Patent Grant
12227581
This application is a National Stage Application and claims priority under 35 U.S.C. § 371 to Patent Cooperation Treaty application PCT/CN2019/095507, filed Jul. 10, 2019, which claims the benefit of CN application No. 201810972054.2, filed Aug. 24, 2018. Priority is claimed to these applications and the disclosures of these prior applications are considered part of the disclosure of this application and to the extent allowed the entire contents of the aforementioned applications are incorporated herein.
The present disclosure belongs to the field of biotechnologies. In particular, the present disclosure relates to a group of single domain antibodies against B cell maturation antigen (BCMA) and use thereof.
BCMA (B cell maturation antigen, BCMA) is a member of tumor necrosis factor receptor (TNFR) superfamily, which can bind to a B cell-activating factor (BAFF) or a B lymphocyte stimulator (BLyS) and a proliferation inducing ligand (APRIL). It is reported that in normal cells. BCMA is mainly expressed by plasmocytes and some mature B cells, but not expressed in most B cells or other organs. Multiple myeloma (MM) is a malignant tumor characterized by massive proliferation of clonal plasmocytes. The RNA of BCMA is generally detected in MM cells, and the BCMA protein can be detected on the surfaces of plasmocytes of a patient with multiple myeloma. Accordingly, a candidate target antigen for immune treatment of MM is BCMA. At present, MM treatment can induce remission, but almost all the patients will eventually relapse and die. Some monoclonal antibody candidate drugs have shown a promise to treat MM in pre-clinical studies and early clinical trials, but have not been universally approved by consensus, and no monoclonal antibody drug has been marketed. Clearly, there is an urgent need of new immunological therapy for MM, and an effective antigen-specific adoptive T cell therapy developed for this disease will be an important research progress.
Single domain antibody (sdAb), also known as nanobody, is a heavy chain antibody found in Alpaca blood in which a light chain is absent. By using the molecular biology technology in combination with nano-particle science, Belgian scientists have developed a novel, low molecular weight fragment of antibody which can bind to an antigen. It has a group of advantages, such as, simple structure, strong penetration, easy expression and purification, high affinity and stability, and no toxic and side reactions, or the like. Single domain antibodies for various target antigens have been researched by use of a single domain antibody platform technology, and then used in the field of biomedicines.
The present disclosure aims to develop a group of promising anti-BCMA single domain antibodies for use in therapeutic antibody candidate drugs and chimeric antigen receptor T cells targeting BCMA.
The technical problem to be solved by the present disclosure is to provide a group of novel anti-BCMA single domain antibodies having good effects.
Another technical problem to be solved by the present disclosure is to develop various uses of anti-BCMA single domain antibodies.
In order to achieve the above objects, the present disclosure provides the following technical solutions:
The present disclosure provides a group of anti-BCMA single domain antibodies composed of a framework region and a complementarity determining region, wherein the complementarity determining region has an amino acid sequence selected from those of SEQ ID NOs: 1-66 (Annex 1: Amino Acid Sequences of Complementarity Determining Regions of Screened BCMA-sdAbs).
In some embodiments, the amino acid sequence of the complementarity determining region has more than 80%, more than 85%, more than 90%, more than 95% or more than 99% identity to the amino acid sequence as set forth in SEQ ID NOs: 1-66.
Preferably, the difference in amino acids is conservative substitution.
In some embodiments, the single domain antibody in the group has an amino acid sequence selected from SEQ ID NOs: 67-132 or is an amino acid sequence selected from SEQ ID NOs: 67-132 (Annex 2: Amino Acid Sequences of Screened BCMA-sdAbs).
In some embodiments, the amino acid sequence of the single domain antibody has more than 80%, more than 85%, more than 90%, more than 95% or more than 99% identity to the amino acid sequence as set forth in SEQ ID NOs: 67-132.
Preferably, the difference in amino acids is conservative substitution, more preferably, one or more conservative substitutions.
The present disclosure provides a group of genes of anti-BCMA single domain antibodies having a nucleotide sequence selected from those of SEQ ID NOs: 133-198 (Annex 3: Nucleotide Sequences of Screened BCMA-sdAbs), or being a nucleotide sequence of SEQ ID NOs: 133-198, or being a nucleotide sequence encoding the above single domain antibodies.
In some embodiments, the nucleotide sequence of the single domain antibody has more than 80%, more than 85%, more than 90%, more than 95% or more than 99% identity to the nucleotide sequence as set forth in SEQ ID NOs: 133-198.
Preferably, the difference in bases is conservative substitution, more preferably, one or more conservative substitutions.
The present disclosure provides a polypeptide having one or more single domain antibodies selected from the group of single domain antibodies as described above.
Preferably, the plurality of single domain antibodies are the same or different.
The present disclosure provides an expression vector including one or more genes selected from the group of genes of single domain antibodies as described above.
Preferably, the expression vector is a prokaryotic cell expression vector, a eukaryotic cell expression vector, or other cell expression vector(s).
The present disclosure provides a host cell including the above expression vector.
Preferably, the host cell is a prokaryotic expression cell, a eukaryotic expression cell, a fungus cell or a yeast cell, wherein the prokaryotic expression cell is preferably Escherichia coli.
The present disclosure provides a chimeric antigen receptor, having one or multiple single domain antibodies selected from the above group of single domain antibodies.
Preferably, the multiple single domain antibodies are the same or different.
The present disclosure provides a T cell modified by a chimeric antigen receptor, which is modified by the above chimeric antigen receptor.
The present disclosure provides a pharmaceutical composition including one or more single domain antibodies selected from the above group of single domain antibodies as active ingredients.
The present disclosure provides a humanized anti-BCMA single domain antibody, which is obtained by humanizing the single domain antibody selected from the above group of single domain antibodies.
The present disclosure provides use of the single domain antibody in the above group of single domain antibodies in detection of BCMA.
The present disclosure provides use of the single domain antibody in the above group of single domain antibodies for blocking an interaction between BAFF and BCMA.
In some embodiments, the single domain antibody is linked to one or more of a cytotoxic agent, an enzyme, a radioisotope, a fluorescent compound or a chemiluminescent compound.
The present disclosure provides use of the single domain antibody in the above group of single domain antibodies in preparation of a drug for treating a disease associated with abnormal BCMA expression.
Preferably, the disease associated with abnormal BCMA expression is a multiple myeloma disease.
The present disclosure has the following beneficial technical effects:
The disclosure screens a group of anti-BCMA single domain antibodies. Compared with the existing antibodies, respective anti-BCMA single domain antibodies in the group have high activity and strong neutralization or binding capability. The group of single domain antibodies can specifically bind to human BCMA antigens or tumor cell strains expressing BCMA on the cell surfaces, effectively block the binding of BAFF antigen to BCMA, and generate a corresponding signal cascade effect. The group of single domain antibodies can be used for detecting and/or treating a plurality of diseases associated with abnormal BCMA expression.
Hereinafter the present disclosure will be described in details by reference to the accompanying drawings and examples, but the scope of the present disclosure is not limited thereto.
The disclosure screens a group of anti-BCMA single domain antibodies by a group of steps, which have potentials of high activity and high neutralization or binding capability. These single domain antibodies have similar structures (composed of a framework region and a complementarity determining region), and similar functional effects. Thus, they can be considered as a group of anti-BCMA single domain antibodies having common structure and common property effects.
The term “BCMA”, as used herein, is a member of tumor necrosis factor receptor (TNFR) superfamily, which can bind to a B cell-activating factor or a B lymphocyte stimulator and a proliferation inducing ligand (APRIL)). Multiple myeloma (MM) is a malignant tumor characterized by massive proliferation of clonal plasmocytes. The RNA of BCMA is generally detected in MM cells, and the BCMA protein can be detected on the surfaces of plasmocytes in a patient with multiple myeloma.
The term “multiple myeloma (MM)” as used herein is a malignant tumor characterized by massive proliferation of clonal plasmocytes. At present. MM treatment can induce remission, but almost all the patients will eventually relapse and die. Some monoclonal antibodies have shown a promise to treat MM in pre-clinical studies and early clinical trials, but have not been universally approved. Clearly, there is an urgent need of new antibodies and new immunological therapy for MM.
New antibodies against BCMA are the development object, and finally the protective object of the present disclosure. The scope of the present disclosure encompasses the obtained anti-BCMA antibodies and various forms thereof (for example, single domain antibodies), as well as substances including the antibody as component (for example, pharmaceutical compositions, kits, vectors, chimeric antigen receptors, a chimeric antigen receptor modified T cells, or the like), uses (for example, uses for diagnosis, treatment or application, etc.). However, it should be understood by those skilled in the art that the protective objects of the present disclosure are not limited to these exemplified contents.
The term “single domain antibody (sdAb)” as used herein refers to a fragment containing a single variable domain in an antibody, and is also known as nanobody. Like a complete antibody, it can selectively bind to a specific antigen. Compared with the mass of the complete antibody (150-160 kDa), the single domain antibody (only about 12-15 kDa) is much smaller. The first single domain antibody was made from alpaca heavy chain antibodies by artificial engineering, and known as “VHH segment”. In a preferred embodiment, the present disclosure uses the single domain antibody of the alpaca, whereas those skilled in the art should understand that the present disclosure can also encompass single domain antibodies derived from other species. Without limitation, the single domain antibody of the present disclosure is an anti-BCMA single domain antibody.
The term “framework region” is also known as a skeleton region. The sequences of about 110 amino acids near the N-terminals of the H chain and the L chain of immunoglobulin vary greatly, while the amino acid sequences in other positions are relatively constant. Accordingly, the light chain and the heavy chain can be divided to a variable region 00 and a constant region (C). The variable region contains an HVR (hypervariable region), also known as complementarity-determining region (CDR) and a frame region (FR). The variability of FR is less than that of CDR. There are four FR molecules in total, that is. FR1, FR2, FR3 and FR4, respectively. During the recognition of antibody, four FR molecules crimp so that CDR molecules are close to each other. It should be understood that the present disclosure is not limited to specific framework region(s), and those skilled in the art can select or obtain appropriate framework region(s) according to practical requirements without departing from the protective scope of the present disclosure.
The term “complementarity determining region (CDR)”, the whole antibody molecule can be divided into a constant region and a variable region. In the variable region, some amino acid residues are highly variable, and the regions in which the compositions and arrangement orders of these amino acid residues are more prone to vary are called hypervariable regions. There are three hyper-variable regions (HVR) in the V regions of the L chain and the H chain, which can form a precise complementation with the antigen determinants in terms of spatial structure, and thus the hyper-variable regions are also called complementarity determining regions.
The term “identity” of sequence as used herein is interchangeably used with “homology”, and refers to a similarity degree between sequences as measured by sequence alignment softwares, such as BLAST. The sequence alignment methods and softwares are well-known by those skilled in the art. Modified nucleotide sequences can be obtained by substitution, deletion and/or addition of one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more) amino acids or bases in a known sequence. For example, by modifying the amino acid sequence or nucleotide sequence as set forth in one or more of sequence SEQ ID NOs: 1-198 of the present disclosure via conventional means (for example, by conservative substitution), it is feasible to obtain sequences having more than 80%, more than 85%, more than 90%, more than 95% or more than 99% sequence identity to these sequences, and having substantially the same properties, which are encompassed within the protective scope of the present disclosure. Preferably, the present disclosure obtains sequence identity by conservative substitution, but is not limited thereto.
The term “amino acid sequence” refers to an arrangement in which amino acids are linked to each other to form a peptide chain (or polypeptide), wherein the amino acid sequence can only be read in one direction. There are more than 100 types of different amino acids, twenty of which are commonly used. The present disclosure does not exclude the case that other substances (e.g., saccharides, lipids, and other modifications) are attached to the amino acid chains, and is not limited to the 20 amino acids that are commonly used, either.
The term “nucleotide sequence” refers to an arrangement of bases in DNAs or RNAs, namely, an arrangement of A, T, G and C in DNA, or an arrangement of A, U, G and C in mRNA. It also includes arrangements of bases in rRNA, tRNA and mRNA. It should be understood that the antibody gene of the present disclosure also encompasses, in addition to DNA sequences, RNA (rRNA, tRNA and mRNA) and their complementarity sequences. It will also be understood that genes encoding the antibodies of the present disclosure are not equivalent to the sequences as set forth in SEQ ID NOs: 133-198 of the present disclosure, and the genes which encode the antibodies of the present disclosure but are different from the nucleotide sequences as set forth in SEQ ID NOs: 133-198 are also within the protective scope of the present disclosure.
In some embodiments, the polypeptide, the pharmaceutical composition, the chimeric antibody receptor or the CART of the present disclosure comprises one single domain antibody, it should be understood that the present disclosure is not limited thereto. The above substances of the present disclosure can contain two, three, or multiple single domain antibodies, wherein the multiple single domain antibodies are the same or different. Moreover, in addition to the single domain antibodies of the present disclosure, other antibodies or single domain antibodies that are not contained in the present disclosure can also be included without going beyond the scope of the present disclosure.
The term “expression vectors” refers to a vector that incorporates expression elements (such as, promoter, RBS, or terminor) on the basis of the basic backbone of a cloning vector so that a target gene can be expressed. The expression vector comprises four parts: a target gene, a promoter, a terminator and a marker gene. The present disclosure includes, but is not limited to, a prokaryotic cell expression vector, a eukaryotic cell expression vector or other cell expression vectors.
“Chimeric antigen receptor (CAR)” is a core component of “chimeric antigen receptor T cell (CART)”, which imparts a T cell with an ability to recognize tumor antigens in an independent manner, so that the T cell modified by CAR is capable of recognizing a broader range of targets as compared with a natural T cell surface receptor. The basic design of CAR comprises a tumor-associated antigen binding region, an extracellular hinge region, a transmembrane region, and an intracellular signal region.
In an embodiment of the present disclosure, the chimeric antigen receptor or chimeric antigen receptor T cell of the present disclosure can contain one, two or more single domain antibodies of the present disclosure, which can be the same or different.
In an embodiment of the present disclosure, the “pharmaceutical composition” of the present disclosure can contain one, two or more single domain antibodies of the present disclosure, which can be the same or different.
The term “humanized” antibody refers to an antibody in which the constant regions (namely CH and CL regions) of the antibodies or the whole antibodies are encoded by human antibody genes. The humanized antibody can greatly reduce the immune side reaction of a heterologous antibody in a human organism. The humanized antibody includes several types including chimeric antibodies, modified antibodies and full human antibodies. It will be appreciated that those skilled in the art can prepare suitable humanized forms of the single domain antibodies of the present disclosure according to the practical requirements, which are within the scope of the present disclosure.
The term “lentivirus” as used herein is one genus of Retroviridae including eight viruses that can infect humans and vertebrates, wherein the primary infection cells are mainly lymphocytes and macrophages, and the infected individuals will eventually develop the diseases. The types of lentiviruses include, for example, human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), equine infectious anemia (EIA), and feline immunodeficiency virus (FIV). The progress of lentiviral vector research is rapid and intensive. This vector can effectively integrate foreign genes into host chromosomes, so as to achieve persistent expression. In terms of infectability, it can effectively infect neurons, hepatocytes, cardiomyocytes, tumor cells, endothelial cells, stem cells and other types of cells, so as to achieve good gene therapeutic effect. In addition, those skilled in the art can also select other suitable vectors other than lentivirus, which are within the protective scope of the present disclosure.
Hereinafter the present disclosure will be described in details by reference to the following examples. However, the present disclosure is not limited to the specific details of these examples, because for persons skilled in the art, other variations are well established, or obvious according to the direct disclosure and the appended claims. Therefore, all the technologies achieved based on the above description of the present disclosure shall fall within the scope of the present disclosure.
Unless otherwise specified, all the experimental methods described in the following examples are conventional methods; and all the reagents and biomaterials are commercially available, unless otherwise specified.
1) Immunization of Alpaca with BCMA Antigen
It is performed according to the conventional immunization method. Briefly, adult healthy alpacas were subject to multipoint subcutaneous injections at their necks and backs with BCMA antigen (Human TNFRSF17/BCMA/CD269 Protein, purchased from Beijing Yiqiao Shenzhou. Product No. 10620-H15H) with a total weight of about 2 mg, in which the antigen and an equal volume of Freund's adjuvant were added. Immunizations were carried out for 4-6 times. The absorption of mass at the injection sites was followed to confirm correct immunization. The immunization interval time was 7-15 days. After the fourth immunization, serum was collected to determine the immune titer of the antigen. When the titer reached 10,000 times or more (ELISA method), about 100 ml of whole blood was collected, and lymphocytes were separated and stored at −80° C., for subsequent use.
2) Separation and RNA Extraction of Peripheral Blood Lymphocytes of Alpaca
Peripheral blood lymphocytes of alpacas were separated by using a QIAGEN kit (QIAamp RNA Blood Mini Kit (50), Product No. 52304) following the instructions. Briefly, to 1 ml of full blood was added 5-10 ml of red blood cell lysate. The mixture was uniformly mixed, and placed in an ice bath for 30 min. It was centrifuged for 10 min at 2000 rpm after red blood cells were lysed. The supernatant was discarded, and an additional 1-2 ml of red blood cell lysate was added and uniformly mixed. The mixture was placed in an ice bath for 10 min to lyse residual red blood cells, and then centrifuged at 2000 rpm for 10 min. The supernatant was discarded, and 0.3 ml of lysate was added to mix with leukocytes uniformly. The resultant mixture was stored at −80° C., for subsequent use.
RNA purification was carried out by using a QIAGEN kit (QIAamp RNA Blood Mini Kit (50), Product No. 52304) following the instructions. Briefly, to 0.3 ml of the separated alpaca lymphocytes was added 0.3 ml of buffer RLT, and the mixture was mixed well with shaking. The mixed liquid from the last step was transferred to a collection tube equipped with an adsorption column (QIAshredderSpinColumn), and centrifuged at 14,000 rpm for 2 min. The filtrate in the collection tube was transferred to a new centrifuge tube. 0.5 ml of 70% ethanol was added into the filtrate, and uniformly mixed upside down. The mixture was centrifuged at 10000 rmp for 15 s, the waste liquid in the collection tube was discarded, and the adsorption column was re-placed into the collection tube. The adsorption column was transferred to a new 2 ml collection tube, 0.7 ml of buffer RWI was added, and the mixture was centrifuged at 10000 rmp for 15 s. The adsorption column was transferred to a new 2 ml collection tube. 0.5 ml of buffer RPE was added, and the mixture was centrifuged for 15 s at 10000 rmp. 0.5 ml of buffer RPE was added, and the mixture was centrifuged at 14000 rmp for 3 min. The adsorption column was transferred to a new 1.5 ml centrifuge tube, and 30-50 μl RNase-free water was added dropwise into the middle of an adsorption membrane in the air. The mixture was placed at room temperature for 2-5 min, and centrifuged at 12,000 rpm for 1 min. The plasmid solution was collected into the centrifuge tube, and measured for the RNA concentration.
3) Variable Region-VHH of Heavy Chain Antibody
Synthesis of a first chain of cDNA: A cDNA synthesis kit (MiniBESTAgarose Gel DNA Extraction Kit ver.4.0, TAKARA Company) was used following the instructions. With this template, two sets of primers were used to perform PCR amplification of the VHH gene fragment of the heavy chain antibody. By using a Nested PCR method, the fragments of greater than 800 bp in the first PCR amplification are common heavy chain gene fragments, and the fragments between 800 bp and 500 bp are heavy chain antibody gene fragments with deletion of light chains (see
4) Ligation of VHH Fragment and Phage Display Vector and Electric Transformation of TG1 Competent Cells
After the VHH fragment and the pHEN6 vector plasmid were subjected to single digestion with Sfl, the VHH fragment and the pHEN6 vector (Conrath, KEM other. Antimicrob Agents Chemother (Antimicrobial Chemotherapy) 2001, 45: (10) 2807-12, Chinese patent ZL20111028003.1)) were ligated by a ligase, and then electrically transformed into TG1 competent cells, which were used to coat a plate, and detected by colony PCR for verification of the antibody insertion rate. Detection of recombinant gene cloning efficiency: an LB/Amp plate was coated with an electrically transformed bacterial solution, cultured overnight at 32° C., and detected by colony PCR for verification of the ligation efficiency of the antibodies on the next day. The ligation efficiency of the phage-antibody library was more than 90%. The LB/Amp plate was coated with the electrically transformed bacterial solution, and cultured overnight at 32° C. The culture was washed with 2YT culture medium, and 15% glycerol was added. The mixture was stored at −80° C.
5) Preparation of VHH Phage Antibody Library
Helper phage M13K07 (Invitrogen) was added into the antibody library for rescue: the phage antibody library was prepared according to a conventional method and stored at −80° C., for subsequent use.
Screening of BCMA-Specific Single Domain Antibody
2. Picking of Positive Clones Via Phage ELISA
A single colony was randomly picked from an agar plate screened for grown colonies in the third round, inoculated and cultured in a 96-well culture plate containing an Amp 2YT liquid culture medium, and subject to superinfection of helper phages to induce the expression of the phage antibody. The expression supernatant was harvested, and then an ELISA assay was carried out with BCMA as an antigen. BCMA-positive wells were selected, and subject to DNA sequencing to identify the gene sequence of the anti-BCMA single domain antibody clones. A series of single domain antibody gene sequences including those in Annex 3 were obtained and used for further expression and screening of the single domain antibodies with high specificity and high activity.
The specific BCMA single domain antibody gene obtained in Example 3 was amplified by PCR to obtain PCR products with restriction enzymes BbsI and BamHI sites. The PCR products and vectors (pSJF2 vector) (kim ls. Biosic Biochem. 2002, 66 (5): 1148-51, Chinese patent ZL 201110280031) were treated with restriction enzymes BbsI and BamHI respectively, and recombined by ligation with T4 ligase to obtain the plasmid sdAb-pSJF2 that can be efficiently expressed in Escherichia coli, which was subject to gene sequencing to determine the correctness of its sequence.
The strains containing the plasmid BCMAsdAb-pSJF2 in example 3 were inoculated on an LB culture plate containing ampicillin at 37° C., overnight. A single colony was picked and inoculated in 15 ml LB medium solution containing ampicillin, and was cultured in a shaker at 37° C., overnight. 10 ml of culture was transferred to 1 L of 2YT culture solution containing ampicillin and cultured in a shaker at 37° C., at 240 rpm/min. After OD value reached 0.4-0.6, 0.5-1.0 mM IPTG was added and additionally incubated overnight. The above solution was centrifuged for collecting bacteria. The bacteria were lysed by adding lysozym and centrifuged, and the soluble single domain antibody protein in the supernatant was collected. A protein with the purity of more than 95% was obtained by Ni ion affinity chromatography.
1) Preparation of Sample
Antigen: Bio-BCMA was diluted to 10 μg/ml with 1× dynamic buffer (1×PBS, containing 0.05% Tween 20, 0.1% BSA, pH7.2):
Single domain antibody was gradually diluted into 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM and 6.25 nM with 1× kinetic buffer:
2) Sample Test
The antigen to be tested was loaded through an SA sensor. The antigen was diluted by 5 gradients, and all the BCMA single domain antibodies had an affinity of 50 nm, 20 nm, 10 nm, 1 nm, 0.1 nm and 0.01 nm.
The BCMA-Fc antigen was diluted to 1 μg/ml with 0.05 M NaHCO3 (pH 9.5). A 96-well place was coated with 100 μl antigen overnight at 4° C. The 96-well plate was blocked with 300 μl 0.5% BSA-PBS for 2 h at 37° C. The purified BCMA single domain antibodies with different dilution concentrations were added in 100 μl/well at 37° C., for 1 h. The plate was washed three times with 0.05% PBST. Mouse anti-His-HRP diluted in 1:5000 fold was added in 100 μl/well at 37° C., for 1 hour. The plate was washed three times with 0.05% PBST. 100 μl of TMB was added and kept in dark place at room temperature for 20 min. 100 μl of 1 mol/L HCl was added to quench the reaction. The OD value of the sample at 450 nm was measured by a microplate reader.
Because BCMA can bind to BAFF, the functional BCMA single domain antibody should be able to competitively inhibit the binding of BAFF to BCMA. BAFF protein coated a detachable ELISA plate according to 1 μg/ml. 100 μl/well and incubated overnight at 4° C. 2% BSA was added for blocking. 300 μl/well, incubated at 37° C., for 2 hours. The BCMA single domain antibody was diluted to a final concentration of 10 μg/ml. 100 μl BCMA (10 μg/ml) single domain antibody was added. 2 μl of BAFF (5 μg/ml) protein was added in each well to be uniformly mixed. Goat anti-rabbit IgG HRP (1:5000) was diluted. 100 μl/well, incubated for 1 h at 37° C. TMB chromogenic solution was added. 100 μl/well, and reacted in dark for 10 min. The reaction was quenched by adding 2M H2SO4 at 50 μl/well. The OD value was measured at 450 nm.
1) Construction of Vector
A BCMA single domain antibody gene and a second-generation CAR structure gene were synthesized. The two genes were spliced by overlapping PCR to obtain a BCMA CAR gene. After the synthetic gene was obtained, molecular cloning was carried out. First. PCR products of two gene fragments were obtained. Then, overlapping PCR was carried out to obtain BCMA CAR gene with the second-generation CAR structure in which two fragments are linked. Through enzyme digestion of Pre vector and BCMA CAR gene, ligation, transformation, cloning, plasmid upgrading and sequencing, the BCMA CAR-expressed lentiviral vector Pre-Lenti-EFI BCMA with a correct sequence was obtained.
2) Packaging of Lentivirus
On the day before virus packaging. 293T cells were digested by trypsin and spread in 150 cm culture dish. The cells were incubated in 5% CO2 culture box for 8-24 h. When the adherent cells reached 80% of the total culture dish area, the 293T cells were transfected. Pre-Lenti-EFI BCMA CAR: psPAX2: pMD2G=4:3:1 was co-transfected with lipofectamine 2000. The virus supernatant was collected after 48 hours, and centrifuged at 4° C., at 1250 rpm for 5 min to remove the dead 293T cells and cell debris. Then, the virus supernatant was filtered, concentrated, sub-packaged, and stored in a refrigerator at −80° C.
3) Preparation of CART Cells
10 ml of fresh blood was taken from healthy volunteers. Peripheral blood mononuclear cells (PBMC) were isolated with lymphocyte isolation solution, and then T cells were isolated and purified by magnetic beads. 2×106 T cells/well were seeded into a 6-well plate, cultured in an x-vivo 15 culture medium containing IL-2 (1000 U/ml) and stimulated with anti-CD3 for 24 h. After 24 hours of stimulation, a BCMA virus solution was added and infected overnight. 2 ml of culture medium was added on the second day. After 6-7 days of infection, the expression of CAR was evaluated by flow cytometry. The positive rate of expressing anti-BCMA-CAR by transfected T cells was analyzed using biotinylated BCMA via flow cytometry.
4) Determination of Killing Vitality
In a cell killing test, an LDH detection kit (Promega) was used for detection. CART cells/T cells: target cells were set with four gradients, which were 0.5:1, 1:1, 2:1 and 4:1, respectively. Daudi cells 3×104/well, and the rest wells were supplemented to 200 μL with an X-VIVO-containing culture medium/1640) culture medium. The 96-well plate was cultured in a 5% CO2 incubator at 37° C. After 17 h, 20 μl of lysate was added into the maximum release well, and the cells were uniformly mixed to be completely ruptured. The 96-well plate was incubated in the CO2 incubator for 2 h. Two hours later, the maximum release well was observed. After target cells were completely lysed. 50 μL of supernatant was sucked from each well to the 96-well plate with a flat bottom, and then 50 μL of substrate solution was added to each well, development was carried out for 30 min in the dark. After 30 min, the mixture was observed for the color change, wherein the colors of the maximum release MM. IS well and the CART cell well should be darker. A microplate reader was used for measurement at a wavelength of 490 nm. The killing results were seen in
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|---|---|---|---|
| 201810972054.2 | Aug 2018 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2019/095507 | 7/10/2019 | WO |
| Publishing Document | Publishing Date | Country | Kind |
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| WO2020/038147 | 2/27/2020 | WO | A |
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| Number | Date | Country | |
|---|---|---|---|
| 20220251226 A1 | Aug 2022 | US |
