CN118206658A

CN118206658A - Chimeric antigen receptor of targeted BCMA based on fully human and murine single chain antibodies and application thereof

Info

Publication number: CN118206658A
Application number: CN202410294964.5A
Authority: CN
Inventors: 黄飞
Original assignee: Shanghai Hengrun Dasheng Biotechnology Co ltd
Current assignee: Shanghai Hengrun Dasheng Biotechnology Co ltd
Priority date: 2021-08-10
Filing date: 2021-08-10
Publication date: 2024-06-18
Also published as: WO2023016576A1; CN115703836A

Abstract

The present invention relates to single chain antibodies targeting BCMA and chimeric antigen receptors based thereon and their uses. The present invention provides fully human and murine single chain antibodies that specifically bind to BCMA. The chimeric antigen receptor comprises an anti-BCMA fully human or murine single chain antibody, a human CD8 alpha hinge region, a human CD8 alpha transmembrane region, a human 41BB intracellular region and a human CD3 zeta intracellular region which are connected in sequence. Further, a CAR-T cell based on an anti-BCMA fully human or murine single chain antibody is provided that is capable of specifically binding to BCMA and effectively killing B lymphocyte lineage malignant cells that express BCMA.

Description

Chimeric antigen receptor of targeted BCMA based on fully human and murine single chain antibodies and application thereof

The invention relates to a chimeric antigen receptor targeting BCMA based on fully human and murine single chain antibodies and application thereof, and a divisional application of the invention patent application with the application number of CN 2021109121618.

Technical Field

The invention belongs to the field of antibodies and chimeric antigen receptors, and in particular relates to an antibody and chimeric antigen receptor targeting an extracellular segment of BCMA and application thereof.

Background

Multiple myeloma is a malignant plasma cell disease, which is manifested by malignant clonal hyperplasia of bone marrow plasma cells, secretion of monoclonal immunoglobulin or fragments thereof (M protein), which causes injury to relevant target organs or tissues such as bones and kidneys, and is frequently manifested clinically by bone pain, anemia, renal insufficiency, infection, etc. [ N Engl J Med,2011.364 (11): 1046-60]. At present, multiple myeloma is the second largest malignant tumor of the blood system, accounts for 10% of malignant tumors of the blood system, frequently occurs in men, the incidence rate of the multiple myeloma increases year by year with the age, and the multiple myeloma has a tendency of younger in recent years [ CA CancerJ Clin,2014.64 (1): 9-29]. The B cell maturation antigen (B-cell maturation antigen, BCMA), also called 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 only one carbohydrate recognition domain is the B cell surface molecule. BCMA, a type I transmembrane signaling protein lacking a signal peptide, is a member of the tumor necrosis factor receptor family (TNFR) that binds to both B cell activating factor BAFF or proliferation-inducing ligand (aproliferation induced ligand, APRIL) [ Curr Opin Struct Biol,2004.14 (2): 154-60]. In normal tissues, BCMA is expressed on the surface of mature B cells and plasma cells, the immune system of a BCMA gene knockout mouse is normal, has normal spleen structure, and the development of B lymphocytes is normal, but the number of plasma cells is obviously reduced, so that BCMA plays an important role in maintaining the survival of the plasma cells, and the mechanism mainly comprises BCMA BAFF protein binding, up-regulates anti-apoptosis genes Bcl-2, mcl-1, bclw and the like, and maintains cell growth [ J Exp Med,2004.199 (1): 91-8]. Similarly, this mechanism also functions in myeloma cells, playing an important role in promoting malignant proliferation of myeloma cells [ Blood,2004.103 (8): 3148-57]. Studies have shown that BCMA is ubiquitously expressed in multiple myeloma cell lines and that detection in multiple myeloma patients also yields consistent results [ Blood,2004.103 (2): 689-94]. Kochenderfer, et al, have studied in depth the expression profile of BCMA on the basis of the reported Q-PCR, flow cytometry and immunohistochemical methods, confirming that BCMA is not expressed in normal human tissues other than mature B cells, plasma cells, and also in cd34+ hematopoietic cells [ CLIN CANCER RES,2013.19 (8): 2048-60]. The high similarity of binding BCMA expression profile to CD19, and the successful progression of anti-CD 19 CAR-T cell therapy, suggest that we BCMA can be used as one of the targets of CAR-T cells for cellular immunotherapy of multiple myeloma. Chimeric antigen Receptor-T cells (CHIMERIC ANTIGEN Receptor-T cells, CAR-T) refer to T cells that, after genetic modification, recognize a specific antigen of interest in an MHC non-limiting manner and continue to activate expansion. The annual meeting of the international cell therapy association in 2012 indicates that biological immune cell therapy has become a fourth means for treating tumors outside surgery, radiotherapy and chemotherapy, and is becoming an essential means for future tumor treatment. CAR-T cell feedback therapy is the most clearly effective form of immunotherapy in current tumor therapy. A large number of researches show that the CAR-T cells can effectively recognize tumor antigens, cause specific anti-tumor immune response and obviously improve the survival condition of patients. Chimeric Antigen Receptors (CARs) are the core component of CAR-T, conferring to T cells the ability to recognize tumor antigens in an HLA-independent manner, which enables CAR engineered T cells to recognize a broader range of targets than native T cell surface receptor TCRs. The basic design of a CAR includes a Tumor Associated Antigen (TAA) binding region (typically an scFv fragment derived from a monoclonal antibody antigen binding region), an extracellular hinge region, a transmembrane region and an intracellular signaling region. The choice of antigen of interest is a critical determinant of the specificity, effectiveness of the CAR and safety of the genetically engineered T cells themselves.

With the continuous development of CAR-T technology, CAR-T is currently divided into four generations. First generation CAR-T cells consist of extracellular binding region-single chain antibodies (SINGLE CHAIN FRAGMENT variable, scFv), transmembrane region (transmembrane region, TM) and intracellular signaling region-immune receptor tyrosine-activating motif (immunoreceptor tyrosine based activation motif, ITAM), wherein the chimeric antigen receptor portions are linked as follows: scFv-TM-CD3 zeta.

Although some specific cytotoxicity can be seen in the first generation of CARs, the clinical trial summary of the first generation of CARs in 2006 shows poor efficacy. The reason for this is that the first generation of CAR-T cells are rapidly depleted in patients and have so poor persistence that CAR-T cells have been apoptotic to a large number of tumor cells that they can elicit an anti-tumor cytotoxic effect, but the cytokine secretion is relatively low, but their survival in vivo is short and not eliciting a long lasting anti-tumor effect [ CANCER RES,2007.67 (22): 11029-11036].

Second generation CAR-T cells optimize CAR design, the T cell activation signaling region remains a hotspot for research. Complete activation of T cells depends on the actions of dual signaling and cytokines. Wherein the first signal is a specific signal initiated by the TCR recognizing an antigen peptide-MHC complex on the surface of an antigen presenting cell; the second signal is a co-stimulatory signal. The second generation CAR [ J Immunol,1998.161 (6) appeared as early as 1998: 2791-7]. The generation 2 CAR adds a co-stimulatory molecule in the intracellular signal peptide region, namely, the co-stimulatory signal is assembled into the CAR, so that an activation signal can be better provided for the CAR-T cell, and the CAR can activate the co-stimulatory molecule and the intracellular signal at the same time after recognizing tumor cells, so that double activation is realized, and the proliferation secretion capacity and the anti-tumor effect of the T cell can be obviously improved. The first T cell costimulatory signaling receptor studied in detail was CD28, which is capable of binding to a B7 family member on the surface of target cells. Co-stimulation of CD28 promotes proliferation of T cells, synthesis and expression of IL-2, and enhances the ability of T cells to resist apoptosis. Co-stimulatory molecules such as CD134 (OX 40) and 41BB (4-1 BB) are then presented to enhance T cell cytotoxicity, proliferative activity, maintain T cell responses, extend T cell survival time, etc. Such second generation CARs produced unexpected effects in subsequent clinical trials, frequently triggering shocks based on clinical reports of second generation CARs since 2010, especially for relapsed, refractory ALL patients, with complete remission rates of up to 90% or more.

The third generation CAR signal peptide region integrates more than 2 co-stimulatory molecules, so that T cells can be continuously activated and proliferated, cytokines can be continuously secreted, and the capability of killing tumor cells by the T cells is more remarkable, namely, a stronger anti-tumor response can be obtained by the novel generation CAR [ Mol Ther,2005.12 (5): 933-941]. Most typically, a 41BB stimulus is added under the influence of the CD28 stimulus.

The fourth generation of CAR-T cells, with the addition of cytokines or co-stimulatory ligands, e.g., the fourth generation of CARs, can produce IL-12, which can modulate the immune microenvironment-increase T cell activation while activating innate immune cells to act to clear target antigen-negative cancer cells, thereby achieving a bi-regulatory effect [ Expert Opin Biol Ther,2015.1 (8): 1145-54].

One great advantage of CAR-T cells is that they are active drugs, and once infused, physiological mechanisms regulate T cell balance, memory formation, and antigen-driven expansion. However, this treatment is not perfect and T cells can be off target and attack other tissues, or the amount of expansion is too high to be desirable for treatment. Given that CAR-T cells have been included in standard therapeutic ranges, it is very useful to design a patient or drug-controlled activation or deactivation mechanism to regulate the presence of CAR-T cells. For technical reasons, the closing mechanism is more easily applied to T cells. As one of these, the iCas9 system is under clinical investigation. When the cells express the iCas9, small molecular compounds can induce the iCas9 precursor molecules to form dimers, and activate apoptosis pathways, so that the purpose of eliminating the cells is achieved. In graft versus host disease, small molecule AP1903 has been used to induce iCas9 dimers and clear T cells, suggesting the feasibility of this approach [ CLIN CANCER RES,2016.22 (8): 1875-84].

In addition, CAR-T cells can also be made to express proteins to which these antibodies are directed simultaneously, such as tgfr, using already clinically used scavenging antibodies, by administering antibody drugs to scavenge the corresponding CAR-T cells after the treatment-related toxic response has occurred or after the treatment has been completed [ SCI TRANSL MED,2015.7:275ra22].

9 Months 2015, carl June's J.In New England published an article of successful treatment of 1 patient with relapsed refractory Multiple Myeloma (MM) using CD19 molecular targeted CAR-T cell therapy [ NEJM,2015.373 (11): 1040-7]. Although multiple myeloma as a B cell line tumor does not normally express CD19, CD19 is not a target for multiple myeloma immunotherapy. A trace of multiple myeloma clones with drug resistance and disease recurrence characteristics have been reported to have a B cell phenotype (i.e., CD19 positive). After defining BCMA as a target of CAR-T cells, the national cancer institute Kochenderfer and the like successfully constructed anti-BCMACAR-T cells, and preclinical studies show that the CAR-T cells specifically recognize BCMA, and greatly expand and secrete cytokines and exert killing functions after being activated by BCMA, and also have anti-tumor effects in a mouse tumor model [ CLIN CANCER RES,2013.19 (8): 2048-60]. A phase I clinical study was conducted by the national cancer institute in 2014 against BCMACAR-T cells to treat multiple myeloma, and the clinical safety and efficacy of the anti BCMACAR-T cells was verified against patients with multiple myeloma who were unresponsive to the current standard treatment regimen (clinical Trials gov Identifier: NCT 02215967). The clinical trial results of CAR-T cell therapy phase I for patients with multiple myeloma were reported by the american national cancer institute medical oncology Syed Abbas Ali professor team at the 57 th american annual meeting at 12 months of 2015. The study is incorporated into 12 patients with refractory relapsing multiple myeloma who fail more than 3 lines of chemotherapy, and some patients have more than or equal to 50% of myeloma cells in the bone marrow. After BCMACAR-T cells were infused into these patients, 1 patient was completely relieved, 3 patients were partially relieved, and the rest were stable, thus the anti-BCMACAR-T cell therapy was first demonstrated to be effective against multiple myeloma without significant side effects, and was evaluated as one of the most influential clinical studies in ASH years [ Late-BreakingAbstracts, meeting abstract No.: LAB-1]. Currently, the ibutglom university of pennsylvania ibutglom cancer center has also registered a phase I clinical trial against BCMACAR-T cells for the treatment of multiple myeloma and was under development in the dense drum (clinical trimals gov Identifier: NCT 02546167).

Summary of The Invention

A plurality of humanized antibodies are obtained as targeting molecules on the surface of chimeric antigen receptor T cells by utilizing a phage display technology-based humanized natural antibody library technology. Similar work was also performed using a murine immune antibody library technology based on phage display technology. An anti-BCMA chimeric antigen receptor constructed based on an anti-BCMA single chain antibody (e.g., scFv), including murine and human (particularly HKl a) is provided that includes an anti-BCMA binding region, a human CD 8a hinge region, a human CD 8a transmembrane region, a human 41BB intracellular region, and a human cd3ζ intracellular region. T cells expressing this chimeric antigen receptor (especially HK 10-based) have the ability to specifically kill BCMA positive tumor cells and are better in terms of ifnγ secretion and CD107a expression than T cells expressing chimeric antigen receptor based on the c11d5.3 clone. T cells expressing the chimeric antigen receptor have better clinical treatment effect. In addition to specific tumor cell killing capacity, superior ifnγ secretion and CD107a expression capacity, when T cells carrying the chimeric antigen receptor (particularly HK 10-based) are used in clinical experiments, immunogenicity can be minimized due to the use of fully human antibody scFv, avoiding clearance of T cells carrying the chimeric antigen receptor, potentially prolonging the survival and improving therapeutic efficacy of T cells carrying the chimeric antigen receptor.

In one aspect, the invention relates to an antibody or antigen binding fragment that specifically binds to human B Cell Maturation Antigen (BCMA), characterized by comprising a heavy chain variable domain and a light chain variable domain, said comprising a heavy chain variable domain and a light chain variable domain:

(1) SEQ ID NO:1, a heavy chain CDR1, SEQ ID NO:2, a heavy chain CDR2, SEQ ID NO:3, a heavy chain CDR3, SEQ ID NO:4, light chain CDR1, SEQ ID NO:5 and the light chain CDR2 shown in SEQ ID NO:6, a light chain CDR3;

(2) SEQ ID NO:7, heavy chain CDR1, SEQ ID NO:8, a heavy chain CDR2, SEQ ID NO:9, heavy chain CDR3, SEQ ID NO:10, light chain CDR1, SEQ ID NO:11 and the light chain CDR2 and SEQ ID NO:12, a light chain CDR3;

(3) SEQ ID NO:13, a heavy chain CDR1, SEQ ID NO:14, a heavy chain CDR2, SEQ ID NO:15, a heavy chain CDR3, SEQ ID NO:16, light chain CDR1, SEQ ID NO:17 and the light chain CDR2 and SEQ ID NO:18, a light chain CDR3;

(4) SEQ ID NO:19, a heavy chain CDR1, SEQ ID NO:20, a heavy chain CDR2, SEQ ID NO:21, heavy chain CDR3, SEQ ID NO:22, light chain CDR1, SEQ ID NO:23 and the light chain CDR2 shown in SEQ ID NO:24, a light chain CDR3; or (b)

(5) SEQ ID NO:25, heavy chain CDRl, SEQ ID NO:26, a heavy chain CDR2, SEQ ID NO:27, heavy chain CDR3, SEQ ID NO:28, light chain CDR1, SEQ ID NO:29 and the light chain CDR2 and SEQ ID NO:30, and a light chain CDR3.

Preferably, the antibody or antigen binding fragment comprises SEQ ID NO:1, a heavy chain CDR1, SEQ ID NO:2, a heavy chain CDR2, SEQ ID NO:3, a heavy chain CDR3, SEQ ID NO:4, light chain CDR1, SEQ ID NO:5 and the light chain CDR2 shown in SEQ ID NO:6, a light chain CDR3.

In one embodiment of the invention, the antibody or antigen binding fragment is chimeric or human.

In another aspect, the invention relates to an antibody or antigen binding fragment that specifically binds to human B Cell Maturation Antigen (BCMA), characterized by comprising a heavy chain variable domain and a light chain variable domain comprising:

(1) SEQ ID NO:31, a heavy chain CDR1, SEQ ID NO:32, a heavy chain CDR2, SEQ ID NO:33, a heavy chain CDR3, SEQ ID NO:34, light chain CDR1, SEQ ID NO:35 and the light chain CDR2 and SEQ ID NO:36, a light chain CDR3;

(2) SEQ ID NO:37, a heavy chain CDR1, SEQ ID NO:38, the heavy chain CDR2, SEQ ID NO:39, heavy chain CDR3, SEQ ID NO:40, light chain CDR1, SEQ ID NO:41 and the light chain CDR2 and SEQ ID NO:42, a light chain CDR3;

(3) SEQ ID NO:43, a heavy chain CDR1, SEQ ID NO:44, a heavy chain CDR2, SEQ ID NO:45, heavy chain CDR3, SEQ ID NO:46, light chain CDR1, SEQ ID NO:47 and the light chain CDR2 and SEQ ID NO:48, a light chain CDR3;

(4) SEQ ID NO:49, heavy chain CDR1, SEQ ID NO:50, a heavy chain CDR2, SEQ ID NO:51, heavy chain CDR3, SEQ ID NO:52, light chain CDR1, SEQ ID NO:53 and the light chain CDR2 and SEQ ID NO:54 light chain CDR3; or (b)

(5) SEQ ID NO:55, heavy chain CDR1, SEQ ID NO:56, heavy chain CDR2, SEQ ID NO:57, a heavy chain CDR3, SEQ ID NO:58, light chain CDR1, SEQ ID NO:59 and light chain CDR2 and SEQ ID NO:60, and a light chain CDR3.

In one embodiment of the invention, the antibody or antigen binding fragment is murine, chimeric or humanized.

In one embodiment of the invention, the antibody or antigen binding fragment specifically binds to the extracellular domain of human B Cell Maturation Antigen (BCMA).

In one embodiment of the invention, the antibody or antigen binding fragment is a scFv, fab or whole antibody. In one embodiment of the invention, the heavy chain of the antibody or antigen binding fragment is IgG, in particular IgG 1. In one embodiment of the invention, the light chain of the antibody or antigen binding fragment is kappa (kappa) or lambda (lambda), in particular kappa.

In a further aspect, the invention relates to one or more polynucleotides, characterized in that it encodes an antibody or antigen binding fragment according to the invention.

In a further aspect, the invention relates to a vector, characterized in that it comprises a polynucleotide according to the invention. In one embodiment of the invention, the vector is a cloning vector or an expression vector, in particular a plasmid vector.

In a further aspect, the invention relates to a host cell, characterized in that it comprises a polynucleotide according to the invention or a vector according to the invention. In one embodiment of the invention, the host cell is a prokaryotic cell, in particular an E.coli cell. In another embodiment of the invention, the host cell is a eukaryotic cell, in particular a chinese hamster ovary cell.

In a further aspect, the invention relates to a method of producing an antibody or antigen-binding fragment, characterized in that a host cell according to the invention is cultured under conditions for expression of the antibody or antigen-binding fragment.

In a further aspect, the invention relates to a pharmaceutical composition characterized by comprising an antibody or antigen-binding fragment according to the invention, and one or more pharmaceutically acceptable carriers.

In yet another aspect, the invention relates to a chimeric antigen receptor characterized by comprising an extracellular region, a transmembrane region, and an intracellular region, wherein the extracellular region comprises an antigen binding region comprising a heavy chain variable domain and a light chain variable domain comprising:

(4) SEQ ID NO:19, a heavy chain CDR1, SEQ ID NO:20, a heavy chain CDR2, SEQ ID NO:21, heavy chain CDR3, SEQ ID NO:22, light chain CDR1, SEQ ID NO:23 and the light chain CDR2 shown in SEQ ID NO:24, a light chain CDR3;

(5) SEQ ID NO:25, heavy chain CDRl, SEQ ID NO:26, a heavy chain CDR2, SEQ ID NO:27, heavy chain CDR3, SEQ ID NO:28, light chain CDR1, SEQ ID NO:29 and the light chain CDR2 and SEQ ID NO:30, a light chain CDR3;

(6) SEQ ID NO:31, a heavy chain CDR1, SEQ ID NO:32, a heavy chain CDR2, SEQ ID NO:33, a heavy chain CDR3, SEQ ID NO:34, light chain CDR1, SEQ ID NO:35 and the light chain CDR2 and SEQ ID NO:36, a light chain CDR3;

(7) SEQ ID NO:37, a heavy chain CDR1, SEQ ID NO:38, the heavy chain CDR2, SEQ ID NO:39, heavy chain CDR3, SEQ ID NO:40, light chain CDR1, SEQ ID NO:41 and the light chain CDR2 and SEQ ID NO:42, a light chain CDR3;

(8) SEQ ID NO:43, a heavy chain CDR1, SEQ ID NO:44, a heavy chain CDR2, SEQ ID NO:45, heavy chain CDR3, SEQ ID NO:46, light chain CDR1, SEQ ID NO:47 and the light chain CDR2 and SEQ ID NO:48, a light chain CDR3;

(9) SEQ ID NO:49, heavy chain CDR1, SEQ ID NO:50, a heavy chain CDR2, SEQ ID NO:51, heavy chain CDR3, SEQ ID NO:52, light chain CDR1, SEQ ID NO:53 and the light chain CDR2 and SEQ ID NO:54 light chain CDR3; or (b)

(10) SEQ ID NO:55, heavy chain CDR1, SEQ ID NO:56, heavy chain CDR2, SEQ ID NO:57, a heavy chain CDR3, SEQ ID NO:58, light chain CDR1, SEQ ID NO:59 and light chain CDR2 and SEQ ID NO:60, and a light chain CDR3.

In one embodiment of the invention, the antigen binding region is an scFv or Fab.

In one embodiment of the invention, the extracellular region further comprises a hinge region. In one embodiment of the invention, the hinge region comprises a human CD 8a hinge region. In one embodiment of the invention, the human CD 8a hinge region has the amino acid sequence of SEQ ID NO: 63.

In one embodiment of the invention, the transmembrane region comprises a human CD8 a transmembrane region. In one embodiment of the invention, the human CD8 a transmembrane region has the amino acid sequence of SEQ ID NO: 65.

In one embodiment of the invention, the intracellular region comprises one or more signal transduction regions. In one embodiment of the invention, the intracellular region comprises a human 41BB intracellular region and/or a human cd3ζ intracellular region. In one embodiment of the invention, the human 41BB intracellular region has the amino acid sequence of SEQ ID NO:67 and/or said human cd3ζ intracellular domain has the amino acid sequence shown in SEQ ID NO: 69.

In a further aspect, the invention relates to a polypeptide characterized by comprising a signal peptide and a chimeric antigen receptor according to the invention. In one embodiment of the invention, the signal peptide is a CD8 a signal peptide. In one embodiment of the invention, the CD8 a signal peptide has the amino acid sequence of SEQ ID NO:61, and a sequence of amino acids shown in seq id no.

In a further aspect, the invention relates to a polynucleotide, characterized in that it encodes a chimeric antigen receptor according to the invention or a polypeptide according to the invention.

In a further aspect, the invention relates to a vector, characterized in that it comprises a polynucleotide according to the invention. In one embodiment of the invention, the vector is a cloning vector or an expression vector. In one embodiment of the invention, the vector is a viral vector.

In a further aspect, the invention relates to an engineered immune effector cell, characterized in that it expresses a polypeptide of a chimeric antigen receptor according to the invention, or comprises a polynucleotide encoding a polypeptide of a chimeric antigen receptor or according to the invention. In one embodiment of the invention, the engineered immune effector cell is selected from T lymphocytes, NK cells, pluripotent stem cells or embryonic stem cells, in particular T lymphocytes.

In a further aspect, the invention relates to a method for engineering an immune effector cell, characterized in that the immune effector cell is infected with a vector according to the invention, in particular a viral vector. In one embodiment of the invention, the immune effector cell is a T lymphocyte.

In a further aspect, the invention relates to the use of an antibody or antigen binding fragment, chimeric antigen receptor, polypeptide, polynucleotide, vector, or engineered immune effector cell according to the invention in the manufacture of a medicament for the treatment of cancer. In a further aspect, the invention relates to the use of an antibody or antigen binding fragment, chimeric antigen receptor, polypeptide, polynucleotide, vector, or engineered immune effector cell according to the invention in the manufacture of a medicament for stimulating immune function in a patient having cancer. In yet another aspect, the invention relates to a method of treating cancer comprising administering an antibody or antigen binding fragment, chimeric antigen receptor, polypeptide, polynucleotide, vector, or engineered immune effector cell according to the invention to a patient having the cancer. In a further aspect, the invention relates to a method of stimulating immune function in a patient having cancer comprising administering to the patient an antibody or antigen binding fragment, chimeric antigen receptor, polypeptide, polynucleotide, vector, or engineered immune effector cell according to the invention. In one embodiment of the invention, the cancer is a B cell-related cancer, particularly multiple myeloma. In one embodiment of the invention, the immune function is IFN-gamma secretion and/or CD107a expression

Brief Description of Drawings

FIG. 1 shows the killing of target cells by anti-BCMACAR-T.

FIG. 2 shows IFN-. Gamma.and CD107a expression against BCMACAR-T CD 3-positive cells.

FIG. 3 shows IFN-gamma expression against BCMACAR-T CD 3-positive cells.

FIG. 4 shows CD107a expression against BCMACAR-T CD3 positive cells.

FIG. 5 shows the heavy chain CDR amino acid sequences of an antibody of the present invention (according to the IMGT system).

FIG. 6 shows the light chain CDR amino acid sequences of the antibodies of the present invention (according to the IMGT system).

Detailed Description

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

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry and immunology. These techniques are well within the skill of the art and are well described in the literature.

It should be understood that embodiments of "comprising" as described herein include embodiments that "consist of … …" and/or "consist essentially of.

I. anti-BCMA antibodies

One aspect of the present invention provides anti-BCMA antibodies that specifically bind to BCMA, such as human BCMA, particularly the extracellular domain of human BCMA. In some embodiments, the anti-BCMA antibodies of the invention are monoclonal antibodies.

B Cell Maturation Antigen (BCMA), also known as CD269 or TNFRSF17, is a member of the tumor necrosis factor receptor superfamily. BCMA is expressed almost exclusively in plasma cells and multiple myeloma cells. BCMA may be a suitable tumor antigen target for immunotherapeutic agents against multiple myeloma.

In some embodiments, the anti-BCMA antibody of the present invention is a full length antibody. The terms "full length antibody," "whole antibody," and "whole antibody" are used interchangeably herein to refer to an antibody in substantially complete form as compared to an antibody fragment. The terms "antibody" and "immunoglobulin" are used interchangeably herein. The basic 4-chain antibody unit is a hetero-tetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. The L chains from any vertebrate species can be classified into two distinct types, called kappa (kappa) and lambda (lambda), based on the amino acid sequence of their constant regions. Antibodies can be categorized into five different classes based on the amino acid sequence of the heavy chain constant region: igA, igD, igE, igG and IgM, which have heavy chains called α, δ, ε, γ and μ, respectively. IgG and IgA classes can be further divided into subclasses such as IgG1, igG2 (including IgG2A and IgG 2B), igG3, igG4, igA1, and IgA2. In the case of IgG, the 4-chain unit is typically about 150,000 daltons. Each H chain has a variable domain at the N-terminus (V _H) followed by three constant domains per gamma chain (C _H). Each L chain has a variable domain at the N-terminus (V _L) followed by a constant domain at the other end (C _L).

In some embodiments, the anti-BCMA antibodies of the invention are IgG, particularly IgG 1. In some embodiments, the anti-BCMA antibodies of the invention are kappa.

In some embodiments, the anti-BCMA antibodies of the invention are antibody fragments. The term "antibody fragment" comprises a portion of an intact antibody, preferably the antigen binding and/or variable regions of an intact antibody. Examples of antibody fragments include Fab, fab '-SH, F (ab') ₂, fv, and scFv fragments; a double body; a linear antibody; a single chain antibody; a single domain antibody; and multispecific antibodies formed from antibody fragments. The term "scFv" is an antibody fragment comprising V _H and V _L domains linked into a single polypeptide chain. Preferably, the scFv further comprises a polypeptide linker between the V _H and V _L domains. The term "Fab" consists of the whole L chain as well as the variable domain of the H chain (V _H) and the first constant domain (C _H 1).

In some embodiments, an anti-BCMA antibody of the present invention is an antigen binding fragment comprising a heavy chain variable domain and a light chain variable domain of a parent antibody, e.g., scFv or Fab. Antibody fragments can be prepared by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies, and production by recombinant host cells.

In some embodiments, the anti-BCMA antibodies of the invention are murine, chimeric, humanized, or human.

In a "chimeric" antibody, a portion of the heavy and/or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the heavy and/or light chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, so long as they exhibit the desired biological activity. In some embodiments, the chimeric antibodies of the invention comprise a murine variable region and a human constant region.

A "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody that contains minimal sequences derived from the non-human antibody. In some embodiments, the humanized antibody is a human antibody (recipient antibody) in which CDR (or HVR) residues of the recipient antibody are replaced with CDR (or HVR) residues of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and/or capacity. In some cases, framework ("FR") residues of a human antibody are replaced with corresponding non-human residues. In addition, the humanized antibody may comprise residues that are not present in either the recipient antibody or the donor antibody. These modifications may be made to further refine antibody properties such as binding affinity.

A "human antibody" is an antibody having an amino acid sequence corresponding to that of a human-produced antibody and/or prepared using any technique for preparing a human antibody. This definition of human antibodies specifically excludes humanized antibodies that comprise non-human antigen binding residues. Human antibodies can be produced using a variety of techniques known in the art. Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge, but whose endogenous loci have been disabled. Human antibodies can also be produced by phage display libraries of human origin. The library of immune sources provides high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, the unexposed pool can be cloned to give antibodies to a variety of non-self and self antigens without any immunization.

In some embodiments, an anti-BCMA antibody or antigen binding fragment of the invention comprises the heavy chain CDRs shown in fig. 5. In some embodiments, an anti-BCMA antibody or antigen binding fragment of the invention comprises the light chain CDRs shown in fig. 6.

In some embodiments, the invention encompasses antibodies that bind to the same BCMA epitope as any of the anti-BCMA antibodies described herein. In some embodiments, the invention encompasses antibodies that compete with any of the anti-BCMA antibodies described herein for binding to BCMA. In some embodiments, competition assays can be used to identify antibodies that bind to the same BCMA epitope as any of the anti-BCMA antibodies described herein or antibodies that compete for binding to BCMA with any of the anti-BCMA antibodies described herein. In some embodiments, two antibodies are considered to bind to the same epitope if one blocks the binding of the other to the antigen by 50% or more.

One aspect of the present invention provides one or more polynucleotides characterized by encoding an anti-BCMA antibody according to the present invention. In one aspect, the invention provides a vector, characterized in that it comprises a polynucleotide according to the invention. In one embodiment, the vector is a cloning vector or an expression vector, in particular a plasmid vector. In one aspect the invention provides a host cell characterized by comprising a polynucleotide according to the invention or a vector according to the invention. In one embodiment, the host cell is a prokaryotic cell, in particular an E.coli cell. In another embodiment, the host cell is a eukaryotic cell, particularly a chinese hamster ovary cell. In one aspect, the invention provides a method for producing an anti-BCMA antibody, characterized by culturing a host cell according to the invention under conditions for expression of the antibody.

Chimeric antigen receptor

One aspect of the invention provides a Chimeric Antigen Receptor (CAR) that targets BCMA. Chimeric antigen receptors are generally composed of an extracellular region, a transmembrane region, and an intracellular region. The extracellular region comprises an antigen binding region, and optionally a hinge region. The intracellular region comprises one or more signaling regions, including a costimulatory signaling region. The polypeptide of the chimeric antigen receptor may also comprise a signal peptide when expressed in a cell.

Signal peptides

When expressed in a cell, the polypeptide of the chimeric antigen receptor of the invention may comprise a signal peptide (also referred to as a signal sequence) at the N-terminus of the polypeptide. In general, a signal peptide is a peptide sequence that targets a polypeptide to a desired site in a cell. In some embodiments, the signal peptide targets the polypeptide to the secretory pathway of the cell and will allow the polypeptide to integrate and anchor to the lipid bilayer.

In some embodiments, the signal peptide used in the present invention is derived from CD8 a. In some embodiments, the CD 8a signal peptide comprises SEQ ID NO:61, and a sequence of amino acids. In some embodiments, the CD 8a signal peptide consists of SEQ ID NO: 62.

Antigen binding region

The chimeric antigen receptor of the invention comprises an antigen binding region that targets BCMA. The antigen binding region may be monovalent or multivalent (e.g., bivalent). The antigen binding region may also be monospecific or multispecific (e.g., bispecific). Bispecific may be against BCMA and another antigen, or against two different epitopes of BCMA.

In some embodiments, the antigen binding regions used in the present invention are in the form of an anti-BCMA antibody or antigen binding fragment described above, particularly scFv.

Hinge region

Optionally, the chimeric antigen receptor of the invention comprises a hinge region between the extracellular antigen binding region and the transmembrane region. The hinge region is a segment of amino acids that typically exist between two domains of a protein and can allow flexibility of the protein and movement of the two domains relative to each other.

The hinge region may be the hinge region of a naturally occurring protein or a portion thereof. The hinge region of an antibody (such as IgG, igA, igM, igE or IgD antibodies) can also be used for the chimeric antigen receptor described herein. Non-naturally occurring peptides can also be used as hinge regions for chimeric antigen receptors described herein. In some embodiments, the hinge region is a peptide linker.

In some embodiments, the hinge region used in the present invention is derived from CD8 a. In some embodiments, the CD 8a hinge region comprises SEQ ID NO: 63. In some embodiments, the CD 8a hinge region consists of SEQ ID NO:64, a nucleic acid sequence encoding a polypeptide.

Transmembrane region

The chimeric antibody receptor of the invention comprises a transmembrane region. The transmembrane region may form an alpha helix, a complex of more than one alpha helix, a beta barrel, or any other stable structure capable of crossing a domain cellular phospholipid bilayer. The transmembrane region may be of natural or synthetic origin. The transmembrane region may be derived from the alpha, beta or zeta chain of T cell receptor 、CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、KIRDS2、OX40、CD2、CD27、LFA-1(CD11a、CD18)、ICOS(CD278)、4-1BB(CD137)、GITR、CD40、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、CD160、CD19、IL-2Rβ、IL-2Rγ、IL-7Ra、ITGA1、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CDlld、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CDllb、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRT AM、Ly9(CD229)、CD160(BY55)、PSGL1、CDIOO(SEMA4D)、SLAMF6(NTB-A、Lyl08)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、PAG/Cbp、NKp44、NKp30、NKp46、NKG2D and/or NKG 2C.

In some embodiments, the transmembrane region used in the present invention is derived from CD8 a. In some embodiments, the CD8 a transmembrane region comprises SEQ ID NO: 65. In some embodiments, the CD8 transmembrane region consists of SEQ ID NO: 66.

Intracellular region

The chimeric antigen receptor of the invention comprises an intracellular region. The intracellular region comprises one or more signaling regions, including a costimulatory signaling region.

The intracellular signaling region is responsible for activation of at least one normal effector function of immune effector cells expressing the chimeric antigen receptor. For example, the effector function of a T cell may be a cell lysis activity or a helper activity, including secretion of cytokines. Although the entire intracellular signaling region is generally available, in many cases, the use of an entire strand is not necessary. In the case of using a truncated portion of the intracellular signaling region, such a truncated portion may be used instead of the complete strand as long as it transduces the effector function signal. Thus, an intracellular signaling region includes any truncated form of the intracellular signaling region sufficient to transduce an effector function signal. In some embodiments, the signaling region is derived from cd3ζ, fcrγ (FCER 1G), fcrβ (fcεrib), cd3γ, cd3δ, cd3ε, CD5, CD22, CD79a, CD79b, and CD66d.

In some embodiments, the signaling region used in the present invention is derived from cd3ζ. In some embodiments, the CD3 zeta signaling region comprises SEQ ID NO: 69. In some embodiments, the CD3 zeta signaling region consists of SEQ ID NO: 70.

In some embodiments, the intracellular regions of the chimeric antigen receptor of the invention further comprise one or more costimulatory signaling regions. In addition to stimulation of antigen specific signals, many immune effector cells require co-stimulation to promote cell proliferation, differentiation and survival, as well as to activate the effector functions of the cells. The "costimulatory signaling region" may be the cytoplasmic portion of a costimulatory molecule. The term "costimulatory molecule" refers to a cognate binding partner on an immune cell (such as a T cell) that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response, such as, but not limited to, proliferation and survival, by the immune cell. The costimulatory signaling region may be derived from a B7/CD28 family member (e.g., B7-1/CD80、B7-2/CD86、B7-H1/PD-L 1、B7-H2、B7-H3、B7-H4、B7-H6、B7-H7、BTLA/CD272、CD28、CTLA-4、Gi24/VISTA/B7-H5、ICOS/CD278、PD-1、PD-L2/B7-DC and PDCD 6); TNF superfamily members (e.g., 4-1BB/TNFSF9/CD137, 4-1BB ligand/TNFSF 9, BAFF/BLyS/TNFSF13B, BAFF R/TNFRSF13C, CD/TNFRSF 7, CD27 ligand/TNFSF 7, CD30/TNFRSF8, CD30 ligand/TNFSF 8, CD40/TNFRSF5, CD40/TNFSF5, CD40 ligand/TNFSF 5, DR3/TNFRSF25, GITR/TNFRSF18, GITR ligand/TNFSF 18, HVEM/TNFRSF14, LIGHT/TNFSF14, lymphotoxin-alpha/TNF-beta, OX40/TNFRSF4, OX40 ligand/TNFSF 4, RELT/TNFRSF19L, TACI/TNFRSF13B, TL A/TNFSF15, TNF-alpha and TNF-gamma/TNFSF 1B); SLAM family members (e.g., ,2B4/CD244/SLAMF4、BLAME/SLAMF8、CD2、CD2F-10/SLAMF9、CD48/SLAMF2、CD58/LFA-3、CD84/SLAMF5、CD229/SLAMF3、CRACC/SLAMF7、NTB-A/SLAMF6 and SLAM/CD 150); and any other costimulatory molecules such as CD2, CD7, CD53, CD82/Kai-1, CD90/Thy1, CD96, CD160, CD200, CD300a/LMIR1, HLA class I, HLA-DR, ikaros, integrin alpha 4/CD49d, integrin alpha 4 beta 1, integrin α4β7/LPAM-1、LAG-3、TCL1A、TCL1B、CRTAM、DAP12、Dectin-1/CLEC7A、DPPIV/CD26、EphB6、TIM-1/KIM-1/HAVCR、TIM-4、TSLP、TSLP R、 lymphocyte function-associated antigen-1 (LFA-1) and NKG2C.

In some embodiments, the costimulatory signaling region used in the present invention is derived from 4-1BB. In some embodiments, the 4-1BB costimulatory signaling region comprises the sequence of SEQ ID NO: 67. In some embodiments, the 4-1BB costimulatory signaling region consists of the sequence set forth in SEQ ID NO:68, and a nucleic acid sequence encoding the same.

In some embodiments, the intracellular region of a chimeric antigen receptor of the invention comprises the 4-1BB costimulatory signaling region described above and the CD3 zeta signaling region described above linked in an N-terminal to C-terminal direction.

In one aspect the invention provides a polynucleotide which encodes a chimeric antigen receptor or polypeptide according to the invention. In one aspect, the invention provides a vector, characterized in that it comprises a polynucleotide according to the invention. In one embodiment, the vector is a cloning vector or an expression vector. In one embodiment, the vector is a viral vector.

Engineered immune effector cells

One aspect of the invention provides cells, such as immune effector cells, engineered to contain or express the BCMA-targeted chimeric antigen receptor of the invention. In some embodiments, the immune effector cells are T cells, NK cells, peripheral Blood Mononuclear Cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, or embryonic stem cell culture differentiated cells (e.g., immune cells). In some embodiments, the immune effector cell is autologous. In some embodiments, the immune effector cell is allogeneic.

Carrier body

One aspect of the present invention provides a vector for cloning and expressing the BCMA-targeted chimeric antigen receptor of the present 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, lentiviral vectors, retrovirus vectors, vaccinia vectors, herpes simplex virus vectors, and derivatives thereof.

Many virus-based systems have been developed for gene transfer to mammalian cells. In some embodiments, lentiviral vectors are used. In some embodiments, self-inactivating lentiviral vectors are used. For example, self-inactivating lentiviral vectors carrying chimeric antigen receptor coding sequences may be packaged using protocols known in the art. The resulting lentiviral vector may be used to transduce mammalian cells (such as primary human T cells) using methods known in the art. Lentiviral derived vectors are suitable tools for achieving long term gene transfer because they allow long term, stable integration of transgenes and their propagation in daughter cells. Lentiviral vectors are also low immunogenic and can transduce non-proliferating cells.

Immune effector cells

An "immune effector cell" 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 mediate ADCC include Peripheral Blood Mononuclear Cells (PBMCs), natural Killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils.

In some embodiments, the immune effector cell is a T cell. In some embodiments, the T cell is CD4+/CD8-, CD4-/CD8+, CD4+/CD8+, CD4-/CD8-, or a combination thereof. In some embodiments, the T cell produces IL-2, IFN, and/or TNF when expressing the chimeric antigen receptor and binding to the target cell. In some embodiments, cd8+ T cells lyse antigen-specific target cells when expressing chimeric antigen receptors and binding to the target cells.

Engineered immune effector cells are prepared by introducing chimeric antigen receptors into immune effector cells, such as T cells. In some embodiments, the chimeric antigen receptor is introduced into the immune effector cell by transfecting a nucleic acid or vector comprising a sequence encoding the chimeric antigen receptor. In some embodiments, the chimeric antigen receptor is introduced into the immune effector cell by inserting the protein into the cell membrane while passing the cell through a microfluidic system.

Methods for introducing nucleic acids or vectors into mammalian cells are known in the art. The vector may 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. Chemical means for introducing nucleic acids or vectors into immune effector cells include colloidal dispersions such as macromolecular complexes, nanocapsules, microspheres, beads and lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles and liposomes). An exemplary colloidal system for use as an in vitro delivery vehicle is a liposome (e.g., an artificial membrane vesicle). Biological methods for introducing nucleic acids or 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 mammalian, e.g., human, cells.

In some embodiments, the transduced or transfected immune effector cells are propagated ex vivo after introduction of the nucleic acid or vector. In some embodiments, the transduced or transfected immune effector cells are further evaluated or screened to select for engineered immune effector cells.

T cell origin

Prior to expansion and genetic modification of T cells, a source of T cells is obtained from an individual. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors.

Activation and expansion of T cells

Whether before or after genetic modification of T cells using the chimeric antigen receptors described herein, T cells can generally be activated and expanded using methods known in the art.

In general, T cells can be expanded by contacting a surface to which are attached reagents that stimulate a CD3/TCR complex-associated signal and ligands that stimulate co-stimulatory molecules on the surface of the T cells. In particular, the anti-CD 3 antibody or antigen-binding fragment thereof or the anti-CD 2 antibody immobilized on a surface may be contacted, or by contacting a protein kinase C activator (e.g., bryostatin) in combination with a calcium ionophore. For co-stimulation of helper molecules on the T cell surface, ligands that bind to the helper molecules are used. For example, a population of T cells may be contacted with an anti-CD 3 antibody and an anti-CD 28 antibody under conditions suitable to stimulate T cell proliferation. To stimulate proliferation of cd4+ T cells or cd8+ T cells, anti-CD 3 antibodies and anti-CD 28 antibodies are used.

In some embodiments, the primary stimulation signal and the co-stimulation signal of the T cells may be provided by different protocols. For example, the reagents providing each signal may be in solution or bound to a surface. When the agent binds to a surface, it can bind to the same surface (i.e., in "cis" form) or a separate surface (i.e., in "trans" form). Or one reagent may be bound to the surface while the other reagent is in solution. In one embodiment, the agent that provides the co-stimulatory signal binds to the cell surface, while the agent that provides the primary activation signal is in solution or bound to the surface. In certain embodiments, both reagents may be in solution. In another embodiment, the agent may be in a soluble form and then crosslinked to the surface.

In some embodiments, T cells are combined with reagent coated beads, followed by separation of the beads and cells, and then culturing the cells. In an alternative embodiment, the reagent coated beads and cells are not separated but are cultured together prior to culturing. In another embodiment, the beads and cells are first concentrated by applying a force (such as a magnetic force) such that the attachment of cell surface markers is increased, thereby inducing cell stimulation.

Suitable conditions for T cell culture include suitable media (e.g., minimal essential media or RPMI media 1640) that may contain factors necessary for proliferation and survival, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF beta, and TNF-alpha, or any other additives known to the skilled artisan for cell growth. Other additives for cell growth include, but are not limited to, surfactants, human plasma protein powder (plasmanate), and reducing agents such as N-acetylcysteine and 2-mercaptoethanol. The culture medium may include RPMI 1640, AIM-V, DMEM, MEM, alpha-MEM, F-12, X-Vivo15 and X-Vivo20 supplemented with amino acids, sodium pyruvate and vitamins, which are serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokines sufficient for T cell growth and expansion. Antibiotics (e.g., penicillin and streptomycin) are included only in the experimental cultures, and not in the cell cultures to be infused into the subject. The cells are maintained under conditions required to support growth, such as an appropriate temperature (e.g., 37 ℃) and atmosphere (e.g., air plus 5% CO ₂). T cells that have been exposed to different stimulation times may exhibit different characteristics. For example, typical peripheral blood mononuclear cell products of blood or apheresis have a helper T cell population (TH, cd4+), which is larger than a cytotoxic or suppressor T cell population (TC, CD 8). Ex vivo expansion of T cells by stimulation of CD3 and CD28 receptors results in a T cell population consisting essentially of TH cells prior to about day 8-9, whereas after about day 8-9, the T cell population comprises a progressively increasing population of TC cells. Thus, depending on the therapeutic purpose, it may be advantageous to infuse the subject with a T cell population comprising predominantly TH cells. Similarly, if an antigen-specific subset of TC cells has been isolated, it may be beneficial to expand that subset to a greater extent.

Furthermore, other phenotypic markers besides the CD4 and CD8 markers are also significantly different, but to a large extent reproducible during cell expansion. Thus, this reproducibility enables the tailoring of the activated T cell product for a specific purpose.

As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably and all of these designations include progeny. It is understood that all progeny may not be exactly identical in DNA content, due to deliberate or inadvertent mutation. Including variant progeny that have the same function or biological activity as the cells selected in the initially transformed cells.

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells" which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. The progeny may not be exactly identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as the cell selected or selected in the originally transformed cell.

Diagnostic and detection methods and uses

One aspect of the invention provides a method for detecting BCMA in a biological sample. The term "detection" encompasses quantitative or qualitative detection. One aspect of the invention provides a method for diagnosing cancer. In one embodiment, the cancer includes, but is not limited to, B cell related cancers, particularly multiple myeloma. In one embodiment, the cancer includes, but is not limited to, hematological malignancies and solid tumors, particularly multiple myeloma.

In some embodiments, the method comprises contacting the biological sample with an anti-BCMA antibody described herein under conditions that allow the anti-BCMA antibody to bind BCMA, and detecting whether a complex is formed between the anti-BCMA antibody and BCMA, wherein the formation of the complex indicates the presence of BCMA in the biological sample or that the subject producing the biological sample has cancer. Such methods may be in vitro or in vivo.

In one embodiment, a labeled anti-BCMA antibody or antigen binding fragment is provided. Labels include, but are not limited to, directly detected labels or moieties (such as fluorescent labels, chromophore labels, electron density labels, chemiluminescent labels, and radioactive labels), and indirectly detected labels or moieties (e.g., by enzymatic reactions or molecular interactions, such as enzymes with substrates, receptors, and ligands).

V. methods of treatment and uses

One aspect of the invention provides methods for treating cancer, or for stimulating immune function in a patient having cancer. In one embodiment, the method comprises administering one or more antibodies or antigen binding fragments of the invention. In one embodiment, the method comprises administering a polynucleotide encoding one or more antibodies or antigen binding fragments of the invention. In one embodiment, the method comprises engineering an immune effector cell to express one or more chimeric antigen receptors of the invention. In one embodiment, the method comprises administering one or more engineered immune effector cells of the invention. In one embodiment, the method is cellular immunotherapy.

In one embodiment, the cancer includes, but is not limited to, B cell related cancers, particularly multiple myeloma. In one embodiment, the cancer includes, but is not limited to, hematological malignancies and solid tumors, particularly multiple myeloma.

Administration may be by any convenient means, including by injection, inhalation, infusion, implantation or implantation. Administration may be intra-arterial, subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intravenous or intraperitoneal. In some embodiments, the administration is systemic or local. Infusion techniques for immunotherapy are known in the art.

The dosage and concentration of the agents of the present invention may vary depending on the particular application. Determination of the appropriate dosage is well within the skill of the ordinarily skilled artisan.

In some embodiments, the pharmaceutical composition is administered in a single administration. In some embodiments, the pharmaceutical composition is administered multiple times, such as2, 3, 4, 5,6, or more times. In some embodiments, the pharmaceutical composition is administered once a week, once a week 3, once a week 4, once a month 2, once a month 3, once a month 4, once a month 6, or once a year.

In some embodiments, the dose may be administered by one or more separate administrations or by continuous infusion. In some embodiments, the pharmaceutical compositions are administered separately, such as in 2, 3, 4, 5, or more administrations. In some embodiments, the divided doses are administered within about one week. In some embodiments, the dose is aliquoted.

The progress of the treatment can be readily monitored by conventional techniques and assays. The optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical arts by monitoring the patient's signs and adjusting the treatment accordingly.

In some embodiments, the amount of the pharmaceutical composition is effective to elicit an objective clinical response in the individual. In some embodiments, the amount of the pharmaceutical composition is effective to cause remission (partial or complete) of the disease in the subject. In some embodiments, the amount of the pharmaceutical composition is effective to prevent recurrence of cancer or disease progression in the subject. In some embodiments, the amount of the pharmaceutical composition is effective to extend survival of the individual (such as disease-free survival). In some embodiments, the pharmaceutical composition is effective to improve the quality of life of the individual. In some embodiments, the amount of the pharmaceutical composition is effective to inhibit the growth of or reduce the size of a solid or lymphoid tumor. In some embodiments, the amount of the pharmaceutical composition is effective to inhibit tumor metastasis in the subject.

VI pharmaceutical composition

One aspect of the invention provides a pharmaceutical composition comprising one or more antibodies or antigen binding fragments, chimeric antigen receptors or engineered immune effector cells of the invention, and one or more pharmaceutically acceptable carriers.

The pharmaceutical compositions may be prepared in the form of lyophilized formulations or aqueous solutions by mixing the active agent of the desired purity with an optional pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants, preservatives, isotonic agents, stabilizers, surfactants, and the like.

In order for pharmaceutical compositions to be useful for in vivo administration, they must be sterile. The pharmaceutical composition may be rendered sterile by filtration through a sterile filtration membrane.

The pharmaceutical compositions may contain more than one active agent as required for the particular indication to be treated, preferably those having complementary activities that do not adversely affect each other. Alternatively or in addition, the pharmaceutical composition may further comprise a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressant, or a growth inhibitory agent. Such molecules are suitably present in combination in an amount effective for the intended purpose.

VII kits and articles of manufacture

One aspect of the invention provides kits and articles of manufacture comprising one or more antibodies or antigen binding fragments, chimeric antigen receptors, or engineered immune effector cells of the invention.

The kit or article of manufacture may comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed from a variety of materials, such as glass or plastic. The label or package insert includes instructions for use, meaning instructions typically included in commercial packages of products, containing information about the indication, usage, dosage, administration, contraindications and/or warnings of use of such products. In addition, the kit or article of manufacture may also include other materials, including diluents, filters, needles and syringes, as desired from a commercial and user standpoint.

The following examples are provided by way of illustration and not by way of limitation as a mere example of the invention and should not be construed as limiting the invention in any way.

Examples

Example 1: construction of expression vector of fusion protein (BCMA-huIgG 1 Fc) of recombinant human B Cell Maturation Antigen (BCMA) extracellular domain and human IgG1 Fc region and eukaryotic expression

1. Gene synthesis of recombinant BCMA extracellular domain and expression vector construction of BCMA-huIgG1 Fc fusion protein

A gene sequence encoding the 1 st methionine to 54 th alanine interval of human B Cell Maturation Antigen (BCMA) (NCBI access No. np_ 001183.2) encoding the amino acid sequence of SEQ ID NO:71, and a nucleotide sequence shown in seq id no. The gene sequence of the 99 th glutamic acid to 330 th lysine amino acid interval of the human IgG1 heavy chain constant region (UniProtKB/Swiss-Prot access No. P01857.1) encoding the sequence of SEQ ID NO:73, and a nucleotide sequence shown in seq id no. Primers containing the fusion protein signal peptide (with the amino acid sequence shown in SEQ ID NO: 75) gene sequence were chemically synthesized for expression vector construction. The BCMA gene fragment was spliced with the human IgG1 Fc gene fragment by molecular cloning. The splice products were cloned into pcdna3.1 (Thermo) using TaKaRa seamless cloning kit.

2. Expression and purification of recombinant BCMA-huIgG1 Fc fusion proteins

After transfection of 293T cells (ATCC) with this expression vector for 5 days, the culture supernatant was collected and the recombinant BCMA-huIgG1 Fc fusion protein was purified with AKTA explorer 100 (GE). Due to glycosylation modification and other reasons, the recombinant BCMA-huIgG1 Fc fusion protein is subjected to reducing SDS-PAGE electrophoresis and then is stained with Coomassie brilliant blue to show that the recombinant BCMA-huIgG1 Fc fusion protein is about 40 kilodaltons in size.

Example 2: construction of chimeric antibody expression vector of anti-human BCMA murine antibody C11D5.3 variable region and human IgG 1/kappa constant region and eukaryotic expression

1.Acquisition of variable region Gene of murine antibody of C11D5.3 and construction of expression vector of murine-human chimeric antibody of C11D5.3

The c11d5.3 antibody light chain variable region gene is synthesized by a chemical synthesis method, and the c11d5.3 antibody light chain variable region has a sequence of SEQ ID NO:77, and the c11d5.3 antibody heavy chain variable region has the amino acid sequence shown in SEQ ID NO:79, and a sequence of amino acids shown in seq id no. The heavy chain variable region gene is used as a template, the heavy chain variable region fragment is amplified by PCR, and the amplified product is cloned into pFUSEss-CHIg-hG1 (invivogen) containing signal peptide and human IgG1 heavy chain constant region gene by using TaKaRa seamless cloning kit. The light chain variable region gene is used as a template, the light chain variable region fragment is amplified by PCR, and the amplified product is cloned into pFUSE2ss-CLIg-hK (invivogen) containing signal peptide and human kappa light chain constant region gene by using a TaKaRa seamless cloning kit.

Expression and purification of chimeric antibodies of C11D5.3

After 5 days of cotransfection of 293T cells (ATCC) in this double plasmid 1:1 ratio, the culture supernatants were collected and the C11D5.3 chimeric antibody was purified with AKTAexplorer (GE). The C11D5.3 chimeric antibody was electrophoresed on non-reducing SDS-PAGE and stained with Coomassie blue to show that it was approximately 150 kDalton in size.

Example 3: ELISA detection of binding of recombinant human BCMA to C11D5.3 chimeric antibody

The binding of recombinant human BCMA to the c11d5.3 chimeric antibody was detected using an enzyme-linked immunosorbent assay (ELISA) which was performed as follows: the BCMA-huIgG1 Fc fusion protein prepared above was added to the microwell plate at 100 ng/well and coated overnight at 4 ℃. The cells were washed three times with PBS, 1% BSA/PBS was added thereto, 200 uL/well, and the cells were blocked at 37℃for 1 hour. 100 ng/well of C11D5.3 chimeric antibody was added and bound for 1 hour at 37 ℃. PBST was washed three times and HRP-goat anti-human IgG (Fab-specific) was added to bind for 1 hour at 37 ℃. PBST was washed three times, 100 uL/well TMB developing solution was added, developed at 37℃for 10 minutes, 100 uL/well ELISA stop solution was added, and the OD450 value was read by an ELISA reader. The OD450 value reflects the binding of the c11d5.3 chimeric antibody to recombinant human BCMA.

Example 4: preparation of anti-human BCMA fully human antibody

1. Construction of high-capacity human-derived Natural antibody phage library

1.1 Collection of human peripheral blood cell resources and cDNA acquisition

Human peripheral blood cells from different individuals who volunteered to donate were collected, and total RNA was extracted from the above peripheral blood cells with Trizol RNA extraction kit (Invitrogen).

First strand cDNA was synthesized using SuperScriptTM IV First-STRAND SYNTHESIS SYSTEM (Invitrogen) kit using the RNA as a template.

1.2 Antibody Gene amplification

The cDNA was used as a template to amplify the heavy chain variable domain and CH1 constant domain genes by PCR using the heavy chain variable domain upstream primer (containing SfiI cleavage site) and the heavy chain constant domain CH1 downstream primer (containing SfiI cleavage site), and the variable domain and constant domain genes of the kappa He Lam chain by PCR using the variable domain upstream primer (containing NheI cleavage site) and the constant domain downstream primer (containing SalI cleavage site) of the kappa He Lam chain. Into a 50uL reaction system, 25uL phusion master mix (Thermo), 2.5uL (25 pmol) of the upstream primer, 2.5uL (25 pmol) of the downstream primer, 1.5uL DMSO,0.5uL cDNA and 18uL of ddH ₂ O were added, respectively. The PCR reaction was performed according to the following procedure: after 1 minute of pre-denaturation at 98 ℃, the mixture enters a temperature cycle, denaturation at 98 ℃ for 30 seconds, annealing at 58 ℃ for 30 seconds, extension at 72 ℃ for 1.5 minutes, and cycle 32 times, and final extension at 72 ℃ for 10 minutes. The amplified heavy chain variable domain and CH1 constant domain genes and variable domain and constant domain genes of kappa or lambda chains were recovered using a DNA gel recovery kit (Invitrogen).

1.3 Construction of a humanized Natural antibody library

The heavy chain variable domain and CH1 constant domain genes were digested with SfiI and the kappa He Lam delta chain variable domain and constant domain genes were digested with NheI (NEB) and SalI (NEB). The digested gene fragment was recovered using a DNA gel recovery kit.

Preparation of a sufficient amount of engineered pComb3XTT phagemids (Scripps institute, usa), wherein: the 5 'side of the light chain variable domain and constant domain gene sequences deleted the SfiI cleavage site, the NheI cleavage site was introduced, and the 3' side of the light chain variable domain and constant domain gene sequences introduced the SalI cleavage site. The 5' side of the heavy chain variable domain and constant domain gene sequences was introduced with SfiI cleavage sites. The amber stop codon of the original phagemid was deleted.

The vector was digested with NheI and SalI, and the digested vector was recovered using a DNA gel recovery kit. The digested phagemid vector was subjected to overnight ligation with the variable and constant domain genes of the digested kappa He Lam-d strand using DNA T4 ligase (NEB). The ligation products were purified using plasmid DNA purification kit (Invitrogen). The recombinant light chain-containing vector was introduced into TG1 competence (Lucigen) by electrotransformation, the recombinant light chain-containing phagemid vector was amplified by culture of escherichia coli, and a sufficient amount of the recombinant light chain-containing phagemid vector was extracted using plasmid extraction kit (Invitrogen). And (3) utilizing SfiI enzyme to cleave the phagemid vector containing the recombinant light chain, and utilizing a DNA gel recovery kit to recover the enzyme-cleaved phagemid vector. The digested phagemid vector was subjected to overnight ligation with the digested heavy chain variable domain and CH1 constant domain genes using DNAT4 ligase. The ligation product was purified using a plasmid DNA purification kit. Vectors containing recombinant light and heavy chains were electrotransformed into SS320 competence using an electrotransformation apparatus (Bio-Rad). 10ul of the electrotransformed bacteria were removed, counted and the phage antibody library size counted by reasonable dilution and streaking on plates containing tetracycline and ampicillin. The remaining electrotransformed bacteria were added to a 2XYT medium containing 5ug/mL tetracycline, 100ug/mL ampicillin and 2% glucose and incubated in a heated incubator. After the completion of the culture, the culture was centrifuged at 4000G for 10 minutes at4℃to supplement the precipitant with an appropriate amount of glycerol and stored at-80℃as an antibody strain library. A large capacity human natural antibody phage library is obtained through multiple electrotransformation accumulation.

2. Screening and identification of antibody phage libraries

2.1 Biotinylation of BCMA-huIgG1 Fc fusion proteins

BCMA-huIgG1 Fc fusion proteins were randomly biotinylated using standard procedures provided by EZ-Link Sulfo-NHS-LC-Biotin (Thermo). ELISA was used to verify the binding activity of biotinylated BCMA-huIgG1 Fc fusion protein to the C11D5.3 chimeric antibody.

2.2 Biopanning

The antibody which is combined with the BCMA-huIgG1 Fc fusion protein (especially the extracellular domain of human B Cell Maturation Antigen (BCMA)) is obtained by using the BCMA-huIgG1 Fc fusion protein as a target protein and using biopanning to carry out biopanning on the human natural antibody library. The antibody strain library was resuscitated and grown to log phase, the antibody library was rescued using M13KO7 helper phage (NEB), centrifuged, resuspended in 2XYT medium containing ampicillin and kanamycin, and amplified overnight at 30 ℃. PEG/NaCl precipitation phage, using glycerol/PBST dissolved phage precipitation to obtain natural library phage suspension. Casein (Thermo) blocked phage input Casein blocked biotinylated PD1-huIgG1 Fc fusion protein (PD 1 part contains the 25 th to 167 th amino acid region of NCBI access No. NP-005009.2, as shown in SEQ ID NO:81, the huIgG1 Fc part differs from the huIgG1 Fc part used to construct BCMA-huIgG1 Fc fusion protein only in that it does not contain glutamic acid 99 th) and casein blocked Dynabeads M-270 streptavidin (Thermo) co-incubation system, and the supernatant phage suspension was collected. Further, the collected phage suspension was put into a casein blocked biotinylated BCMA-huIgG1 Fc fusion protein and casein blocked Dynabeads M-270 streptavidin co-incubation system and the beads were washed with PBST to remove phages that could not bind to BCMA-huIgG1 Fc fusion protein. Phage bound to the magnetic beads were eluted with 100mM triethylamine (Thermo) and then neutralized with 1M Tris-HCl (pH-6.4). 10ul of eluted phage solution was left for determination of total phage output, the remaining phage solution was used to infect logarithmically growing SS320, and after overnight amplification was considered as antibody library for the next round of panning. Biopanning was performed three times together, with BCMA-huIgG1 Fc antigen concentration starting at 100nM and screening antigen concentration decreasing at 3-fold gradient per round.

2.3 Screening of BCMA ectodomain specific binding clones

The antibody library obtained after the completion of the third biopanning was diluted and plated on plates containing tetracycline and ampicillin to obtain a monoclonal, and the monoclonal was selected and cultured overnight in a deep well plate. The deep-hole plate is repeatedly frozen and thawed three times by using a refrigerator at the temperature of-20 ℃ in the next day, and the supernatant is used for the subsequent ELISA reaction. ELISA reactions were screened for positive clones using goat anti-human IgG (Fab-specific) overnight coating, followed by addition of the centrifugal supernatant, BCMA-huIgG1 Fc fusion protein and streptavidin-HRP. This screening step was repeated twice for independent experiments to ensure data accuracy.

To exclude clones binding to the huIgG1 Fc region, the following ELISA reactions were performed: screening of huIgG1 Fc region binding positive clones was performed using goat anti-human IgG (Fab specific) overnight coating, followed by addition of the centrifugal supernatant, PD1-huIgG1 Fc fusion protein and streptavidin-HRP. This screening step was repeated twice for independent experiments to ensure data accuracy.

BCMA ectodomain specific binding clones were selected from the two above steps.

2.4 Affinity ranking Using dissociation Rate constant k _off

The freeze-thaw supernatants of the BCMA extracellular domain specific binding clones screened in the above step were analyzed using an Octet K2 (ForteBio) molecular interaction analyzer. Biotinylated BCMA-huIgG1 Fc fusion proteins were immobilized on SA probes (ForteBio) and affinity assays were performed with freeze-thaw supernatants as analytes. In the analysis step, only the dissociation curve is selected to calculate the k _off value, and the k _off value is used as a reference basis to order from small to large. Preferably, clones k _off, such as clones HK10, HA08, HD10, HA05, and HD07, are ranked first five.

Example 5: preparation of anti-human BCMA murine antibody

1. Immunization of animals

2Mg/mL BCMA-huIgG1 Fc fusion protein was mixed and emulsified as antigen with an equal volume of complete Freund's adjuvant (Sigma-Aldrich) and 56 week old female Balb/c mice (Shanghai Nannon model animal research center) were immunized subcutaneously. After the primary immunization, booster immunization was performed every ten days, four subcutaneous immunizations were performed in total, and spleen impact immunization was performed directly with BCMA-huIgG1 Fc fusion protein at the fifth immunization.

2. Serum titer detection

Blood was collected from the anterior-caudal vein after each boost for 50uL, and the cells were removed by centrifugation, leaving serum. ELISA microwells were added with 50ng of recombinant BCMA per well and coated overnight at 4 ℃. The cells were washed three times with PBS, 1% BSA/PBS was added thereto, 200 uL/well, and the cells were blocked at 37℃for 1 hour. The mouse serum was added in gradient dilution and combined at 37℃for 1 hour. PBST was washed three times, and HRP-goat anti-mouse IgG was added for binding for 1 hour at 37 ℃. PBST was washed three times, 100 uL/well TMB developing solution was added, developed at 37℃for 10 minutes, 100 uL/well ELISA stop solution was added, and the OD450 value was read by an ELISA reader.

3. Construction of an immune library

3.1 Total cDNA acquisition of mouse spleen cells

The BCMA-huIgG1 Fc fusion protein was directly used for the intraperitoneal injection for impact immunization, and four days later, mice were sacrificed and spleens were taken. The whole spleen was ground with a cell screen (BD) to obtain spleen cells. After washing twice with PBS, 1000g was centrifuged for 10 minutes to obtain spleen cells. Total RNA was extracted using Trizol RNA extraction kit.

First strand cDNA was synthesized using the SuperScript ^TM IV First-STRAND SYNTHESIS SYSTEM kit using the RNA as template.

3.2 Antibody Gene amplification and light-heavy chain splicing

The cDNA was used as a template to PCR amplify the heavy chain variable domain gene using the heavy chain variable domain upstream primer and downstream primer (VH-F, VH-R), and the light chain variable domain upstream primer and downstream primer (VK-F, VK-R) to PCR amplify the kappa chain variable domain gene. Into a 50uL reaction system, 25uL phusion master mix uL (25 pmol) of the upstream primer, 2.5uL (25 pmol) of the downstream primer, 1.5uL DMSO,0.5uL cDNA and 18uL of ddH ₂ O were added, respectively. The PCR reaction was performed according to the following procedure: after 1 minute of pre-denaturation at 98 ℃, the mixture enters a temperature cycle, denaturation at 98 ℃ for 30 seconds, annealing at 58 ℃ for 30 seconds, extension at 72 ℃ for 1 minute, 30 times of cycle and final extension at 72 ℃ for 10 minutes.

The amplified VH gene and VK gene were recovered using a DNA gel recovery kit. Equal amounts of VH gene and VK gene were mixed and used as templates, and scFv genes were amplified by overlap PCR using the upstream primer scFv-F and the downstream primer scFv-R. Into a 50uL reaction system, 25uL phusion master mix uL (25 pmol) of the upstream primer, 2.5uL (25 pmol) of the downstream primer, 1.5uL DMSO,0.5uL cDNA and 18uL of ddH ₂ O were added, respectively. The PCR reaction was performed according to the following procedure: after 1 minute of pre-denaturation at 98 ℃, the mixture enters a temperature cycle, denaturation at 98 ℃ for 30 seconds, annealing at 58 ℃ for 30 seconds, extension at 72 ℃ for 1 minute, 30 times of cycle and final extension at 72 ℃ for 10 minutes.

And (3) recovering the amplified scFv gene fragment by using a DNA gel recovery kit.

3.3 Construction of immune library

The scFv gene fragment and pcomb XTT vector (Scripps institute in the United states) were digested with SfiI DNA endonuclease, respectively. Into the 50uL reaction system, sfiI 2uL,10 Xbuffer 5uL, DNA 3ug and ddH ₂ O were added to 50uL, respectively. After thoroughly mixing, incubation was carried out at 50℃for 3 hours.

The digested scFv gene fragment and pcomb X vector were recovered using a DNA gel recovery kit. The digested scFv gene fragment was cyclized using T4 ligase and the digested pcomb X vector. In a 50uL reaction system, 1uL of T4 ligase, 5uL of 10 Xbuffer, 100ng of scFv gene, 500ng of pComb3X vector and ddH ₂ O to 50uL were added, respectively. After thoroughly mixing, incubation was carried out at 4℃for 16 hours. A small amount of the product was taken and the ligation efficiency was verified by agarose gel electrophoresis.

10UL of the ligation cyclized product was added to the homemade TGI electrotransformation competence and then subjected to electrotransformation using an electrotransformation apparatus. 10ul of the electrotransformed bacteria were removed, counted and the phage antibody library size counted by reasonable dilution and streaking on ampicillin-containing plates. The remaining electrotransformed bacteria were added to 2XYT medium containing 100ug/mL ampicillin and 2% glucose and incubated in a heated incubator. After the completion of the culture, the culture was centrifuged at 4000G for 10 minutes at 4℃to supplement the precipitant with an appropriate amount of glycerol and stored at-80℃as an antibody strain library. scFv immune libraries were obtained by multiple electrotransformation accumulation.

4. Screening and identification of murine immune antibody phage library

4.1 Biotinylation of BCMA-huIgG1 Fc fusion proteins

BCMA-huIgG1 Fc fusion proteins were randomly biotinylated using standard procedures provided by EZ-Link Sulfo-NHS-LC-Biotin. ELISA was used to verify the binding activity of biotinylated BCMA-huIgG1 Fc fusion protein to the C11D5.3 chimeric antibody.

4.2 Biopanning

The antibody binding to the BCMA-huIgG1 Fc fusion protein (especially the extracellular domain of human B Cell Maturation Antigen (BCMA)) is obtained by biopanning the murine immune antibody library by using the BCMA-huIgG1 Fc fusion protein as a target protein and applying biopanning. The antibody strain library was resuscitated and grown to log phase, the antibody library was rescued using M13KO7 helper phage, centrifuged, resuspended in 2XYT medium containing ampicillin and kanamycin and amplified overnight at 30 ℃. PEG/NaCl precipitation phage, using glycerol/PBST dissolved phage precipitation to obtain immune library phage suspension. The casein blocked phage were placed into a casein blocked biotinylated PD1-huIgG1 Fc fusion protein and casein blocked Dynabeads M-270 streptavidin co-incubation system and the supernatant phage suspension was collected. Further, the collected phage suspension was put into a casein blocked biotinylated BCMA-huIgG1 Fc fusion protein and casein blocked Dynabeads M-270 streptavidin co-incubation system and the beads were washed with PBST to remove phages that could not bind to BCMA-huIgG1 Fc fusion protein. Phage bound to the beads were eluted with 100mM triethylamine and then neutralized with 1M Tris-HCl (pH=6.4). 10ul of eluted phage solution was left for determination of total phage output, the remaining phage solution was used to infect log-growing TG1, and after overnight amplification was considered as antibody library for the next round of panning. Biopanning was performed in two rounds with BCMA-huIgG1 Fc antigen concentrations of 50nM and 5nM, respectively.

4.3 Screening of extracellular Domain-specific binding clones of BCMA

The antibody library obtained after the second round of biopanning was diluted and spread on ampicillin-containing plates to obtain monoclonal antibodies, and the monoclonal antibodies were selected and cultured overnight in deep well plates. The deep-hole plate is repeatedly frozen and thawed three times by using a refrigerator at the temperature of-20 ℃ in the next day, and the supernatant is used for the subsequent ELISA reaction. ELISA reactions were screened for positive clones using goat anti-human IgG (Fab-specific) overnight coating, with sequential addition of supernatant, biotinylated BCMA-huIgG1 Fc fusion protein and streptavidin-HRP. This screening step was repeated twice for independent experiments to ensure data accuracy.

To exclude clones binding to the huIgG1 Fc region, the following ELISA reactions were performed. Screening of huIgG1 region binding positive clones was performed using goat anti-human IgG (Fab specific) overnight coating, sequentially adding supernatant, biotinylated PD1-huIgG1 Fc fusion protein and streptavidin-HRP. This screening step was repeated twice for independent experiments to ensure data accuracy.

BCMA ectodomain specific binding clones were selected from the two above steps.

4.4 Affinity ranking Using dissociation Rate constant k _off

The freeze-thaw supernatant of the BCMA extracellular domain specific binding clone screened in the above step was analyzed using an Octet K2 molecular interaction analyzer. Biotinylated BCMA-huIgG1 Fc fusion protein was immobilized on SA probes and affinity assays were performed using freeze-thaw supernatants as analytes. In the analysis step, only the dissociation curve is selected to calculate the k _off value, and the k _off value is used as a reference basis to order from small to large. Preferably, clones k _off, such as clones MC12, MB09, MB11, MA04, and MD07, order the top five.

Example 6: preparation of chimeric antigen receptor-T cells against human BCMA and determination of chimeric antigen receptor positive rate

1. Preparation of lentivirus packaging master plasmid containing CAR element

The antibody sequences of clones MB09, MC12, MD07 and HK10 and clone C11D5.3 were selected to construct chimeric antigen receptors. Construction of a CAR element lentiviral packaging master plasmid comprising the following structure from N-terminal to C-terminal by molecular cloning: a CD8 a signal peptide, an scFv antibody, a CD8 a hinge region, a CD8 a transmembrane region, a 41BB cytoplasmic region, and a CD3z cytoplasmic region. Clones sequenced correctly were selected, inoculated with bacterial solution into 300ml of 2YT medium, shaken overnight and the large plasmid was completed according to NucleoBond Xtra Maxi EF kit instructions.

2. Lentivirus package

Packaging the lentivirus-containing package with cationic polymer PEI, the procedure is as follows: PEI and lentiviral packaging plasmids (lentiviral master plasmid, RRE-SIV, REV, VSVG) were diluted with serum-free DMEM, respectively; adding PEI/DMEM into the plasmid/DMEM mixture, vortex shaking and mixing uniformly, and standing for 15 minutes at room temperature; the plasmid-PEI complex was added to pre-plated 293T cells (China academy of sciences cell bank). The liquid is changed 16h after transfection, virus supernatant is collected 48h later, filtered by a 0.45um filter, partial stock solution is reserved, and the rest is concentrated 30 times.

3. Virus titer assay

3.1 Biological titer determination

The biological titer of a virus solution refers to the number of infectious virus particles contained per milliliter.

Taking a 24-hole culture plate, adding a gradient diluted virus stock solution into each hole, wherein the initial infection volume is 1ml, the ratio of three times, five gradient dilutions, the volume is less than 1ml, and supplementing 1ml with a culture medium. After each well 100 μl of a cell suspension containing 1x10 ⁵ 293T cells (polybrene (Santa Cruz) at a final concentration of 8 μg/ml) was added, and 3 days later the efficiency of cell infection was measured by flow cytometry, the first staining using biotin-goat anti-mouse Fab (or biotin-goat anti-human IgG Fab), the second staining using PE-streptavidin to label the test cells, and uninfected, simultaneous culture of 293T cells as a negative control (293T). The titer calculation formula is: titer (TU/ml) =number of infected cells x flow assay CAR expression ratio/virus solution volume. The titer should be no less than 1X10 ⁵ TU/ml.

The biological titer determination results were: the biological titer of the lentiviral stock solution containing the MB09 antibody is 24.6x10 ⁶ TU/ml, the biological titer of the lentiviral stock solution containing the MC12 antibody is 3.06x10 ⁶ TU/ml, the biological titer of the lentiviral stock solution containing the MD07 antibody is 2.10x10 ⁶ TU/ml, the biological titer of the lentiviral stock solution containing the HK10 antibody is 8.82x10 ⁶ TU/ml, and the biological titer of the lentiviral stock solution containing the C1 1D5.3 antibody is 2.65x10 ⁶ TU/ml.

3.2 Physical titre determination

The physical titer of the virus solution is determined by the number of virus particles in the virus solution, including virus particles with infectious capability and defective virus particles without infectious capability. Lentiviral genetic material is known to be two single-stranded RNA genomes, and physical titer detection takes the CAR gene copy number in virus liquid as an evaluation index, and theoretically, two CAR gene copy numbers correspond to one virus particle.

Specifically, the present experiment uses the 41BB sequence of lentiviral gene as the amplification target to determine the physical titer of lentivirus. The virus supernatant was first DNA-treated and then subjected to one-step RT-PCR (TaKaRa) using 41BB specific fluorescent quantitative PCR primers to determine the copy number of the 41BB gene to determine the lentiviral genome copy number.

The physical titer test results were: the physical titer of the lentiviral stock solution containing the MB09 antibody is 4.65x10 ⁶ copies/ul, the physical titer of the lentiviral stock solution containing the MC12 antibody is 5.99x10 ⁶ copies/ul, the physical titer of the lentiviral stock solution containing the MD07 antibody is 6.89x10 ⁶ copies/ul, the physical titer of the lentiviral stock solution containing the HK10 antibody is 3.21x10 ⁶ copies/ul, and the physical titer of the lentiviral stock solution containing the C11D5.3 antibody is 4.26x10 ⁶ copies/ul.

4. Preparation of chimeric antigen receptor-T cells and determination of chimeric antigen receptor-positive Rate

4.1CD3+T cell isolation and activation

Relatively pure CD3+ T cells were isolated using Ficcol% AB serum X-VIVO (LONZA) medium to a cell density of 1X10 ⁶/mL. Cells were inoculated at 1 ml/well into cells previously infected with anti-human 50ng/ml CD3 antibody (Beijing co-dried sea cell) and 50ng/ml CD28 antibody (Beijing co-dried sea cell), followed by addition of 100IU/ml IL2 (Beijing Shuanglu), and after 48 hours of culture stimulation.

4.2 Infection with Virus stock and culture

The activated T cells were adjusted to 5X10 ⁵/mL, 1mL of T cells and 1mL of virus stock were added to each well of a 24-well plate, and 1ul polybrene was added to each well, and centrifuged at 32℃and 2500rpm for 1.5 hours. The supernatant was discarded and 1ml of T cell medium (containing IL-250 IU/ml) was added to each well. The plates were placed in a 37℃5% CO ₂ incubator for cultivation. 24h after infection, transferring to a 6-hole plate, observing the density of cells every day, and timely supplementing a T cell culture solution containing IL-250IU/ml to maintain the density of the T cells to be about 5x10 ⁵/ml so as to expand the cells.

4.3CAR Positive Rate detection

CAR positive rate was detected 72h after virus infection. The chimeric antigen receptor group containing MB09, MC12, MD07, C11D5.3 clone and the negative control group are selected from biotin-goat anti-mouse IgG F (ab') 2 fragment as a primary antibody to incubate cells after lentivirus infection. The cells after lentiviral infection were incubated with biotin-goat anti-human IgG F (ab') 2 fragments as primary antibodies against the chimeric antigen receptor group containing the HK10 clone and the negative control group. After this time, the cells were incubated with Brilliant Violet 421. TM. Streptavidin (BioLegend) secondary antibody, and the 5 CAR-T positives were all substantially identical, at about 80%.

Example 7: functional verification of anti-human BCMA chimeric antigen receptor-T cells

1. Target cell construction

1.1 Packaging of lentiviruses

Luciferase and green fluorescent protein genes in tandem with Internal Ribosome Entry Sites (IRES) were synthesized by chemical synthesis. A lentivirus packaging master plasmid (PCCL-LUC-GFP) was constructed by molecular cloning and lentiviruses were packaged.

1.2K562-LUC-GFP and L363-LUC-GFP target cell construction

K562 (BCMA is not expressed, cell bank of China academy of sciences) and L363 (BCMA is expressed, cell bank of China academy of sciences) are respectively transfected by corresponding lentiviruses, and then the K562-LUC-GFP target cells and the L363-LUC-GFP target cells which are high in expression of luciferase and green fluorescent protein are screened out by taking green fluorescent protein as a marker.

2. Anti-human BCMA chimeric antigen receptor-T cell killing experiments

CAR-T killing experiments CAR-T cell in vitro function was assessed by detecting the killing effect of CAR-T cells on target cells in vitro. T cells were co-cultured with K562-LUC-GFP target cells and L363-LUC-GFP target cells, respectively, at different potency target ratios (3 x10 ⁴ target cells with 1.5x10 ⁵ effector cells and 3x10 ⁴ target cells with 6x10 ⁴ effector cells), while negative control groups of target cells mixed with untransfected CAR element T cells were set. After 18 hours, a luciferase reaction substrate was added to the culture system, and the fluorescence value was detected, and the killing efficiency was calculated by the following formula: killing efficiency = (1-experimental Kong Yingguang value/control Kong Yingguang value) x100%. The experimental groupings and results are shown in the following table.

In the cell killing experiments, 5 CAR-T and negative control T cells were essentially non-functional for K562. MB09, MC12, HK10 clones performed well for L363, with small differences in value compared to the positive control clone C11D5.3, but with weak MD07 clone. The results are shown in FIG. 1.

3. Anti-human BCMA chimeric antigen receptor-T cell IFN-gamma secretion and CD107a expression assay

After co-incubation of effector cells (CAR-T and negative control T cells without transfected CAR elements) with target cells (both effector and target cells are 3x10 ⁵), their IFN- γ and CD107a expression were flow tested to evaluate CAR-T cell effector function in vitro after stimulation by target cells. The experimental groups are shown in the table, and after the effector cells and the target cells are mixed and placed in a 37 ℃ and co-incubated in a 5% CO ₂ incubator for 4 hours, the proportion of cells secreting IFN-gamma and expressing CD107a in the CD3+ cells in each group of samples is detected, and the experimental groups and the results are shown in the table below.

Group of	CAR-T clone number	Target cells	CD3+IFN-γ+	CD3+CD107a+
					1	MB09	K562	1.30％	0.91％
2	MC12	K562	2.51％	0.78％
					3	MD07	K562	7.97％	6.83％
4	C11D5.3	K562	2.45％	0.82％
					5	HK10	K562	2.54％	0.83％
6	NT	K562	1.33％	0.68％
					7	MB09	L363	19.30％	26.90％
8	MC12	L363	18.90％	25.00％
					9	MD07	L363	11.60％	7.25％
10	C11D5.3	L363	24.80％	34.00％
					11	HK10	L363	34.40％	44.60％
12	NT	L363	2.45％	0.50％

After co-incubation of CAR-T cells with target cells, MD07 clones stimulated non-specifically for K562 and L363, and both IFN- γ and CD107a expression were seen in CD3 positive cells. The remaining CAR-T and negative control T cells were non-functional for K562. HK10 was functionally stronger for L363 than the positive control clone C11D5.3, with MB09, MC12 being slightly weaker than the control. The results are shown in FIGS. 2-4.

Claims

1. An antibody or antigen-binding fragment that specifically binds to human B Cell Maturation Antigen (BCMA), comprising a heavy chain variable domain and a light chain variable domain, said heavy chain variable domain and light chain variable domain comprising:

(5) SEQ ID NO:25, a heavy chain CDR1, SEQ ID NO:26, a heavy chain CDR2, SEQ ID NO:27, heavy chain CDR3, SEQ ID NO:28, light chain CDR1, SEQ ID NO:29 and the light chain CDR2 and SEQ ID NO:30, and a light chain CDR3.

2. An antibody or antigen binding fragment that specifically binds to human B Cell Maturation Antigen (BCMA), comprising a heavy chain variable domain and a light chain variable domain comprising:

3. A chimeric antigen receptor comprising an extracellular region, a transmembrane region, and an intracellular region, wherein the extracellular region comprises an antigen binding region comprising a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain and the light chain variable domain comprising:

(5) SEQ ID NO:25, a heavy chain CDR1, SEQ ID NO:26, a heavy chain CDR2, SEQ ID NO:27, heavy chain CDR3, SEQ ID NO:28, light chain CDR1, SEQ ID NO:29 and the light chain CDR2 and SEQ ID NO:30, a light chain CDR3;

4. The chimeric antigen receptor according to claim 3, wherein the extracellular region further comprises a polypeptide having the amino acid sequence of SEQ ID NO:63, and a hinge region of the amino acid sequence shown in seq id no.

5. The chimeric antigen receptor according to claim 3 or 4, wherein the transmembrane region has the amino acid sequence of SEQ ID NO: 65.

6. The chimeric antigen receptor according to any one of claims 3 to 5, wherein the intracellular region has the sequence of SEQ ID NO:67 and/or the amino acid sequence shown in SEQ ID NO: 69.

7. A polynucleotide encoding an antibody or antigen binding fragment according to claim 1 or 2 or a chimeric antigen receptor according to any one of claims 3 to 6.

8. An engineered immune effector cell characterized in that it expresses the chimeric antigen receptor according to any one of claims 3 to 6, or comprises a polynucleotide encoding the chimeric antigen receptor according to any one of claims 3 to 6.

9. Use of an antibody or antigen binding fragment according to claim 1 or 2, a chimeric antigen receptor according to any one of claims 3 to 6, a polynucleotide according to claim 7, or an engineered immune effector cell according to claim 8 in the manufacture of a medicament for the treatment of cancer.

10. Use of an antibody or antigen binding fragment according to claim 1 or 2, a chimeric antigen receptor according to any one of claims 3 to 6, a polynucleotide according to claim 7, or an engineered immune effector cell according to claim 8 in the manufacture of a medicament for stimulating immune function in a patient with cancer.