The present application claims priority of "anti-human BCMA nanobody having hydrophilic amino acid residues and methods for preparing and using the same" and "2022115801736" based on bispecific antibody of anti-BCMA nanobody having hydrophilic amino acids, CAR-T formulation and use "in chinese patent application [ 2022115574272 ] filed in 12 month 6 of 2022, both priority patent applications of the invention are incorporated by reference in their entirety.
Background
B cell maturation antigen (B-cell maturation antigen, BCMA), also known as tumor necrosis factor receptor superfamily member 17 (TNFRS 17) or CD269, is a protein specifically expressed on the surface of plasma cells and differentiated plasma cells, whose extracellular domain is composed of 185 amino acid residues, plays a major role in B cell maturation and differentiation into plasma cells. BCMA is a receptor for proliferation-inducing ligand (APRIL) and B cell activating factor (B-cell activating factor, BAFF) of the tumor necrosis factor superfamily. Both can trigger NF- κB signaling, increase the expression of pro-survival BCL-2 family genes and down-regulate pro-apoptotic genes, in addition, BCMA can activate JNK and MAPK8 signaling pathways, and these signaling pathways are involved in regulating humoral immunity, B cell development and homeostasis, promote B cell survival in different development stages, and are key to regulating B cell proliferation, maturation and differentiation into plasma cells.
Multiple Myeloma (MM) is the second largest hematological malignancy next to non-hodgkin's lymphoma, and is characterized by abnormal proliferation of malignant plasma cells in the bone marrow and osteolytic bone lesions. Related clinical symptoms include osteolytic lesions, anemia, renal insufficiency, hypercalcemia, infections and other related organ dysfunctions. BCMA protein and mRNA are almost exclusively found on plasma cells in normal human tissues, but are selectively overexpressed during malignant transformation of plasma cells, are highly expressed on the surface of multiple myeloma cells, and elevated BCMA levels in serum of MM patients promote tumor cell growth, survival and development of drug resistance, mainly by activating NF- κ B, AKT, phosphatidylinositol 3 kinase (PI 3K), STAT3 and MAPK intracellular signaling cascades. The current major treatment regimens include chemotherapy, autologous Stem Cell Transplantation (ASCT) and novel drugs (proteasome inhibitors, immunomodulatory drugs and monoclonal antibodies (such as daratumumab or elotuzumab), which, while delaying the progression of the disease, remain largely incurable, MM, and most patients relapse after multiple line therapy.
In 1993, an antibody having only a heavy chain variable region (variabledomain of the heavy chain of HCAb, VHH) and two CH2 and CH3 regions, called a single domain antibody (singledomain antibody, sdAb), also called a nanobody, was isolated from serum of camelids and sharks, which is superior to conventional antibodies in terms of molecular size, stability, solubility, penetrability, and the like. Such nanobodies alone express VHH structures, but still retain antigen specificity and binding affinity for the original heavy chain intact, being the smallest antibody fragment known to date to bind antigen. Nanobodies may therefore play an advantage in vaccine development, disease diagnosis and treatment. While nanobodies have numerous advantages over conventional antibodies, how nanobodies perform better and more stable functions in practical clinical treatments and applications is widely proven, and many doubts remain incompletely understood. And what types of nanobodies have good effects currently have no accurate answer, and still require considerable time and effort from researchers to explore.
In view of the foregoing, there remains a need in the art to develop novel nanobodies against BCMA and related applications to alleviate the deficiencies of current medical techniques in the treatment of multiple myeloma.
Disclosure of Invention
In view of the above, the present invention aims to provide a series of anti-human BCMA nanobodies with hydrophilic amino acid residues, CAR-T and application thereof, and the specific technical scheme is as follows.
An anti-human BCMA nanobody comprising a heavy chain variable region comprising a framework region and a complementarity determining region comprising a plurality of residues ranging from hydrophobic amino acids to hydrophilic amino acids mutated at positions 37-47 of the amino acid sequence of the complementarity determining region; the sequence of the heavy chain variable region is shown in SEQ ID NO. 1-SEQ ID NO. 41.
Further, the complementarity determining region amino acid sequence at positions 37, 44, 45 and 47 are simultaneously hydrophilic mutations (in conventional antibodies, the above sites are usually hydrophobic amino acids, usually including V37, G44, L45 and W47), the mutations into hydrophilic amino acids including F37 (phenylalanine) or Y37 (tyrosine), E44 (glutamic acid) or Q44 (glutamine), R45 (arginine) and G47 (glycine).
Further, the anti-human BCMA nanobody has an affinity KD value ranging from 1.5×10-9 ～10.0×10-12 。
It is understood that the larger the KD value, the smaller the nanobody affinity.
Further, the CAR-T cells also include a CD8 signal peptide, a hinge region (hinge), a human CD8 transmembrane region (TM), a human 4-1BB costimulatory signal region, and a human CD3 zeta signal domain.
The preparation method of the anti-human BCMA nano antibody comprises the following steps:
step 1: taking plasmid containing full-length sequence of human BCMA gene as a template, and preparing recombinant protein of specific targeted BCMA by using a eukaryotic expression system;
step 2: immunizing an animal by using the recombinant protein prepared in the step 1, extracting total RNA of peripheral blood lymphocytes of the immunized animal, reversely transcribing the total RNA into cDNA, performing two-round amplification by nested PCR to obtain nanobody genes, and cloning the nanobody genes to a phage display carrier to construct a phage nanobody library;
step 3: panning the nanobody constructed in the step 2 by using the specific targeted BCMA recombinant protein prepared in the step 1 by using a phage display technology, and identifying the nanobody capable of specifically binding to BCMA protein;
step 4: affinity detection is carried out on the elutriated nano antibody which specifically targets BCMA.
Further, the primers for the first round of nested PCR amplification are shown as SEQ ID NO.42 and SEQ ID NO. 43; the primers for the second round of nested PCR amplification are shown as SEQ ID NO.44 and SEQ ID NO. 45.
Further, the nano-antibodies which are specifically targeted to BCMA and are elutriated in the step 4 are detected, and the nano-antibodies with the hydrophilic mutation at the 37 th, 44 th, 45 th and 47 th positions of the amino acid sequence of the complementarity determining region are screened.
Preferably, the mutated hydrophilic amino acids include F37 (phenylalanine) or Y37 (tyrosine), E44 (glutamic acid) or Q44 (glutamine), R45 (arginine) and G47 (glycine).
The application of the anti-human BCMA nano antibody in preparing medicaments for targeting multiple myeloma, B lymphocyte malignant tumor, chronic lymphocytic leukemia, diffuse large B cell lymphoma, neuromyelitis optica (NGL) pedigree diseases, human rheumatoid arthritis, multiple sclerosis or systemic lupus erythematosus.
The CAR-T cells are applied to the preparation of medicines for targeting multiple myeloma, B lymphocyte malignant tumor, chronic lymphocytic leukemia, diffuse large B cell lymphoma, neuromyelitis optica (NGL) lineage diseases, human rheumatoid arthritis, multiple sclerosis or systemic lupus erythematosus.
Beneficial technical effects
In the complementarity determining regions, four amino acids in positions 37-47 of the amino acid sequence of a VHH of the invention are simultaneously subject to hydrophilic mutations, as compared to conventional antibody VH. The 4 hydrophobic amino acid residues V37, G44, L45 and W47 in the conventional antibody are quite conserved in evolution, in the VHH of the invention, the four sites are simultaneously mutated into hydrophilic amino acid residues F37 or Y37, E44 or Q44, R45 and G47, and the water solubility of the VHH is correspondingly increased, so that the VHH can enter the inside of the solid tumor more easily. Experiments further prove that the CAR-T prepared by the VHH with hydrophilic mutation has the killing effect of targeting solid tumor cells, and even better than that of the conventional CAR-T. In particular, taking the CAR prepared by VHH shown in SEQ ID No.25 as an example, when the 44 th amino acid is mutated into Q, the tumor cell targeting killing ability is strongest.
Finally, the nano antibody prepared by the method has good affinity and can play a great clinical potential in the treatment of multiple myeloma and spontaneous immune diseases. Experiments of the invention prove that the affinity of the nano-antibody is not positively correlated with the killing capacity of the nano-antibody. When the affinity of the nanobody is too high, the nanobody can be tightly combined with the surface antigen of a single tumor cell, so that the nanobody cannot be flexibly separated to kill other tumor cells, and the final tumor killing effect is general. Thus, VHH with better affinity for tumor cells, but moderate affinity, needs to be screened to perform better and more stable functions in practical clinical treatments and applications.
In conclusion, the screened nano antibody and the prepared CAR-T can play a stable anti-tumor effect in BCMA positive tumor treatment such as multiple myeloma and the like.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Herein, "and/or" includes any and all combinations of one or more of the associated listed items.
Herein, "plurality" means two or more, i.e., it includes two, three, four, five, etc.
It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
As used in this specification, the term "about" is typically expressed as +/-5% of the value, more typically +/-4% of the value, more typically +/-3% of the value, more typically +/-2% of the value, even more typically +/-1% of the value, and even more typically +/-0.5% of the value.
In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be appreciated that such a description of "within a certain range" is merely for convenience and brevity and should not be construed as a inflexible limitation on the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values within that range. For example, a rangeThe description of (c) should be taken as having specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within such ranges, e.g., 1,2,3,4,5, and 6. The above rule applies regardless of the breadth of the range.
Example 1
The embodiment provides a method for constructing, expressing and purifying BCMA recombinant protein expression vector
1.1 construction of eukaryotic expression vectors
The full-length sequence of human BCMA gene is synthesized into a pMD19-T vector, a plasmid containing the full-length BCMA gene is used as a template, a primer is designed for amplification to obtain a BCMA extracellular region (ECD) gene, the BCMA extracellular region (ECD) gene is cloned into a pVax-mFc vector subjected to double enzyme digestion by restriction enzymes PstI and XbaI in a digestion connection mode, the pVax-mFc vector is transformed into DH5 alpha competent cells, screening is carried out through a kanamycin resistance plate, monoclonal sequencing is selected for identification, and the successfully constructed vector is named as pVax-mFc-BCMA-His.
1.2 recombinant protein expression and purification
The successfully constructed recombinant plasmid pVax-mFc-BCMA-His is transfected into HEK293T cells by using a transfection reagent PEI, the supernatant is collected after 5 days of expression, and the recombinant protein BCMA-mFc with high purity is obtained by purifying by using Ni gel through an affinity chromatography mode, and the result is shown in figure 1. The recombinant protein has the effect of specifically targeting a BCMA target.
Example 2
Screening and preparation method of anti-BCMA protein nano antibody
2.1 protein emulsification and animal immunization
The BCMA recombinant protein purified in example 1 (for example, 1 mg) was mixed with an equal volume of aluminum hydroxide adjuvant and the camel was immunized subcutaneously through the neck for the first time, then immunized continuously 4 times every 2 weeks, and peripheral anticoagulation was collected on day 7 after impact immunization.
2.2 construction of phage antibody library and antibody screening
2.2.1 isolation of peripheral blood lymphocytes
200mL of peripheral blood, containing a large number of B lymphocytes, was collected aseptically from the neck vein of the camel, and diluted with an equal volume of PBS buffer. And separating by Ficoll-Paque Plus lymphocyte separating liquid to obtain peripheral blood lymphocyte, wherein the obtained lymphocyte can be directly used for extracting total RNA or frozen at-80 ℃ for standby.
2.2.2 nanobody Gene amplification
Firstly extracting total RNA of lymphocytes, carrying out reverse transcription by taking the RNA as a template to obtain cDNA, then taking the cDNA as the template, amplifying VHH genes by using nested PCR, wherein the first round of amplification primers are CALL001 and CALL002 (see table 1), and separating and recovering about 700bp fragments by agarose gel electrophoresis (figure 2A); a second round of PCR amplification was then performed using the recovered 700bp product as template, the second round of amplification primers VHH-FOR, VHH-REV (see Table 1), and the 400bp fragment was separated and recovered by agarose gel electrophoresis (FIG. 2B).
TABLE 1 primer sequences required for VHH Gene amplification
| Primer name | Primer sequence (5 '-3') | Sequence numbering |
| CALL001 | GTCCTGGCTGCTCTTCTACAAGG | 42 |
| CALL002 | GGTACGTGCTGTTGAACTGTTCC | 43 |
| VHH-FOR | CAGGTGCAGCTGCAGGAGTCTGGGGGAG | 44 |
| VHH-REV | CTAGTGCGGCCGCTGAGGAGACGGTGACCTGGGT | 45 |
2.2.3 construction of phage display vectors
Both the PCR product amplified and recovered in the above step and the phage display vector pMECS were digested with the restriction enzymes Pst I and Not I, and then ligated using T4 DNA ligase.
2.2.4 electrotransformation and harvesting of phage antibody libraries
Adding the ligation product obtained in the above steps into TG1 competent cells, transforming the cells into TG1 by using an electrotransformation instrument, adding an SOC culture medium after electrotransformation is completed, culturing and activating the cells for 1h at 37 ℃ and 220rpm, uniformly coating the culture on an LB/Amp-Glu plate, culturing the cells for 6-8 h at 37 ℃, collecting the cells, adding sterile glycerol to reach the final concentration of 15%, and storing the prepared phage library at-80 ℃ or directly using the phage library for subsequent antibody screening.
2.2.5 screening of specific nanobodies
Taking the prepared phage library as an antibody source, taking a phage library with 20 times of library capacity, adding an auxiliary phage M13K07 to obtain recombinant phage for expressing the antibody, incubating the recombinant phage with BCMA recombinant protein coated in an ELISA plate, eluting with 0.1M Glycine (pH 1.5), re-infecting TG1 strain with the eluent, culturing at 37 ℃ and 220rpm to logarithmic phase, and adding the auxiliary phage to display the antibody on the surface of the phage. After repeating the above steps for 3 rounds of screening, a monoclonal colony is selected from the colony plate prepared by the third round of screening for amplification culture, a nanobody capable of specifically binding to BCMA protein is identified by monoclonal ELISA, and the colony is subjected to sequencing analysis to obtain 41 specific nanobodies against BCMA, as shown in Table 2.
TABLE 2 nanobody complete sequence
2.2.6 preparation of nanobodies
The VHH gene is amplified by taking a recombinant plasmid pMECS-Nbs containing the nanobody gene in 2.2.5 as a template, cloned into a eukaryotic expression vector pcDNA3.1-hFc in a way of enzyme digestion connection, and the plasmid is extracted and transfected into HEK293T cells after the construction is successful, the supernatant is collected after the expression is carried out for 5 days, and the recombinant nanobody is obtained by purifying the supernatant by utilizing a Ni column in an affinity chromatography way.
Example 3
3.1Biacore detection of nanobody affinity
The binding affinity of the nanobodies of this batch was measured with the antigen BCMA-mFc coated on the CM5 chip using a Biacore 8k instrument, and the results are shown in Table 3. The invention screens 41 strains of BCMA targeting nano antibodies with specific site hydrophilic mutation, and the KD value range is 1.5x10-9 ～10.0×10-12 In between, the greater the KD value, the lower the affinity.
TABLE 3 nanobody affinity data
Remarks: 1e+04=1×104 ；1E-04＝1×10-4 The method comprises the steps of carrying out a first treatment on the surface of the Other things and the like
3.2 specific analysis of anti-BCMA protein nanobodies
The affinity of the nano antibody and BCMA antigen is compared by an indirect ELISA (enzyme-linked immunosorbent assay) method, the BCMA recombinant protein is coated in a 96-hole ELISA plate, the prepared nano antibody (0.1 mug/mL) is added into the ELISA plate after the sealing is finished, and the detection is carried out by using an HRP-marked goat anti-human secondary antibody, and the result is shown in figure 4, the obtained 41 nano antibodies can be specifically combined with the BCMA protein and do not react with other irrelevant antigens, so that the nano antibodies have good specific binding activity with the BCMA protein, and can specifically target multiple myeloma.
Example 4
In vitro killing activity assay of chimeric antigen receptor modified T cells targeting BCMA on myeloma cells
Taking the nano sequences shown as SEQ ID No.18, SEQ ID No.23 and SEQ ID No.25 as an example, a chimeric antigen receptor lentiviral expression vector pWPXLD-BCMA-CAR expressing the targeted BCMA is constructed by using a molecular cloning method, and the CAR fragment further comprises a CD8 signal peptide, BCMA VHH, a hinge region (hinge), a human CD8 transmembrane region (TM), a human 4-1BB co-stimulatory signal region and a human CD3 zeta signal domain.
In the embodiment, pWPXLD-BCMA-CAR is used as a core plasmid, psPAX2 and pMD2.G are used as auxiliary plasmids, 293T cells are transfected by using a calcium phosphate transfection technology, viruses are packaged, the viruses are used for preparing BCMA-CAR-T cells, and the positive expression rate of the CAR is shown in figure 5.
Taking a multiple myeloma cell line MM.1S-mcherry expressing fluorescent protein mcherry as a target cell and BCMA-CAR-T cells as effector cells, co-culturing 2 cells according to an effective target ratio of 1 (CAR-T: target cell=2.5:1), and using a living cell workstation to monitor a fluorescence value to characterize the killing condition of the BCMA-CAR-T cells, wherein the result is shown in figure 6, the CAR-T prepared by the nano antibody has good in vitro killing activity on a natural MM cell line and is enhanced along with the enhancement of the effective target ratio.
The embodiments of the present invention have been described above with reference to the accompanying drawings, but the present invention is not limited to the above-described embodiments, which are merely illustrative and not restrictive, and many forms may be made by those having ordinary skill in the art without departing from the spirit of the present invention and the scope of the claims, which are to be protected by the present invention.