WO2021031113A1 - Anti-bcma antibody and use thereof in car-t field - Google Patents

Abstract

The present invention relates to the field of biomedicine, in particular to an anti-BCMA antibody and the use thereof in the CAR-T field. The anti-BCMA antibody comprises at least one of the following: (1) a heavy chain variable region with amino acid sequences as shown in GYTFTSYV, IIPYNDDT and ARWNYDGYFDV, and a light chain variable region with amino acid sequences as shown in QSLVHSNGNTY, YKVS and SQITHVPYT; and an amino acid sequence with at least one conservative amino acid substitution compared with point (1). The anti-BCMA antibody can specifically bind to a BCMA antigen, and therefore same is used for targeted therapy of tumors.

Description

Anti-BCMA antibody and its application in CAR-T field Technical field

The present invention relates to the field of biomedicine, in particular to an anti-BCMA antibody and its application in the CAR-T field.

Background technique

Chimeric antigen receptor T cell (CAR-T, Chimeric antigen receptor T cell) refers to a T cell into which chimeric antigen receptor (CAR, Chimeric antigen receptor) genes are transferred through gene modification technology. CAR-T immunotherapy belongs to adoptive immune cell therapy. The peripheral blood lymphocytes of the patient are extracted and modified in vitro to express the CAR gene, and then amplified in vitro and then transferred back to the patient. The modified CAR-T cells can be MHC Recognize and activate specific antigens on the surface of tumor cells in a non-restricted way, and a large number of them are amplified in the body. The tumor cells are directly killed by releasing perforin and granzyme B, and at the same time by releasing cytokines IFN-γ, IL-2, etc. Recruit human endogenous immune cells to kill tumor cells, so as to achieve the purpose of treating tumors.

The key factor for CAR-T cells to function is the transferred chimeric antigen receptor (CAR) gene. CAR is a fusion protein, including extracellular target binding domain, extracellular hinge domain, transmembrane domain and intracellular domain. The extracellular target binding region is usually derived from the single-chain variable fragment (scFv, Single-chain variable fragment) of an antibody that recognizes tumor-associated antigen (TAA, Tumor-associated antigen), and its binding affinity to tumor antigen is much higher than that of MHC polypeptide The affinity of the complex with T cell receptor (TCR, T cell receptor) improves the response ability of T cells. The sequence of the extracellular hinge region is derived from the constant region of IgG or CD8, etc. Its sequence and length have varying degrees of influence on the binding of scFv to the target, and are related to the epitope of the binding target. The transmembrane region generally comes from the transmembrane region of receptors such as CD8, CD28, or CD3, which has a certain impact on the stability of the CAR structure.

The use of Car-T cells for tumor immunotherapy needs further improvement.

Summary of the invention

The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, an object of the present invention is to provide an anti-BCMA antibody and its application in the CAR-T field. For example, the present invention provides an anti-BCMA antibody or antigen-binding fragment, isolated polynucleotides, expression vectors, recombinant cells, kits containing anti-BCMA antibodies or antigen-binding fragments, pharmaceutical compositions, and an anti-BCMA Chimeric antigen receptors and Car-T cells, etc., also provide a method for treating tumors in the subject.

B-cell maturation antigen (BCMA, B-cell maturation antigen), also known as CD269, is a member of the tumor necrosis factor (TNF, Tumor necrosis factor) receptor family, and can be combined with B-cell activating factor (BAFF, B-cell activating factor). ) Or proliferation inducing ligand (APRIL, A proliferation inducing ligand). BCMA is mainly expressed on the surface of normal and diseased plasma cells, and some B cell subgroups, while BCMA RNA or protein expression cannot be detected in hematopoietic stem cells and cells of major human organs. BCMA is a promising target in the treatment of multiple myeloma. A number of CAR-T treatment clinical trials using targeted BCMA have reported relatively high remission rates. At the 2017 American Clinical Oncology Conference (ASCO), Nanjing Legend reported the clinical data of a CAR-T therapy targeted to BCMA. According to data, in a clinical trial involving 35 patients with relapsed or drug-resistant multiple myeloma, the objective remission rate of this therapy reached 100%. Among the 19 patients who received treatment at the earliest, Nanjing Legend has been observed and followed up for more than 4-14 months. Among them, 14 patients continued to meet the strict diagnostic criteria for complete response, and 5 patients experienced partial remission. At the ASCO annual meeting in 2018, Celgene and bluebird bio announced the latest data of their CAR-T therapy bb2121 at the 2018 ASCO conference. The test results showed that this CAR-T therapy is late recurrent. Patients with refractory multiple myeloma brought deep and lasting relief. Among the 43 patients who received different doses of CAR-T treatment, the overall remission rate reached 33%-95%, especially among the 18 patients who received high-dose CAR-T infusion, the overall remission rate reached 94%, with a median The progression-free survival (PFS) reached 11.8 months. CAR-T therapy targeting BCMA is expected to become the second CAR-T therapy approved after targeting CD19CAR-T. The screening of antibodies that can target BCMA has also become the key to Car-T therapy.

To this end, according to the present invention, an anti-BCMA antibody or antigen-binding fragment thereof, as well as isolated nucleotides, expression vectors, recombinant cells, etc. are provided. The anti-BCMA antibody or its antigen-binding fragment provided by the present invention can specifically bind to the BCMA antigen. It can be applied to the targeted therapy of tumors, or made into a kit for specific detection of BCMA antigen, etc., which has important value.

Specifically, this application provides the following technical solutions:

According to the first aspect of the present invention, the present invention provides an anti-BCMA antibody or antigen-binding fragment, comprising at least one of the following: (1) a heavy chain variable region having the amino acid sequence shown in GYTFTSYV, IIPYNDDT and ARWNYDGYFDV, and The light chain variable region of the amino acid sequence shown in QSLVHSNGNTY, YKVS and SQITHVPYT; compared with (1), an amino acid sequence with at least one conservative amino acid substitution.

In some embodiments of the present invention, the aforementioned anti-BCMA antibody or antigen-binding fragment may further include the following technical features:

In some embodiments of the present invention, the anti-BCMA antibody or antigen-binding fragment includes at least one of the following: (a) a heavy chain variable region having an amino acid sequence shown in SEQ ID NO: 1 and a heavy chain variable region shown in SEQ ID NO: 2. The variable region of the light chain showing the amino acid sequence; compared with (a), the amino acid sequence has at least one conservative amino acid substitution.

According to the second aspect of the present invention, the present invention provides an isolated polynucleotide encoding the antibody or antigen-binding fragment of the first aspect of the present invention.

According to an embodiment of the present invention, the isolated polynucleotide described above may further include the following technical features:

In some embodiments of the present invention, the polynucleotide is a nucleotide sequence having at least one of the following: the heavy chain variable region nucleotide sequence shown in SEQ ID NO: 3 and the nucleotide sequence shown in SEQ ID NO: 4 The nucleotide sequence of the variable region of the light chain shown in SEQ ID NO: 3, a sequence with more than 90% homology, optionally with more than 95% homology The original sequence is preferably a sequence with more than 98% homology, and more preferably a sequence with more than 99% homology; compared with the nucleotide sequence of the light chain variable region shown in SEQ ID NO: 4 , A sequence with 90% or more homology, optionally a sequence with 95% or more homology, preferably a sequence with 98% or more homology, and more preferably a sequence with 99% or more homology.

According to the third aspect of the present invention, the present invention provides an expression vector comprising the polynucleotide according to the second aspect of the present invention.

According to an embodiment of the present invention, the above-mentioned expression vector may further include the following technical features:

In some embodiments of the present invention, the above-mentioned expression vector may further include a control element operably linked to the polynucleotide for controlling the expression of the polynucleotide in the host cell.

In some embodiments of the present invention, the control element includes at least one of the following: a promoter, an enhancer, and a terminator.

In some embodiments of the present invention, the host cell is a mammalian cell.

According to the fourth aspect of the present invention, the present invention provides a recombinant cell comprising the expression vector according to the third aspect of the present invention.

According to the fifth aspect of the present invention, the present invention provides a kit comprising the antibody or antigen-binding fragment according to the first aspect of the present invention.

In some embodiments of the present invention, the kit is used for the diagnostic detection of BCMA antigen. The antibodies or antigen-binding fragments in the kit can be used for the diagnosis and detection of BCMA antigen, for example, it can be applied to immunoblotting, immunoprecipitation or ELISA detection and so on.

According to the sixth aspect of the present invention, the present invention provides a pharmaceutical composition, comprising: the antibody or antigen-binding fragment of the first aspect of the present invention and a pharmaceutically acceptable carrier.

According to the seventh aspect of the present invention, the present invention provides the use of an antibody or antigen-binding fragment in the preparation of a medicament for the treatment of tumors, and the antibody or antigen-binding fragment is the one described in the first aspect of the present invention Antibody or antigen-binding fragment.

In some embodiments of the present invention, the tumor is multiple myeloma.

According to the eighth aspect of the present invention, the present invention provides an anti-BCMA chimeric antigen receptor, comprising: an extracellular target binding region, an extracellular hinge region, a transmembrane region and an intracellular region, the extracellular target binding Region, the extracellular hinge region, the transmembrane region, and the intracellular region are sequentially connected; the extracellular target binding region includes an antigen or antigen-binding fragment, and the antibody or antigen-binding fragment is a single chain, The antibody or antigen-binding fragment is the antibody or antigen-binding fragment described in the first aspect of the present invention.

According to an embodiment of the present invention, the above-mentioned anti-BCMA chimeric antigen receptor may further include the following technical features:

In some embodiments of the present invention, the antibody or antigen-binding fragment is a single-chain fragment shown in SEQ ID NO: 5.

In some embodiments of the present invention, the extracellular hinge region further includes a human CD8 extracellular hinge region, and the amino acid sequence of the human CD8 extracellular hinge region is preferably as shown in SEQ ID NO: 6.

In some embodiments of the present invention, the transmembrane region is a human CD28 transmembrane region, and the amino acid sequence of the human CD28 transmembrane region is preferably as shown in SEQ ID NO:7.

In some embodiments of the present invention, the intracellular region includes a human CD28 intracellular region and a human CD3ζ intracellular region. The amino acid sequence of the human CD28 intracellular region is preferably as shown in SEQ ID NO: 8, the human CD3ζ The amino acid sequence of the intracellular region is preferably as shown in SEQ ID NO: 9.

In some embodiments of the present invention, the nucleic acid sequence encoding the antibody or antigen-binding fragment is shown in SEQ ID NO: 10; the nucleic acid sequence encoding the human extracellular hinge region is shown in SEQ ID NO: 11; encoding The nucleic acid sequence of the human CD28 transmembrane region and the human CD28 intracellular region is shown in SEQ ID NO: 12; the nucleotide sequence encoding the human CD3ζ intracellular region is shown in SEQ ID NO: 13.

According to the ninth aspect of the present invention, the present invention provides a Car-T cell that expresses the anti-BCMA chimeric antigen receptor of the eighth aspect of the present invention. The antibody or antigen-binding fragment provided by the present invention is used as a single-chain antibody to perform gene recombination in vitro with the extracellular hinge region, transmembrane region and intracellular region, such as immunoreceptor tyrosine activation motif protein (which can be CD3ζ), Recombinant plasmids are generated, and then transfected into the patient’s T cells by transfection technology in vitro to allow the patient’s T cells to express tumor antigen receptors. After transfection, the purified and large-scale amplified T cells are called chimeric antigen receptors. Somatic T cells (Car-T cells). Infusing these Car-T cells back into the patient's body can achieve the therapeutic effect of identifying and killing cancer cells, just like installing a GPS navigation system on the patient's T cells to accurately identify and kill cancer cells.

According to the tenth aspect of the present invention, the present invention provides a method for treating tumors in a subject, comprising administering to the subject an effective amount of Car-T cells, the Car-T cells being the ninth aspect of the present invention Car-T cells.

In some embodiments of the present invention, the tumor is multiple myeloma. Multiple myeloma (MM, Multiple myeloma) is a malignant B-cell lymphoma caused by malignant clonal proliferation of bone marrow plasma cells, accompanied by monoclonal immunoglobulin or light chain (M protein) overproduction. The common clinical manifestations are Anemia, bone pain, renal insufficiency, infection, bleeding, neurological symptoms, hypercalcemia, amyloidosis, etc. Traditional treatment methods such as chemotherapy, radiotherapy, and corticosteroids can alleviate the condition, but it will almost eventually relapse, and the survival rate within five years is only 20-30%. Although the transplantation of hematopoietic stem cells can remove myeloma cells, the difficulty and risk of surgery are not suitable for patients of all ages. The immunotherapy of Car-T technology can effectively treat multiple myeloma and has broad application prospects.

The additional aspects and advantages of the present invention will be partly given in the following description, and part of them will become obvious from the following description, or be understood through the practice of the present invention.

Description of the drawings

Fig. 1 is a graph showing the binding and dissociation curves of BCMA antigen and the biosensor loaded with mouse anti-human BCMA antibody at 25, 50, and 100 nM concentration after homogenization with a negative control sample provided by an embodiment of the present invention.

Fig. 2 is a flow cytometry result diagram of an anti-BCMA antibody provided according to an embodiment of the present invention after incubation with the MM.1S cell line.

Fig. 3 is a flow cytometry result diagram after incubating the anti-BCMA antibody and the RPMI8226 cell line according to an embodiment of the present invention.

Fig. 4 is a flow cytometry result diagram of anti-BCMA antibodies after incubation with negative control K562 cells according to an embodiment of the present invention.

Figure 5 is a flow cytometer according to an embodiment of the present invention to detect the expression of CAR protein on the surface of T cells after infection.

Fig. 6 is a graph showing the secretion results of IFN-gamma after different cells are co-cultured with three MM target cells and negative control cells according to an embodiment of the present invention.

Fig. 7 is a graph showing the secretion of IL-2 after different cells are co-cultured with three MM target cells and negative control cells according to an embodiment of the present invention.

Fig. 8 is a graph showing the killing effect of different cells on CHO-BCMA cells according to an embodiment of the present invention.

Fig. 9 is a graph showing the killing effect of different cells on negative CHO cells according to an embodiment of the present invention.

Fig. 10 is a graph showing changes in tumor size in mice after injection of different cells according to an embodiment of the present invention.

detailed description

The embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings, in which the same or similar reference numerals indicate the same or similar elements or elements with the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary, and are intended to explain the present invention, but should not be construed as limiting the present invention.

antibody

Herein, the term "antibody" is an immunoglobulin molecule capable of binding to a specific antigen. It includes two light chains with a lighter molecular weight and two heavy chains with a heavier molecular weight. The heavy chain (H chain) and the light chain (L chain) are connected by disulfide bonds to form a tetrapeptide chain molecule. Among them, the amino terminal (N-terminal) amino acid sequence of the peptide chain has great changes, called the variable region (V region), and the carboxyl terminal (C-terminal) is relatively stable with little change, and is called the constant region (C region). The V regions of the L chain and H chain are called VL and VH, respectively.

In the variable region, the amino acid composition and arrangement sequence of certain regions have a higher degree of variation, which is called hypervariable region (Hypervariable region, HVR). The hypervariable region is the position where antigen and antibody bind, so it is also called determinant complementation Complementarity-determining region (CDR). There are three CDR regions on both the heavy chain variable region and the light chain variable region.

The anti-BCMA antibody provided by the present invention can specifically bind to BCMA antigen, and can be used to prepare BCMA CAR-T cells to realize targeted therapy of tumors.

In some embodiments, the present invention provides an anti-BCMA antibody or antigen-binding fragment. The CDR regions on the heavy chain variable region of the antibody or antigen-binding fragment are respectively CDR1 being GYTFTSYV, CDR2 being IIPYNDDT and CDR3 being ARWNYDGYFDV; The CDR regions on the light chain variable region are respectively CDR1 for QSLVHSNGNTY, CDR2 for YKVS and CDR3 for SQITHVPYT. "Antigen-binding fragment" herein refers to an amino acid fragment that has the ability to specifically bind to BCMA antigen. Herein, anti-BCMA antibodies can also be referred to as BCMA antibodies, both of which refer to immunoglobulin molecules that can bind to BCMA antigen.

In some embodiments, the antibody or antigen-binding fragment has a heavy chain variable region shown in SEQ ID NO: 1 and a light chain variable region shown in SEQ ID NO: 2. In other embodiments, the heavy chain variable region sequence of the antibody or antigen-binding fragment has more than one conservative amino acid substitution, such as one conservative amino acid substitution, compared with the amino acid sequence shown in SEQ ID NO:1. In some embodiments, the light chain variable region sequence of the antibody or antigen-binding fragment has more than one conservative amino acid substitution compared to the amino acid sequence shown in SEQ ID NO: 2, for example, has one conservative amino acid substitution, and has two A conservative amino acid substitution, or even three conservative amino acid substitutions. Of course, these conservative amino acid substitutions will not change the biological function of the antibody or antigen-binding fragment. In some embodiments, these conservative amino acid substitutions can occur on amino acids other than the CDR regions in the heavy chain variable region and the light chain variable region.

As used herein, "conservative amino acid substitution" refers to the substitution of an amino acid with a residue that is biologically, chemically or structurally similar to another amino acid. Biologically similar means that the substitution does not destroy the biological activity of the BCMA antibody or the BCMA antigen. Structurally similar means that amino acids have side chains of similar length, such as alanine, glycine or serine, or side chains of similar size. Chemical similarity means that the amino acids have the same charge or are both hydrophilic or hydrophobic. For example, the hydrophobic residues isoleucine, valine, leucine or methionine are substituted for each other. Or use polar amino acids such as arginine for lysine, glutamic acid for aspartic acid, glutamine for asparagine, serine for threonine and so on.

Among them, the amino acid sequence of the variable region of the heavy chain (SEQ ID NO:1)

Light chain variable region amino acid sequence (SEQ ID NO: 2)

Polynucleotide, expression vector, recombinant cell

In the process of preparing or obtaining these antibodies, polynucleotides expressing these antibodies can be connected to different vectors to obtain corresponding antibodies.

To this end, the present invention also provides an isolated polynucleotide encoding the antibody or antigen-binding fragment described above.

The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 of the variable region of the heavy chain is (SEQ ID NO: 3):

The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2 of the light chain variable region is (SEQ ID NO: 4):

In some embodiments, the isolated polynucleotide has at least 90% or more homology with the nucleotide sequence shown in SEQ ID NO: 3, preferably more than 95% homology, more preferably 98%, 99% or more homology. In at least some embodiments, the isolated polynucleotide has at least more than 90% homology, preferably more than 95%, compared with the nucleotide sequence shown in SEQ ID NO: 4 above , More preferably 98%, 99% or more homology. These sequences that have homology with the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 can express amino acids similar to SEQ ID NO: 1 and SEQ ID NO: 2, so that they can be specific to the BCMA antigen Sexual binding to achieve the targeted function of antibodies.

The present invention also provides an expression vector comprising the above-mentioned isolated polynucleotide. When the isolated polynucleotide is connected to the vector, the polynucleotide can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the polynucleotide. Of course, these control elements can come directly from the carrier itself, or they can be exogenous, that is, not from the carrier itself. Of course, the polynucleotide can be operably linked to the control element. In this context, "operably linked" refers to the connection of a foreign gene to a vector, so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their intended role in regulating the transcription and translation of the foreign gene. Function. Of course, the polynucleotides used to encode the heavy chain and light chain of an antibody can be inserted into different vectors independently, and it is common to insert into the same vector. Commonly used vectors can be, for example, plasmids, phages, lentiviruses, and so on.

The present invention also provides a recombinant cell, which contains the expression vector. The expression vector can be introduced into mammalian cells to construct recombinant cells, and then use these recombinant cells to express the antibodies or antigen-binding fragments provided by the present invention. By culturing the recombinant cells, corresponding antibodies can be obtained. These usable mammalian cells can be, for example, 293F cells, CHO cells and the like.

Pharmaceutical compositions, kits, pharmaceutical uses and uses in the preparation of kits.

The present invention also provides a pharmaceutical composition, which comprises the aforementioned antibody or antigen-binding fragment and a pharmaceutically acceptable carrier.

The anti-BCMA antibodies provided herein can be incorporated into pharmaceutical compositions suitable for administration to a subject. Generally, these pharmaceutical compositions include the anti-BCMA antibodies provided herein and a pharmaceutically acceptable carrier. The "pharmaceutically acceptable carrier" may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Specific examples may be one or more of water, saline, phosphate buffered saline, glucose, glycerol, ethanol, etc., and combinations thereof. In many cases, isotonic agents are included in the pharmaceutical composition, such as sugars, polyalcohols (such as mannitol, sorbitol), or sodium chloride. Of course, the pharmaceutically acceptable carrier may also include minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, to extend the shelf life or efficacy of the antibody.

For example, the antibodies of the invention may be incorporated into pharmaceutical compositions suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These pharmaceutical compositions can be prepared in various forms. For example, liquid, semi-solid and solid dosage forms, including but not limited to liquid solutions (for example, injection solutions and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. Typical pharmaceutical compositions are in the form of injection solutions or infusion solutions. The antibody can be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.

Of course, the anti-BCMA antibodies herein can also be made into a part of kits or other diagnostic reagents as needed. According to an embodiment of the present invention, the present invention also provides a kit including the above-mentioned BCMA antibody. Using the kit provided by the present invention, for example, it can be used for immunoblotting, immunoprecipitation, etc., which involve the use of the specific binding properties of BCMA antigen and antibody for detection. These kits may contain any one or more of the following: antagonists, anti-BCMA antibodies or drug reference materials; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents; instructions or literature, etc. Anti-BCMA antibodies can be used in different types of diagnostic tests, for example, it can detect the presence of various diseases or drugs, toxins or other proteins in vitro or in vivo. For example, it can be used to test related diseases by testing the subject's serum or blood. Such related diseases may include BCMA related diseases, such as various cancers or tumors. Of course, the antibodies provided herein can also be used for radioimmunoassay and radioimmunotherapy of cancer.

These cancers or tumors can be any unregulated cell growth. Specifically, it may be lung cancer, stomach cancer, pancreatic cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, and so on.

When using the anti-BCMA antibody provided by the present invention to treat cancer, the anti-BCMA antibody provided by the present invention can be provided to the subject. To this end, the present invention provides a method for treating cancer, which comprises administering the antibody or antigen-binding fragment thereof provided by the present invention to a subject in need.

The solution of the present invention will be explained below in conjunction with examples. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the examples, the procedures shall be carried out in accordance with the techniques or conditions described in the literature in the field or in accordance with the product specification. The reagents or instruments used without the manufacturer's indication are all conventional products that are commercially available.

Through the following research, this application has developed antibody-based immunotherapy methods based on BCMA antigens against multiple tumor cells and even tumor stem cells, including monoclonal antibodies against BCMA and Car-T cell therapy technology based on the antibodies.

Example 1

By immunizing mice with human BCMA protein antigen and screening monoclonal hybridomas, monoclonal hybridomas capable of producing anti-BCMA antibodies are obtained. The anti-BCMA antibody produced by one of the monoclonal hybridomas was sequenced, and the nucleotide sequence of the heavy chain variable region was shown in SEQ ID NO: 3, and the nucleotide sequence of the light chain variable region was shown in SEQ ID NO: 4 shown. Correspondingly, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is SEQ ID NO: 2.

Example 2

In Example 2, the affinity of the anti-BCMA antibody obtained in Example 1 to the antigen and the affinity to the MM cell line were measured.

1. Biofilm layer interference technology detects the affinity of anti-BCMA antibody and antigen

The biofilm layer interference technology combines biomolecules on the surface of the sensor to form a biofilm. The biofilm will cause interference with the light waves passing through the sensor. This interference will be detected in a phase shifting manner, so that the binding can be detected. The changes in the number of molecules on the sensor surface can be used to study the effects of antibodies and antigens.

The specific experimental process of using ForteBio Blitz instrument (ForteBio) to detect the affinity of antibody and antigen is as follows:

1) Take 4 biosensors (ForteBio) that bind to the anti-mouse constant region and soak them in buffer (PBS, 0.1% Tween, 0.05% BSA) for more than 10 minutes in advance.

2) Dilute the mouse anti-human BCMA antibody obtained in Example 1 to 0.1 mg/mL in a buffer solution, incubate with the sensor for more than 30 minutes, and then put the sensor in the buffer solution and wash three times for 10 minutes each.

3) Dilute the human BCMA antigen to 25, 50, 100 nM in buffer respectively. Control (negative control) is a buffer without antigen.

4) Select the "Basic Kinetics" mode on the Blitz instrument, and set the baseline duration to 300 seconds, the binding duration to 300 seconds, and the dissociation duration to be 900 seconds.

5) Load the biosensor combined with mouse anti-human BCMA antibody on the instrument, incubate it with Control according to the set mode, and record the binding signal on the sensor; then incubate the sensor with the human BCMA antigen diluted to 25nM 300 seconds (combination), then put the sensor in the Control for 900 seconds (dissociation), and record the signal on the sensor during the whole process.

6) Replace with a new biosensor that binds mouse anti-human BCMA antibody, and record the binding signal with other diluted antigens according to the steps in 5).

7) Use Blitz's supporting software to analyze the data, and after normalizing the signal curve bound to the antigen with the Control sample, the curve is fitted and calculated.

Figure 1 shows the binding and dissociation curves of BCMA antigen and the biosensor loaded with mouse anti-human BCMA antibody at 25, 50, and 100 nM concentration after normalization with a negative control (Control) sample. The X axis represents the entire time course of the sensor from the baseline to the combination and then to dissociation. The Y axis represents the strength of the combined signal.

The fitting calculation results are shown in Table 1 below:

Table 1 The effect of anti-BCMA antibody and antigen

sample name	Concentration (nM)	KD(M)	Ka(1/Ms)	Kd(1/s)	Rmax	R equilibrium	R 2
Human BCMA antigen	25	9.986e-9	5.276e5	5.269e-3	0.1203	0.08596	0.9885
Human BCMA antigen	50	2.491e-9	3.371e5	8.395e-4	0.2553	0.2432	0.9895
Human BCMA antigen	100	2.822e-9	1.539e5	4.342e-4	0.3864	0.3758	0.994

In Table 1, the corresponding concentration refers to the sample loading concentration, KD represents the affinity constant, Ka represents the binding rate constant, Kd represents the dissociation rate constant, Rmax represents the maximum response obtained by data fitting, and R equilibrium represents equilibrium. The maximum response at time, R ² represents the correlation coefficient.

The detection result of biofilm layer interference technology shows that the affinity constant value KD of anti-BCMA antibody and antigen is in the order of 10 ^-9 M.

2. Flow cytometry to detect the binding of anti-BCMA antibody to MM cell line and negative control cell line

Multiple myeloma (MM, Multiple myeloma) is a malignant B-cell lymphoma caused by malignant clonal proliferation of bone marrow plasma cells, accompanied by monoclonal immunoglobulin or light chain (M protein) overproduction. The common clinical manifestations are Anemia, bone pain, renal insufficiency, infection, bleeding, neurological symptoms, hypercalcemia, amyloidosis, etc. Use flow cytometry to detect the binding ability of anti-BCMA antibodies to MM cell lines.

Take the MM cell line and the negative control cell line, each 2×10 ⁵ cells, wash once with flow buffer (PBS+0.1% BSA), discard the supernatant, add the corresponding anti-BCMA antibody and incubate for 30 minutes and wash with buffer. Resuspend, then incubate with fluorescently labeled secondary antibody for 30 minutes in the dark, wash in buffer, resuspend, and finally detect by flow cytometry.

The results of flow cytometry are shown in Figure 2 to Figure 4. Figure 2 shows that the anti-BCMA antibody specifically recognizes the cell line MM.1S with high expression of BCMA; Figure 3 shows that the anti-BCMA antibody specifically recognizes the cell line RPMI8226 with low expression of BCMA; Figure 4 shows that the anti-BCMA antibody does not recognize cells that do not express BCMA. Department of K562. The curve marked with an arrow in the figure is the fluorescence intensity curve detected after the anti-BCMA antibody is incubated with the corresponding cell line, and the curve not marked with an arrow is the staining situation of the isotype control and the corresponding cell line, that is, using the anti-BCMA antibody extract Immunoglobulins of the same species source, the same subtype, the same dose, and the same immunoglobulin and subtype are used to eliminate background staining due to the non-specific binding of antibodies to the cell surface.

From the results shown in Figures 2 to 4, it can be seen that the anti-BCMA antibody of the present invention specifically binds to the MM cell line (MM.1S, RPMI8226) but does not recognize the control cell line K562.

Example 3 Design of chimeric antigen receptors targeting BCMA

The targeted BCMA CAR designed in the present invention contains an anti-BCMA single-chain antibody, a human CD8 extracellular hinge region, a human CD28 transmembrane region, a human CD28 intracellular region and a human CD3ζ intracellular region connected in sequence.

Among them, the amino acid sequence of the anti-BCMA single-chain antibody is as follows (SEQ ID NO: 5):

The amino acid sequence of the extracellular hinge region of human CD8 is as follows (SEQ ID NO: 6):

The amino acid sequence of human CD28 transmembrane region is as follows (SEQ ID NO: 7):

The amino acid sequence of human CD28 intracellular region is as follows (SEQ ID NO: 8):

The amino acid sequence of the intracellular region of human CD3ζ is as follows (SEQ ID NO: 9):

Correspondingly, the nucleotide sequences encoding these amino acid sequences are:

The nucleotide sequence encoding the amino acid sequence of the anti-BCMA single-chain antibody is (SEQ ID NO: 10):

The nucleotide sequence encoding the amino acid sequence of the extracellular hinge region of human CD8 is (SEQ ID NO: 11):

The nucleotide sequence encoding the amino acid sequence of the transmembrane and intracellular regions of human CD28 is (SEQ ID NO: 12):

The nucleotide sequence encoding the amino acid sequence of the intracellular region of human CD3ζ is (SEQ ID NO: 13):

2. BCMA CAR lentiviral shuttle plasmid construction

Synthesized BCMA CAR nucleic acid sequence (sequence includes BCMA scFv, human CD8 extracellular hinge region, human CD28 penetrating region and intracellular region, and human CD3ζ intracellular region, each sequence is connected in sequence) about 1.5kb, with NheI at both ends And EcoRI restriction endonuclease digestion sites, the BCMA CAR nucleic acid fragment with sticky ends was obtained by double digestion, and the pCDH-EF1α-MCS vector was double digested with NheI and EcoRI restriction enzymes (vector from System The vector fragments obtained by Biosciences) were ligated, transformed into DH5α competent cells, selected single clones for sequencing identification, and selected the vector with the correct sequence.

3. Lentiviral vector packaging

1) Day 1: The 293T cells should be in good condition, less than 20 generations, and not overgrown. Plate the plate at 0.3-0.4×10 ⁶ cells/mL, add 10 mL of DMEM+10% FBS complete medium to a 10 cm culture dish, mix the cells thoroughly, and place them in a 37°C, 5% CO ₂ incubator overnight.

2) The second day: 293T cells reach a confluence of about 60-70% for transfection; take Lipofectamine3000 transfection reagent as an example, prepare a tube of plasmid and P3000 complex, the amount of each plasmid is 12ug BCMA CAR shuttle plasmid, PsPAX2 7.8ug, pMD2.G 4.2ug, P3000 48uL, add DMEM medium to make up to 300uL; another tube is diluted 36uL Lipofectamine3000 to 300uL with DMEM (serum-free) medium. Mix the two tubes separately, combine them, mix well, incubate at room temperature for 15-20 minutes, gently add the mixture to the 293T Petri dish. After incubating at 37°C for 6 hours, remove the medium and re-add the pre-warmed fresh medium.

3) Day 4-5: Collect the supernatant for 48-72 hours after transfection, remove floating cells by centrifugation at 3000 rpm for 10 minutes, filter with 0.45um filter, ultracentrifugation at 35000 rpm for 90 minutes, discard the supernatant, and use 100uL DMEM medium Resuspend the virus pellet, and freeze it at -80℃ after aliquoting.

4. Lentivirus infects human PBMC

1) Use Ficoll-paque plus (GE healthcare) to obtain purer PBMC, adjust the cell density to 1x106/mL with 10% FBS AIM-V (Thermo Fisher Scientific) medium, and inoculate the cells in T25 or T75 culture flasks , Add 50ng/mL anti-human CD3 antibody (ebiosciences) and 50ng/mL anti-human CD28 antibody (ebiosciences), then add 200IU/mL interleukin 2 (Peprotech), stimulate the culture for 48 hours.

2) After T cells are activated, count the cells again. According to the count results, inoculate 2x106 cells/well in a six-well plate. Add 2ml AIM-V medium + 10% FBS (containing 50ng/mL anti-human CD3 antibody, 50ng/mL anti-human CD28 antibody, 200IU/mL interleukin 2) medium, the virus solution prepared in item 8 and polybrene (Sigma), pipet and mix well. Move the above-mentioned six-well plate into a plate centrifuge, centrifuge at 800g for 30 minutes, and transfer it to an incubator to continue culturing.

3) After 12 hours of infection, collect the cells in each well of the six-well plate into a 15ml centrifuge tube, centrifuge at 400g for 5min, and discard the supernatant.

4) After centrifugation, inoculate the cells in T25 or T75 culture flasks at a density of 1x106/ml, add an appropriate amount of AIM-V medium containing 10% FBS, and add 200IU/mL IL-2, 10ng/mL IL-7 ( Peprotech), transferred to the incubator to continue cultivation. Observe the cell density every day, and add cytokine-containing culture medium at the right time to maintain the T cell density at ^{about 0.5-1x10 6} /mL to expand the cells.

5. Flow cytometry to detect the expression of CAR protein on the surface of T cells after infection

Centrifuge the BCMA CAR-T cells prepared above 3-5 days after infection, mock CAR-T cells (mock CAR-T cells, that is, CAR-T cells that target intracellular antigens and have the same skeleton as BCMA CAR Cells) and uninfected T cells (NT), wash once with flow buffer (PBS+0.1% BSA), discard the supernatant, add BCMA recombinant antigen and avoid light for 30 minutes, wash with buffer, resuspend, and detect by flow cytometry . The result is shown in Figure 5. Flow cytometry results showed that the expression efficiency of BCMA CAR reached 31.1%, and the BCMA protein could specifically detect the expression of BCMA CAR.

Example 4

1. Detection of cytokine secretion after CAR-T cells and MM target cells are co-cultured

(1) Take the prepared BCMA CAR-T cells, mock CAR-T cells, and NT cells, and resuspend them in cytokine-free RPMI1640+10% FBS medium, and adjust the cell concentration to 1×10 ⁵ /mL.

(2) Resuspend the three MM target cells (H929, MM.1S, RPMI8226) and the negative control cell (K562) in the same medium, and adjust the cell concentration to 1x10 ⁵ /mL.

(3) in a V-bottom 96 well plates containing one experimental group of target cells per well or negative control cells 1x10 ⁴ th, CAR-T th 1x10 ⁵ cells per well in a volume of 200uL. In the control group, NT cells or mock CAR-T cells were added. After mixing well, incubate at 37C for 12 hours and collect the supernatant.

(4) According to the instructions of the cytokine detection kit, detect the cytokine content in the supernatant of the experimental group and the control group.

Among them, NT cells and mock CAR-T cells are defined in Example 3. The test results are shown in Figure 6 and Figure 7. Figure 6 represents the results of interferon-γ (IFN-gamma) secretion after co-cultivation of different cells with three MM target cells and negative control cells; Figure 7 represents the co-cultivation of different cells with three MM target cells and negative control cells Later, the secretion of interleukin-2 (IL-2) results. Among them, Target cell alone in Figure 6 and Figure 7 refers to only culturing target cells or negative control cells, without using NT cells, mock CAR-T cells or BCMA CAR-T cells to co-culture. The test results showed that compared with the control group, the BCMA CAR-T cells in the experimental group were incubated with MM target cells and the secretion of IFN-gamma and interleukin-2 increased significantly, while the secretion of cytokines did not increase after incubation with the negative control cell line , Indicating that BCMA CAR-T cells can specifically recognize target cells and are activated to release corresponding cytokines.

2. Detect the tumor-specific cell killing effect after CAR-T cells are co-cultured with target cells

(1) Take CHO cells (CHO-BCMA cells) and negative CHO cells that overexpress BCMA, and resuspend them in 1640+10% FBS medium without cytokines, adjust the cell concentration to 1x10 ⁵ /mL, and 1x10 ⁴ cells per well are plated on E-Plate 96-well plates (ACEA Biosciences), divided into experimental group (co-cultured with BCMA CAR-T), control group (co-cultured with mock CAR-T cells or NT cells), each experiment Condition to make three auxiliary holes. Put the well plate back into the xCELLigence real-time cell analyzer (ACEA Biosciences), 37C, 5% CO ₂ , make the cells adhere to the wall, and record the cell index changes (see 3 for principle).

(2) After 24 hours, take the prepared BCMA CAR-T cells, mock CAR-T cells, and NT cells and resuspend them in cytokine-free RPMI1640+10% FBS medium, and adjust the cell concentration to 1x10 ⁶ /mL. Take out the E-Plate with target cells and negative cells, discard the supernatant medium, add BCMA CAR-T cells, mock CAR-T cells, or NT cells according to the experimental group, 1x10 ⁵ cells per well (effector cells: target The cell ratio is 10:1), put the E-Plate back into the culture instrument, 37C, 5% CO ₂ , and co-culture with target cells or negative cells.

(3) Real-time detection and recording of the adherence status of target cells or negative cells during the co-cultivation process. Since the killed cells are floating and no longer adhere to the wall, their activity is reflected by the adherence status of the cells, and the cells before and after the co-culture are obtained The graph of normalized cell index over time (generally record the cell index 24-70 hours after co-cultivation).

As shown in Figure 8, the killing effect of different cells on CHO-BCMA cells is shown. The results in Figure 8 show that the target cells continued to increase in cell index before CAR-T cells were added. After adding BCMA CAR-T cells (pointed by the arrow in the figure), most of the CHO-BCMA target cells were killed within 24 hours. The index continued to decrease; while the control mock CAR-T cells or NT cells had no killing effect. Each graph line shows the average value of three-well experiment data.

As shown in Figure 9, the results of the killing effect of different cells on negative CHO cells are shown. The above figure shows that compared with the control group, BCMA CAR-T has no killing effect on negative CHO cells, and has no effect on the growth status of negative CHO cells, thus proving the specific killing effect of BCMA CAR-T on target cells CHO-BCMA. Each graph line shows the average value of three-well experiment data.

3. Evaluation of BCMA CAR-T in vivo efficacy

Based on the ability of BCMA CAR-T in the present invention to kill multiple myeloma cell lines and target cells engineered to express BCMA in vitro, the RPMI8226 cell line was used to evaluate the resistance of CAR-T in a preclinical animal model of multiple myeloma. Tumor activity. Among them, the RPMI8226 cell line is a human multiple myeloma cell line, purchased from ATCC.

Fifteen NSG mice (NODscid gamma mice, purchased from Jackson Laboratory) were divided into three groups, 5 mice in each group, and 1×10 ⁷ RPMI8226 tumor cells were inoculated subcutaneously; on the 18th and 22nd day after tumor inoculation Day, the three groups of mice were injected intravenously with PBS (PBS group), 2×10 ⁷ mock CAR-T cells (mock CAR-T group), 2×10 ⁷ BCMA CAR-T cells (BCMA-CAT-T group, according to Prepared by infection with the method described in item 8, CAR-positive cells are about 25-30%). The tumor volume (unit: mm ³ ) was measured every four days. Compare the tumor growth status of the control group, mock CAR-T group and BCMA-CAR-T group. As shown in Figure 10.

In Figure 10, the abscissa represents the days of injection of RPMI8226 tumor cells into mice, and the abscissa represents the tumor volume. The results in Figure 10 show that compared with the PBS group and the mock CAR-T group, the tumor growth of the mice infused with BCMA CAR-T was significantly inhibited. On the 36th day, the tumor size of the mice in each group was detected. The tumor size of mice in the mock CAR-T group reached about 2500-3000 mm ³ , while the tumors in the BCMA-CAR-T group did not grow and were significantly reduced.

In addition, the terms "first" and "second" are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Therefore, the features defined with "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise specifically defined.

In the present invention, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", "fixed" and other terms should be understood in a broad sense. For example, they may be directly connected or indirectly through an intermediary. The connection may be the internal communication between two elements or the interaction relationship between the two elements, unless specifically defined otherwise. When two nucleic acid sequences or nucleotide sequences are connected, they can be connected by a 3'-5' phosphodiester bond. For those of ordinary skill in the art, the specific meaning of the above-mentioned terms in the present invention can be understood according to specific circumstances.

In the description of this specification, descriptions with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" etc. mean specific features described in conjunction with the embodiment or example , Structure, materials or features are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms are not necessarily directed to the same embodiment or example. Moreover, the described specific features, structures, materials or characteristics can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine the different embodiments or examples and the characteristics of the different embodiments or examples described in this specification without contradicting each other.

Although the embodiments of the present invention have been shown and described above, it can be understood that the above-mentioned embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can comment on the above-mentioned The embodiment undergoes changes, modifications, substitutions and modifications.

Claims (23)

1. An anti-BCMA antibody or antigen-binding fragment characterized by comprising at least one of the following:

   (1) Heavy chain variable regions with amino acid sequences shown in GYTFTSYV, IIPYNDDT and ARWNYDGYFDV, and light chain variable regions with amino acid sequences shown in QSLVHSNGNTY, YKVS and SQITHVPYT;

   Compared with (1), an amino acid sequence with at least one conservative amino acid substitution;
2. The antibody or antigen-binding fragment according to claim 1, characterized in that it comprises at least one of the following:

   (a) A heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 2;

   Compared with (a), the amino acid sequence has at least one conservative amino acid substitution.
3. An isolated polynucleotide, characterized in that the polynucleotide encodes the antibody or antigen-binding fragment of claim 1 or 2.
4. The polynucleotide of claim 3, wherein the polynucleotide is a nucleotide sequence having at least one of the following:

   The nucleotide sequence of the heavy chain variable region shown in SEQ ID NO: 3 and the nucleotide sequence of the light chain variable region shown in SEQ ID NO: 4;

   Compared with the heavy chain variable region nucleotide sequence shown in SEQ ID NO: 3, a sequence with more than 90% homology, optionally a sequence with more than 95% homology, preferably a sequence with more than 98% homology A source sequence, more preferably a sequence with more than 99% homology;

   Compared with the nucleotide sequence of the light chain variable region shown in SEQ ID NO: 4, a sequence with more than 90% homology, optionally a sequence with more than 95% homology, preferably with more than 98% homology The original sequence is more preferably a sequence with 99% or more homology.
5. An expression vector characterized by comprising the polynucleotide of claim 3 or 4.
6. The expression vector of claim 5, further comprising a control element operably linked to the polynucleotide for controlling the expression of the polynucleotide in the host cell.
7. The expression vector of claim 6, wherein the control element comprises at least one of the following: a promoter, an enhancer and a terminator.
8. The expression vector of claim 6, wherein the host cell is a mammalian cell.
9. A recombinant cell characterized by comprising the expression vector of any one of claims 5-8.
10. A kit, characterized in that it comprises the antibody or antigen-binding fragment of claim 1 or 2;
11. The kit according to claim 10, wherein the kit is used for diagnostic detection of BCMA antigen.
12. A pharmaceutical composition, characterized by comprising: the antibody or antigen-binding fragment of claim 1 or 2 and a pharmaceutically acceptable carrier.
13. The use of the antibody or antigen-binding fragment of claim 1 or 2 in the preparation of a medicament for the treatment of tumors.
14. The use according to claim 13, wherein the tumor is multiple myeloma.
15. An anti-BCMA chimeric antigen receptor, which is characterized by comprising: an extracellular target binding region, an extracellular hinge region, a transmembrane region, and an intracellular region, the extracellular target binding region and the extracellular hinge region , The transmembrane region and the intracellular region are sequentially connected;

    The extracellular target binding region includes an antibody or antigen-binding fragment, the antibody or antigen-binding fragment is a single chain, and the antibody or antigen-binding fragment includes the antibody or antigen-binding fragment of claim 1 or 2.
16. The anti-BCMA chimeric antigen receptor according to claim 15, wherein the antibody or antigen-binding fragment is a single-chain fragment shown in SEQ ID NO: 5.
17. The anti-BCMA chimeric antigen receptor according to claim 15 or 16, wherein the extracellular hinge region further comprises a human CD8 extracellular hinge region, and the amino acid sequence of the human CD8 extracellular hinge region is preferably SEQ ID NO: 6.
18. The anti-BCMA chimeric antigen receptor according to any one of claims 15 to 17, wherein the transmembrane region is a human CD28 transmembrane region, and the amino acid sequence of the human CD28 transmembrane region is preferably as SEQ ID NO: 7 shows.
19. The anti-BCMA chimeric antigen receptor according to any one of claims 15 to 18, wherein the intracellular region comprises a human CD28 intracellular region and a human CD3ζ intracellular region, and the human CD28 intracellular region The amino acid sequence of the region is preferably shown in SEQ ID NO: 8, and the amino acid sequence of the human CD3ζ intracellular region is preferably shown in SEQ ID NO: 9.
20. The anti-BCMA chimeric antigen receptor according to any one of claims 15 to 19, wherein the nucleic acid sequence encoding the antibody or antigen-binding fragment is shown in SEQ ID NO: 10; encoding the human The nucleic acid sequence of the extracellular hinge region is shown in SEQ ID NO: 11; the nucleic acid sequence encoding the human CD28 transmembrane region and the human CD28 intracellular region is shown in SEQ ID NO: 12; encoding the human CD3ζ intracellular region The nucleotide sequence of is shown in SEQ ID NO: 13.
21. A Car-T cell, wherein the Car-T cell expresses the anti-BCMA chimeric antigen receptor according to any one of claims 15 to 20.
22. A method for treating tumors in a subject, which is characterized in that it comprises administering to the subject an effective amount of the Car-T cell of claim 21.
23. The method for treating a tumor in a subject according to claim 22, wherein the tumor is multiple myeloma.

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