptide molecules (SEQ ID NO: 56-72). Inserting BCMA-CAR nucleotide sequence into multiple cloning site of lentiviral expression vector pK1 by homologous recombination to obtain pK1-BCMA-CAR, and verifying that lentiviral expression vector sequence is successfully constructed by electrophoresis and sequencing results.

(2) Packaging of lentiviral vectors

Resuscitate 293T cells and culture in DMEM medium containing 10% FBS; after 2-3 generation cell expansion culture, the method is carried out according to 4 multiplied by 10 ⁴ Individual/cm ² Is seeded into a 2-layer cell factory; plasmid transfection was performed 3 days after cell inoculation; plasmid transfection 40mL Optim-MEM was added to a sterile 50mL centrifuge tube and the tube was then sterilized according to pK1-BCMA-CAR: pLP1: pLP2: pLP-vsvg=5: 4:3:1, adding a virus packaging vector and a virus envelope vector in proportion, then adding 800 mu LPEI transfection reagent, immediately mixing, incubating for 15min at room temperature, and then adding the plasmid/vector/transfection reagent compound dropwise into a culture flask of 293T cells; collecting the virus supernatant after 24 hours into a 50mL centrifuge tube, centrifuging 250g for 5 minutes, filtering the supernatant after centrifugation by a 0.45 mu m filter, and ultracentrifugating the filtered supernatant (25000 g,4 ℃ C., 3 hours) to obtain concentrated BCMA-CAR lentivirus; centrifuging, discarding supernatant, re-suspending lentivirus with PBS pre-cooled at 4deg.C, packaging the re-suspended BCMA-CAR lentivirus liquid, and storing at-80deg.C.

(3) Resuscitation and activation of T cells

Setting the temperature of the water bath kettle to 38 ℃, and preheating the culture medium in advance; taking out the freezing bag from the liquid nitrogen tank, immediately immersing the bag in a water bath, and taking out the freezing bag when the navel blood to be frozen is completely melted in a transparent state; wiping the outside of the freezing bag with dry cotton ballsSpraying 75% alcohol for disinfection, and transferring to a biosafety cabinet after alcohol is completely volatilized; taking out umbilical blood, placing the umbilical blood into a 50mL centrifuge tube, adding a proper amount of RPMI 1640 culture medium, uniformly mixing, and sampling and counting; centrifuging at 300g for 5min, collecting lower layer cell, and re-suspending with complete culture medium to T lymphocyte density of 1×10 ⁶ Adding an activated antibody Anti-human CD3 Anti-ibody and an activated antibody Anti-human CD28 Anti-ibody according to the re-suspension volume, wherein the concentration of CD3 is 0.15 mug/mL, the concentration of CD28 is 0.625 mug/mL, and culturing in a 5% carbon dioxide incubator at 37 ℃; culturing for 4hr, adding complete culture medium to adjust T lymphocyte density to 4×10 ⁵ Culture activation was continued for a predetermined time at a volume of each mL.

(4) Sorting and purification of T cells

After 36hr of cell activation, mixing uniformly, sampling 20 μl, adding fluorescent labeled antibody, staining for 10min, diluting 10 times with PBS, detecting and counting by using a flow cytometer, recording CD3+, CD4+ and CD8+ T cell density, and observing CD69 and CD25 molecule expression conditions; recording the cell volume and confirming the cell quantity; transferring the cell suspension to a centrifuge tube for centrifugation at 300g for 5min, and collecting the lower layer cells after discarding the supernatant; adding MACS Buffer for washing, centrifuging again, collecting lower cells, centrifuging under the same conditions, and re-suspending cells with appropriate amount of MACS Buffer; the amount of the magnetic beads was calculated from the amount of cells per 1X 10 ⁶ mu.L of CD4+ magnetic beads were added to each 1X 10 CD4+ T cell ⁶ mu.L of CD8+ magnetic beads were added to the CD8+ T cells; adding magnetic beads, uniformly mixing, incubating for 20min at room temperature in a dark place, adding MACS Buffer for washing after incubation is completed, centrifuging for 300g for 5min, discarding supernatant, and re-suspending with a proper amount of MACS Buffer; placing the LS sorting column on a MACS magnetic sorting frame, washing the column with 1mLBuffer, passing the cell suspension through the column after washing, and continuously adding Buffer 9mL through the column; taking the LS column out of the MACS magnetic frame, adding 5mLBuffer, and flushing out the cells trapped on the LS column; and finally, uniformly mixing the cell suspension, sampling, dyeing and counting, recording the densities of the CD4+ T cells and the CD8+ T cells respectively, and calculating the sorting rate and the purity.

(5) Preparation of BCMA-targeted CAR-T cells

To be obtainedCd8+ T cell density was adjusted to 2×10 ⁵ Each group of concentrated slow virus solution expressing the BCMA-CAR objective gene (slow virus addition amount: MOI=25) was added per mL, and the negative control group (UnT) was T cells transduced without slow virus, and then cultured in RPMI 1640 medium (Hyclone, logan) containing recombinant IL-7 at a final concentration of 10ng/mL, recombinant IL-15 at a final concentration of 10ng/mL, L-glutamine at 2mM (Gibco, US), and 2-mercaptoethanol at 55. Mu.M (Gibco, US) for 72hr to obtain T cells expressing the BCMA-CAR objective gene. Then the fluid infusion is carried out every 2-3 days, and the density of the fluid infusion is 4 multiplied by 10 ⁵ /mL. When lentiviral infected T cells were cultured for 5 days, CAR expression amount detection was performed on T cells expressing the target gene, BCMA expression (Acrobiosystems, beijin) in CAR-T cells was detected using conjugated Phycoerythrin (PE) -labeled BCMA protein, data was collected with a flow cytometer (Agilent, california) and analyzed with NovoExpress software, and the results are shown in fig. 1.

Example 3 validation of tumor cell killing Effect of BCMA-targeted CAR-T cells

(1) Measurement of expression of BCMA antigen on target cell surface

MM.1S cells (purchased from ATCC, CRL-2974), RPMI 8226 cells (purchased from ATCC, CRM-CCL-155), nalm6 cells (purchased from ATCC, CRL-3273) and Nalm6 BCMA positive cells were used as target cells, each of which was cultured using RPMI 1640 medium, and after 3 consecutive generations of culture, appropriate amounts of the cells were suspended in a dye of PE-conjugated anti-BCMA antibody, and after incubation for 10 minutes in the absence of light, the expression of the cell surface BCMA antigen was detected by flow cytometry, and the results are shown in FIG. 2. The results show that: mm.1s cells and RPMI 8226 cells were BCMA-expressed moderately, nalm6 cells were BCMA-expressed low, and Nalm6 BCMA-positive cells were BCMA-expressed high.

Preparation of Nalm6 BCMA positive cells: obtaining lentiviruses expressing BCMA antigen, first transiently transfecting 293T cells with plasmid, and then subjecting the cells to 2X 10 transfection ⁵ Density/cm 2 in T-shaped vessel, plasmid transfection into sterile 50mL centrifuge tube after adding 40mL Optim-MEM, according to pK1-BCMA: pLP1: pLP2: pLP-vsvg=5: 4:3:1, and then 800. Mu.L of a viral packaging vector and a viral envelope vector were addedThe PEI transfection reagent is immediately and evenly mixed, incubated for 15min at room temperature, and then the plasmid/vector/transfection reagent complex is added into a culture flask of 293T cells drop by drop; collecting the virus supernatant after 24 hours into a 50mL centrifuge tube, centrifuging 250g for 5 minutes, filtering the supernatant after centrifugation by a 0.45 mu m filter, and ultracentrifugating the filtered supernatant (25000 g,4 ℃ for 3 hours) to obtain concentrated BCMA lentivirus; centrifuging, discarding supernatant, re-suspending lentivirus with PBS pre-cooled at 4deg.C, packaging the re-suspended BCMA lentivirus liquid, and storing at-80deg.C. Then the normal Nalm6 cells are proliferated according to the ratio of 4×10 ⁴ The density of individual cells/wells was plated in 96-well plates, BCMA slow virus solution was added at moi=25, the remaining volume was complemented by fresh growth medium, and the total volume was 200 μl; detecting BCMA antigen expression of cells on the 3 rd day after virus infection, when the BCMA expression of the cells is stable for more than 5 generations and the infection efficiency is higher than 90%, using a monoclonal plate-spreading machine (Cytena, model f.signal) to perform plate-spreading, and observing the monoclonal growth state of the cells in 8h, 24h and 72h after plate-spreading; after the Nalm6 cells are confirmed to be monoclonal, the cells are cultured for more than 2 months, and the BCMA antigen expression of the cells is detected by using a flow cytometry, and after the expression is stable, a Nalm6 BCMA positive cell strain is obtained.

(2) In vitro killing effect assay of BCMA-targeted CAR-T cells

In a 24-well plate, each group of CAR-T cells (group: TN1, TN62, TN65, TN79, TN81, TN84, TN85, TN87, TN88, TN89, TN90, TN91, TN92, TN93, TN94, TN107, TN109, added in an amount of 7.5X10) was added ⁴ Number/well), mm.1s cells were added in the corresponding cell amounts at the effective target ratio (E: T) =1:3 or 1:1, and the negative control group (UnT) was also added with the same amounts of T cells and target cells at the corresponding effective target ratio, and medium was added to 500 μl/well as an experimental group; simultaneously, the same number of effector cells as that of each experimental group are independently added into an orifice plate as an effector cell spontaneous release group, target cells are independently added as a target cell spontaneous release group and a target cell maximum release group, and culture medium is supplemented to ensure that the total volume is 500 mu L and the mixture is placed at 37 ℃ and 5% CO ₂ Culturing in an incubator; after 18hr of co-culture, the samples were assayed using a non-radioactive cytotoxicity assay kit (Promega, US) and pressed againstInjury rate= (experimental group OD-effector cells spontaneous OD-target cells spontaneous OD)/(target cells maximum release OD-target cells spontaneous OD) ×100%, and D1 (18 hr) kill rate was calculated, and the results are shown in fig. 3A and 3B.

In a 24-well plate, CAR-T cells of each group (groups: TN1, TN62, TN65, TN79, TN81, TN84, TN85, TN87, TN88, TN89, TN90, TN91, TN92, TN93, TN94, TN107, TN109, in an amount of 5×10) were added ⁴ RPMI 8226 cells were added in the corresponding cell amount at the effective target ratio (E: T) =1:3, and the negative control group (UnT) was also added with the same amount of T cells and target cells at the corresponding effective target ratio, and medium was added to 500 μl/well as an experimental group; simultaneously, the same number of effector cells as that of each experimental group are independently added into the pore plate as an effector cell spontaneous release group, the same number of target cells as that of the experimental group are independently added as a target cell spontaneous release group and a target cell maximum release group, and the culture medium is supplemented to ensure that the total volume is 500 mu L, and the mixture is placed at 37 ℃ and 5% CO ₂ Culturing in an incubator; after 18hr of co-culture, the killing rate of D1 (18 hr) was calculated as killing rate= (experimental group OD-effector spontaneous OD-target cells spontaneous OD)/(target cells maximum release OD-target cells spontaneous OD). Times.100% by measurement using a non-radioactive cytotoxicity assay kit (Promega, US). When the target ratio (E: T) =1:1, each group of CAR-T cells was added to adjust to 1.5X10 cells per well ⁵ The remaining conditions were the same as above, and the kill rate of D1 (18 hr) was calculated. The results are shown in FIGS. 4A and 4B.

In 24-well plates, each group of CAR-T cells (TN 62, TN65, TN88, TN90, TN94, 5X 10 were added) ⁴ Number of cells/well), nalm6 cells were added at a corresponding cell amount according to an effective target ratio (E: T) =1:9, and medium was added to 500. Mu.L/well, and placed at 37℃in 5% CO ₂ Culturing in an incubator; after co-culturing for 18hr, the amount of Nalm6 cells in each well was measured by flow cytometry, and the killing rate of D1 (18 hr) was calculated as killing rate=target cell decrease amount/target cell plating cell amount×100%. When the target ratio (E: T) =1:3 was further calculated, each group of CAR-T cells was added to adjust the ratio to 1.5X10 per well ⁵ The remaining conditions were the same as above, and the kill rate of D1 (18 hr) was calculated. The results are shown in FIGS. 5A and 5B.

In 24-well plates, each group of CAR-T cells (groupTN1, TN62, TN65, TN79, TN81, TN84, TN85, TN87, TN88, TN89, TN90, TN91, TN92, TN93, TN94, TN107, TN109, in an amount of 5X 10 ⁴ Number of cells/well), nalm6 BCMA positive cells were added at a corresponding cell amount according to an effective target ratio (E: T) =1:9, and a negative control group (UnT) was also added with the same amount of T cells and target cells according to the corresponding effective target ratio, and medium was added to 500. Mu.L/well, and placed at 37℃and 5% CO ₂ Culturing in an incubator; after co-culturing for 18hr, nalm6 BCMA positive cells in each well were detected by flow cytometry, and the killing rate of D1 (18 hr) was calculated as killing rate=target cell reduction amount/target cell plating cell amount×100%. When the target ratio (E: T) =1:3 was further calculated, each group of CAR-T cells was added to adjust the ratio to 1.5X10 per well ⁵ The remaining conditions were the same as above, and the kill rate of D1 (18 hr) was calculated. The results are shown in FIGS. 6A and 6B.

(3) Cytokine release assay of BCMA-targeted CAR-T cells

After 18hr of plating in vitro killing experiments, the supernatant of each group of CAR-T cells (TN 1, TN62, TN65, TN79, TN81, TN84, TN85, TN87, TN88, TN89, TN90, TN91, TN92, TN93, TN94, TN107, TN 109) was collected and centrifuged (500 g, 5 min) with the RPMI 8226 cell co-culture solution, and the supernatant was subjected to CBA detection (CBA detection kit: LEGENDplex) ^TM Human CD8/NK Panel(13-plex)with V-bottom Plate,Biolegend,Cat.No.741065；LEGENDplex ^TM Human macrogel/Microglia Panel (13-plex) with V-bottom Plate, biolegend, cat. No. 740503) and the release of each group of cytokines Granzyme B, TNF- α, IFN- γ, IL-2 was examined.

Claims

1. A single domain antibody targeting BCMA, wherein said antibody comprises CDR1, CDR2, and CDR3;

wherein the CDR1 is an amino acid sequence shown as SEQ ID NO. 1, wherein the CDR2 is an amino acid sequence shown as SEQ ID NO. 6, and wherein the CDR3 is an amino acid sequence shown as SEQ ID NO. 17;

wherein the CDR1 is an amino acid sequence as shown in SEQ ID NO. 2, wherein the CDR2 is an amino acid sequence as shown in SEQ ID NO.7, wherein the CDR3 is an amino acid sequence as shown in SEQ ID NO. 18;

Wherein the CDR1 is an amino acid sequence as shown in SEQ ID NO. 2, wherein the CDR2 is an amino acid sequence as shown in SEQ ID NO. 8, wherein the CDR3 is an amino acid sequence as shown in SEQ ID NO. 19;

wherein the CDR1 is an amino acid sequence as shown in SEQ ID NO. 3, wherein the CDR2 is an amino acid sequence as shown in SEQ ID NO. 9, wherein the CDR3 is an amino acid sequence as shown in SEQ ID NO. 20;

wherein the CDR1 is an amino acid sequence shown as SEQ ID NO. 2, wherein the CDR2 is an amino acid sequence shown as SEQ ID NO. 10, wherein the CDR3 is an amino acid sequence shown as SEQ ID NO. 21;

wherein the CDR1 is an amino acid sequence as shown in SEQ ID NO. 4, wherein the CDR2 is an amino acid sequence as shown in SEQ ID NO. 11, wherein the CDR3 is an amino acid sequence as shown in SEQ ID NO. 22;

wherein the CDR1 is an amino acid sequence as shown in SEQ ID NO. 2, wherein the CDR2 is an amino acid sequence as shown in SEQ ID NO. 12, wherein the CDR3 is an amino acid sequence as shown in SEQ ID NO. 23;

wherein the CDR1 is an amino acid sequence as shown in SEQ ID NO. 2, wherein the CDR2 is an amino acid sequence as shown in SEQ ID NO. 13, wherein the CDR3 is an amino acid sequence as shown in SEQ ID NO. 24;

Wherein the CDR1 is an amino acid sequence as shown in SEQ ID NO. 2, wherein the CDR2 is an amino acid sequence as shown in SEQ ID NO. 12, wherein the CDR3 is an amino acid sequence as shown in SEQ ID NO. 25;

wherein the CDR1 is an amino acid sequence as shown in SEQ ID NO. 2, wherein the CDR2 is an amino acid sequence as shown in SEQ ID NO. 12, wherein the CDR3 is an amino acid sequence as shown in SEQ ID NO. 26;

wherein the CDR1 is the amino acid sequence shown as SEQ ID NO. 1, wherein the CDR2 is the amino acid sequence shown as SEQ ID NO. 13, wherein the CDR3 is the amino acid sequence shown as SEQ ID NO. 27;

wherein the CDR1 is an amino acid sequence as shown in SEQ ID NO. 2, wherein the CDR2 is an amino acid sequence as shown in SEQ ID NO. 10, wherein the CDR3 is an amino acid sequence as shown in SEQ ID NO. 28;

wherein the CDR1 is the amino acid sequence shown as SEQ ID NO. 1, wherein the CDR2 is the amino acid sequence shown as SEQ ID NO. 12, wherein the CDR3 is the amino acid sequence shown as SEQ ID NO. 29;

wherein the CDR1 is an amino acid sequence as shown in SEQ ID NO. 2, wherein the CDR2 is an amino acid sequence as shown in SEQ ID NO. 14, wherein the CDR3 is an amino acid sequence as shown in SEQ ID NO. 30;

Wherein the CDR1 is an amino acid sequence as shown in SEQ ID NO. 1, wherein the CDR2 is an amino acid sequence as shown in SEQ ID NO. 15, wherein the CDR3 is an amino acid sequence as shown in SEQ ID NO. 31;

wherein the CDR1 is an amino acid sequence as shown in SEQ ID NO. 1, wherein the CDR2 is an amino acid sequence as shown in SEQ ID NO. 12, wherein the CDR3 is an amino acid sequence as shown in SEQ ID NO. 32; or (b)

Wherein the CDR1 is the amino acid sequence shown as SEQ ID NO. 5, wherein the CDR2 is the amino acid sequence shown as SEQ ID NO. 16, and wherein the CDR3 is the amino acid sequence shown as SEQ ID NO. 33.

2. The single domain antibody of claim 1, wherein the determination of CDR1, CDR2 and CDR3 is according to the IMGT numbering scheme.

3. The single domain antibody of claim 1 or 2, wherein the antibody is an amino acid sequence as set forth in SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49 or SEQ ID NO 50.

4. A chimeric antigen receptor comprising:

(a) An extracellular antigen-binding domain,

(b) A transmembrane domain, and

(c) An intracellular signaling domain;

wherein the extracellular antigen binding domain comprises the single domain antibody of any one of claims 1-3 that targets BCMA.

5. The chimeric antigen receptor according to claim 4, wherein the transmembrane domain is derived from CD8 a, CD28, cd3ζ, cd3γ, cd3δ, cd3ε, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, tcra, tcrβ, tcrγ, tcrδ, tcrζ, OX40, ICOS, LAG-3, 2B4, BTLA, CTLA-4, PD-1, or any combination thereof.

6. The chimeric antigen receptor according to claim 5, wherein the transmembrane domain is derived from CD8 alpha, which is the amino acid sequence shown in SEQ ID NO. 53.

7. The chimeric antigen receptor according to claim 4, wherein the intracellular signaling domain is derived from cd3ζ, cd3γ, cd3δ, cd3ε, CD22, CD66d, CD79a, CD79b, fcrβ, fcrγ, or any combination thereof.

8. The chimeric antigen receptor according to claim 7, wherein the intracellular signaling domain is derived from cd3ζ, which is the amino acid sequence depicted as SEQ ID No. 55.

9. The chimeric antigen receptor according to claim 7, wherein the intracellular signaling domain further comprises a costimulatory signaling domain, wherein the costimulatory signaling domain is derived from 4-1BB (CD 137), CD28, OX40, ICOS, ICAM, LFA-1, TLR1-10, CARD11, CD2, CD3, CD7, CD8 a, CD27, CD30, CD40, CD83, HVEM, BTLA, B7-H3, GITR, DAP10, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, ZAP70, CD83 ligand, or any combination thereof.

10. The chimeric antigen receptor according to claim 9, wherein the costimulatory signaling domain is derived from 4-1BB (CD 137), which is the amino acid sequence depicted as SEQ ID No. 54.

11. The chimeric antigen receptor according to claim 4, further comprising a hinge region between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain, wherein the hinge region is derived from CD8 a, CD28, CD137, igG1, igG4, or any combination thereof.

12. The chimeric antigen receptor according to claim 11, wherein the hinge region is derived from CD8 a, which is the amino acid sequence shown in SEQ ID No. 52.

13. The chimeric antigen receptor according to claim 4, further comprising a signal peptide at the N-terminus of the chimeric antigen receptor polypeptide, wherein the signal peptide is derived from HLA-A, CD8 a, CD4, CD33, CD137, GM-csfra, igG1, igκ, IL-2, or any combination thereof.

14. The chimeric antigen receptor according to claim 13, wherein the signal peptide is derived from HLA-A, which is the amino acid sequence shown in SEQ ID No. 51.

15. A chimeric antigen receptor is characterized by an amino acid sequence shown as SEQ ID NO. 56, SEQ ID NO. 57, SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 71 or SEQ ID NO. 72.

16. An isolated nucleic acid comprising a nucleic acid sequence encoding the chimeric antigen receptor of any one of claims 4-15.

17. The isolated nucleic acid of claim 16, wherein the nucleic acid is the nucleic acid sequence set forth in SEQ ID NO. 73-89.

18. A vector comprising the nucleic acid of claim 16 or 17.

19. An engineered immune effector cell comprising the chimeric antigen receptor of any one of claims 4-15, the isolated nucleic acid of claim 16 or 17, or the vector of claim 18.

20. The engineered immune effector cell of claim 19, wherein the immune effector cell is selected from T cells, B cells, NK cells, macrophages, dendritic cells, or any combination thereof.

21. A pharmaceutical composition comprising the single domain antibody of any one of claims 1-3 that targets BCMA or the engineered immune effector cell of claim 19 or 20 and a pharmaceutically acceptable carrier or excipient.

22. Use of the single domain BCMA-targeting antibody according to any one of claims 1-3, the engineered immune effector cell according to claim 19 or 20, or the pharmaceutical composition according to claim 21 for the preparation of a medicament for the treatment of a B-cell related malignancy, a B-cell related autoimmune disease and/or an inflammatory disease.

23. The use of claim 22, wherein the B-cell related malignancy is selected from marginal zone lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, primary central nervous system lymphoma, primary mediastinum large B-cell lymphoma, small lymphocytic lymphoma, B-cell prolymphocytic leukemia, follicular lymphoma, burkitt's lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, hairy cell leukemia, precursor B-lymphocytic leukemia, and/or multiple myeloma.