CN112159476A - Bispecific antibody for multiple myeloma and application thereof - Google Patents

Abstract

The invention discloses a bispecific antibody for multiple myeloma and application thereof, belonging to the technical field of genetic engineering antibodies. The invention takes an antibody F00 targeting human BCMA and an antibody D00 targeting human PD-1 as parent antibodies, respectively introduces 'knob' and 'hole' ends on two heavy chain Fc by using a genetic engineering technology according to a Crossmab platform, and a mammal eukaryotic expression system expresses DF 00. The invention also provides a method for expressing and purifying the bispecific antibody DF00, which is used for obtaining the target protein through HEK293 cell secretory expression and affinity chromatography purification. This bispecific antibody DF00 was able to specifically bind to both BCMA molecules on the surface of Multiple Myeloma (MM) cells and activated T cell surface immune checkpoint molecules PD-1, inhibit the immunosuppressive microenvironment in tumors and recruit T cells to the periphery of multiple myeloma cells, remodeling the function of T cells to kill MM cells.

Description

Bispecific antibody for multiple myeloma and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering antibodies, and particularly relates to a novel bispecific antibody DF00 capable of simultaneously targeting a human B Cell Maturation Antigen (BCMA) and an immunosuppressive molecule programmed death receptor 1(PD-1), which can recruit a T cell expressing the PD-1 antigen to the periphery of a multiple myeloma cell expressing the BCMA by utilizing double targeting, inhibit an immunosuppressive microenvironment and reshape the killing effect of the T cell on the multiple myeloma cell.

Background

The tumor immunotherapy is a treatment method which applies immunological principles and methods, specifically eliminates tumor tiny residual focuses, inhibits tumor growth and breaks immune tolerance by activating immune cells in vivo and enhancing anti-tumor immune response of organisms. Tumor immunotherapy is intended to overcome the mechanism of tumor immune escape, thereby re-awakening immune cells to eliminate cancer cells. Due to its small side effects and obvious therapeutic effects, it is becoming the development direction of future tumor therapy, and is called the fourth major tumor therapy technique following surgery, radiotherapy and chemotherapy. The effectiveness of immunomodulatory strategies depends on the presence of a baseline immune response and pre-existing immune release, and thus a general consensus has been reached that effector T cells play a central role in anti-tumor responses.

Multiple myeloma and tumor associated antigen BCMA

Multiple Myeloma (MM) is a malignant tumor of terminally differentiated plasma cells, and patients are mostly exposed to marrow infiltration of clonal plasma cells and monoclonal proteins in serum or urine. Multiple myeloma is diagnosed when a clear end organ damage is attributable to a plasma cell proliferative disorder or when found to indicate a high likelihood of its development. It is important to identify symptomatic multiple myeloma in need of treatment from among the prodromal stages of monoclonal immunoglobulin increase (MGUS) and smoldering multiple myeloma of unknown significance, as observations are the norm for these cases. Over the last decade, great progress has been made in the understanding of disease biology and personalized treatment approaches. Some new drugs, such as proteasome inhibitors and immunomodulatory drugs, have been added to traditional therapies (corticosteroids, alkylating agents and anthracyclines) and have led to deeper and lasting clinical responses along with high dose therapies and autologous hematopoietic stem cell transplantation. Most MM eventually recurs due to the development of resistance. Therefore, new therapeutic strategies are urgently needed, especially in high-risk relapsed and refractory multiple myeloma.

BCMA is a transmembrane glycoprotein and non-tyrosine kinase receptor, belonging to the Tumor Necrosis Factor Receptor (TNFR) superfamily, expressed almost exclusively in plasmablasts and plasma cells. In non-malignant lymphoid tissues, BCMA protein was detected in the interfollicular region of the germinal center, but not in the follicular coat region. Unlike the other two functionally related TNFR B cell activator receptor (BAFF-R) and transmembrane activator, as well as calcium modulator and cyclophilin ligand interactor (TACI), BCMA is uniquely induced in late-stage memory B cells committed to plasma cell differentiation and is present in all plasma cells. In contrast, other normal tissues did not detect BCMA transcripts except plasmacytoid dendritic cells (pdcs) with significantly lower BCMA expression compared to plasma cells of the same individual. Multiple independent studies using tissue gene expression profiling and immunohistochemistry and flow cytometry analysis showed strong expression of BCMA transcripts and proteins in plasma cells, whereas in normal tissues expression was detected weakly due to strong expression of plasma cells. BCMA-dependent biological effects in MM cells are regulated primarily by a key growth and survivin kinase B (akt), MAPK and Nuclear Factor (NF) - κ B signaling cascade. BCMA overexpression not only induces survival and proliferation of MM cells, but also induces upregulation of immunosuppressive factors (PD-L1, IL10, TGF β, etc.) on MM cells, forming an immunosuppressive microenvironment. BCMA has therefore become an ideal antigen for targeting new immunotherapeutic modalities of multiple myeloma.

(II) PD-1/PD-L1 Immunity checkpoint and bone marrow microenvironment immunosuppression

Programmed death receptor 1(PD-1) and PD-L1, a ligand for PD-1, play important roles in T cell activation, immunity and tolerance. Under normal conditions, in order to prevent the activated T cells from destroying normal human cells, the immune system can control the activation process of the T cells by activating immune check points such as PD-1/PD-L1 and the like, so as to prevent the T cells from falsely attacking the normal cells. However, cancer cells specifically recognize PD-1 on the surface of T cells by expressing the surface protein PD-L1, stealing this control mechanism, and activating immune checkpoints to suppress T cell immune activity, which leads cancer cells to escape immune recognition and thrive. The interaction of PD-1/PD-L1 also results in selective inhibition of tumor-specific T cells, since T cells, when activated upon encountering tumor antigens, induce the expression of PD-1 by T cells, thereby forming a "molecular barrier" that allows the tumor to evade the immune response.

There have been numerous studies showing that PD-L1 is expressed on pathological Plasma Cells (PC) of myeloma patients but not on normal plasma cells of healthy donors and that PD-L1 is expressed on PC in MM higher than in undefined monoclonal immunoglobulin deficiency (MGUS), and there are also reports showing a correlation between PD-L1 expression and increased risk of clinical MM progression, with significant increases in CD4 and CD 8T cell PD-1 levels detected in persistent MRD and relapsed MM patients. Therefore, the interaction between PD-L1 on MM cells and PD-1 inhibits tumor-specific cytotoxic T cells, avoids the killing effect of T cells on multiple myeloma cells, and forms an immunosuppressive microenvironment.

(III) bispecific genetically engineered antibody

Monoclonal antibody therapy is an important means for treating hematological malignancies, but monoclonal antibodies are less active. However, bispecific antibody immunotherapy does not rely on the cytotoxic mechanisms of traditional therapies, can redirect immune cells to tumors for subsequent lysis, preclinical and accumulated clinical data support the single agent efficacy of these drugs against hematological malignancies, and compared to using t-cell therapy, bispecific antibodies (BsAbs) are a ready-to-use product, easily scalable, suggesting that the development of new bispecific formats will enter an exciting era.

However, bispecific antibodies are structurally more complex than monoclonal antibodies, and bispecific antibodies face greater challenges in antibody drug development. Unlike antibody molecules in the general sense that bispecific antibodies do not exist in nature, the main challenge in the generation of bispecific IgG antibodies is the correct association of the light and heavy chains. The most primitive way in the past was that the random association of light and heavy chains into asymmetric heterodimeric IgG antibodies produced a number of unwanted by-products that had to be artificially produced by recombinant DNA or cell fusion techniques. Therefore, the molecular structure design of the double antibody is a very important key point, namely, the technical platform of the double antibody is the technical core of the double antibody.

Based on the theoretical basis research, the invention designs a bispecific antibody DF00 which simultaneously targets BCMA and PD-1 according to a Crossmab platform, and the growth of multiple myeloma and the expression of immunosuppressive factors are inhibited by utilizing that one end F00 of the bispecific antibody can recognize and combine with BCMA on the surface of multiple myeloma cells; meanwhile, the other end D00 can recognize and combine with the PD-1 antigen on the surface of the T cell, recruit the T cell to the periphery of the multiple myeloma cell, and simultaneously reshape the killing effect of the T cell on the tumor cell, thereby avoiding the immune escape of the tumor in the conventional treatment and enhancing the anti-tumor effect. Provides a new inspiration and thought for the treatment of multiple myeloma.

Disclosure of Invention

The purpose of the invention is as follows: the invention provides a bispecific antibody DF00 which has tumor immunity curative effect and can simultaneously and specifically target human BCMA and PD-1.

The technical scheme is as follows: a bispecific antibody targeting human BCMA and reactivating T cells comprising a parent antibody F00(knob end) targeting human BCMA at one end and a parent antibody D00(hole end) targeting human PD-1 at the other end. According to a CrossMab platform, a gene engineering technology is utilized to introduce ends of 'knob' (T366W, S354C) and 'hole' (T366S, L368A, Y407V and Y349C) on heavy chain Fc of two parent antibodies respectively to form a bispecific antibody DF 00.

The amino acid sequence of the heavy chain of the bispecific antibody anti-human BCMA parent antibody is shown as SEQ ID NO.1, and the nucleotide sequence of the heavy chain of the anti-human BCMA parent antibody is shown as SEQ ID NO. 2; the amino acid sequence of the light chain of the anti-human BCMA parent antibody is shown as SEQ ID NO.3, and the nucleotide sequence of the light chain of the anti-human BCMA parent antibody is shown as SEQ ID NO. 4; the amino acid sequence of the heavy chain of the anti-human PD-1 parent antibody is shown as SEQ ID NO.5, and the nucleotide sequence of the heavy chain of the anti-human PD-1 parent antibody is shown as SEQ ID NO. 6; the amino acid sequence of the light chain of the anti-human PD-1 parent antibody is shown as SEQ ID NO.7, and the nucleotide sequence of the light chain of the anti-human PD-1 parent antibody is shown as SEQ ID NO. 8.

CrossMab is a bispecific antibody technology independently developed by roche, which is capable of producing bispecific antibody molecules with correct folding and assembly using traditional therapeutic antibody production processes, and by which roche developed bispecific antibodies targeting Ang2 and VEGF simultaneously. The bispecific antibody designed and constructed in the invention is mainly based on the Roche Crossmab technology platform, and the use of the Knobs-into-holes (KiH) method in the technology platform solves the correct association of heavy chain heterodimers, but the correct association of similar light chains is still a problem for decades; the advent of Crossover technology ensured the correct association of light chains in bispecific heterodimeric IgG antibodies, which was based on the exchange of the Fab arm domains of bispecific IgG antibodies.

The two parent monoclonal antibodies F00 and D00 of the bispecific antibody have the characteristics that one end F00 is characterized by being capable of specifically targeting the BCMA (BCMA antigen) with high expression on the surface of multiple myeloma and high affinity; the other end D00 can specifically target the immune checkpoint molecule PD-1 on the surface of the T cell, block an immune suppression pathway and reshape the killing effect of the T cell. The Fc segments of two parent antibodies are mutated by using a 'Knob intuhole' technology to promote the two parent heavy chains to form heterodimers, and the light chain CL of D00 is exchanged with the reconnected CH1 by using a 'Crossover', so that the heterologous mismatch between the light chain and the heavy chain in the assembly process of the bispecific antibody is avoided, the structure of the antibody is not changed, and the affinity of the antibody and the antigen can be maintained; the expression system of the mammalian cell expresses the protein to ensure the biological activity of the bispecific antibody.

An expression vector comprising said bispecific antibody targeting human BCMA and PD-1 simultaneously.

A host cell comprising said bispecific antibody targeting human BCMA and PD-1 simultaneously.

The application of the bispecific antibody simultaneously targeting human BCMA and PD-1 in preparing a medicine for treating multiple myeloma.

Application mode of bispecific antibody DF00 targeting human BCMA and PD-1 simultaneously: the parent antibody F00 plays a targeting role to position the bispecific antibody around the multiple myeloma cells with high BCMA expression, inhibits the survival and growth of the multiple myeloma cells by blocking BCMA signal pathways, and can reduce the expression of immunosuppressive factors in a tumor microenvironment; the parent antibody D00 plays another end targeting role, and can not only recruit the activated T cells to the periphery of the multiple myeloma cells through the PD-1 antigen expressed on the T cells, but also can reshape the immune monitoring function of the T cells on the tumor cells through blocking the PD-1/PD-L1 axis of an immune checkpoint pathway.

The invention further describes:

the bispecific antibody DF00 in the invention is composed of four chains, wherein one end of the four chains is a parent antibody F00 targeting BCMA, and the Fc segment of the parent antibody F00 is transformed into a heavy chain and a light chain at a 'knob' end; the other end is a parent antibody D00 which is converted by a Crossover and targets PD-1, and the Fc section of the parent antibody D00 is transformed into a heavy chain and a light chain at a hole end.

One of the purposes of the invention is to construct a novel bispecific antibody DF00 capable of simultaneously targeting BCMA and PD-1, which has the core action force of reactivating T cells to kill tumor cells, wherein the double-antibody DF00 eliminates the microenvironment of immunosuppression on the basis that the end F00 can specifically target BCMA, and simultaneously recruits T cells to the vicinity of tumor cells to play a killing function; the second purpose is to form a set of process flow for expressing the bispecific antibody; the third objective is to evaluate the therapeutic feasibility of the bispecific antibody on MM, and to provide a new elicitation and reference scheme for the market that is currently difficult to solve the therapeutic bottleneck of MM.

The bispecific antibody is constructed by adopting a molecular biological method. Relying on an amino acid sequence of an anti-BCMA single-chain antibody 2A9 obtained by screening a phage library and optimized by a company, constructing and obtaining a knob-end anti-BCMA full-length antibody F00 heavy and light chains by PCR and overlap PCR technologies and IgG4 compression splicing, carrying out cloning and recombination, and constructing a bispecific antibody DF00 recombinant vector by utilizing hole-end anti-PD-1 full-length antibody D00 heavy and light chain sequences preserved in a laboratory; extracting plasmids containing four heavy and light chain sequences of the bispecific antibody while preserving bacteria, and transiently transfecting HEK293 cells with the four plasmids by using a transient reagent for fermentation expression; centrifuging at low temperature to obtain supernatant, separating and purifying the supernatant with Protein A column, and identifying whether expression and assembly of the expression product is correct by SDS-PAGE (sodium didecyl sulfate polyacrylamide gel electrophoresis) and WB (Western blotting); ELISA (enzyme linked immunosorbent assay) experiment analyzes the affinity ability of the antibody and antigen, FCM (flow cytometry) detects the affinity of the antibody at the cellular level, and further reveals the binding condition of the bispecific antibody and BCMA + MM cell line (NCI-H929, RPMI 8226); the identification of these two parts is the basis for the bispecific antibody DF00 to exert its function of targeting and reactivating T cells to kill target cells; the LDH lactate dehydrogenase cytotoxicity detection method shows that the PBMCs have killing effect on myeloma cells through double-antibody mediation, and further reveals that DF00 plays a drug effect mainly through enabling T cells to become a core acting force for killing tumor cells, so that the killing function is further enhanced.

Has the advantages that:

the invention discloses a novel bispecific antibody DF00, which consists of two heavy light chains of an IgG4 type antibody respectively targeting BCMA and PD-1. The DF00 can specifically target BCMA, and simultaneously recruit activated T cells to the periphery of multiple myeloma cells of high expression BCMA through a PD-1 end, so that the T cells become a core acting force for killing tumor cells, and the killing function is further enhanced.

Drawings

Figure 1 is a F00-based bispecific antibody DF00 recombinant gene map. The size of the heavy chain gene of the bispecific antibody DF00 is about 1400bp, and the size of the light chain gene is about 700 bp. VH and VL represent the heavy and light chain variable region sequences, respectively, of DF00, and CH and CL represent the heavy and light chain constant region sequences, respectively, of DF 00; SP is the signal peptide sequence. FIGS. 1A and 1B depict the construction of F00 and DF00 gene expression vectors.

FIG. 2 shows the identification of the expression purification of F00 and DF 00. FIGS. 2A and 2B are SDS-PAGE results of non-reducing (8% gel) and reducing (12% gel) SDS-PAGE of fermentatively expressed F00 and DF00 through ProteinA column, showing that F00 and DF00 have correct molecular weights, the non-reducing protein has a molecular weight of about 150kD, and the reducing protein has bands of about 50kD and 25kD, respectively; FIGS. 2C and 2D are the non-reduced and reduced WB results of F00 with DF00 after purification, demonstrating the correct assembly of F00 with DF 00; the primary antibodies incubated in FIGS. 2C and 2D were goat anti-human IgG (H + L); and M is a protein Marker.

FIG. 3 is an ELISA demonstrating the binding affinity of DF00 to BCMA and PD-1. FIG. 3A is a graph of the concentration-dependent ELISA binding curves for F00 and DF00, respectively, to antigen BCMA, half maximal effector concentration EC₅₀10.43nM and 21.34nM, respectively; FIG. 3B is a graph of the concentration-dependent ELISA binding curves for D00 and DF00, respectively, to antigen PD-1 at half maximal effector concentration EC₅₀14.81nM and 30.21nM, respectively; the double antibody DF00 was slightly down-regulated in affinity compared to the two naked antibodies F00 and D00, but the overall decrease was not large.

Figure 4 is a flow chart demonstrating binding affinity at the DF00 cell level. Figures 4A-C depict flow-binding of DF00 to HEK293, HEK293-BCMA and HEK293-PD-1, respectively, with binding rates of 4.29%, 99.3% and 99.9%, respectively, showing that dual anti-DF 00 also shows better binding affinity to BCMA, PD-1 at the cellular level.

FIG. 5 shows the binding of DF00 to different BCMA-expressing human myeloma cell lines by flow. FIG. 5A depicts F00 and BCMA⁺Myeloma cell line NCI-H929 and RPMI8226 are combined, the combination rate is 95.8 percent and 42.7 percent respectively, and the combination rate is different from that of BCMA^-Raji cell binding Rate of11.2%, essentially no binding; FIG. 5B depicts DF00 and BCMA⁺Myeloma cell line NCI-H929 and RPMI8226 are combined, the combination rate is 86.3 percent and 35.7 percent respectively, and the combination rate is different from that of BCMA^-The Raji cells are basically not combined, and the combination rate is only 4.58%; meanwhile, DF00 showed comparable binding rates to F00 for the same myeloma cell line.

FIG. 6 is a graph showing the results of DF 00-mediated killing of myeloma cells NCI-H929 and RPMI8226 by concentration-dependent PBMCs. FIGS. 6A and 6B depict DF 00-mediated killing of NCI-H929 and RPMI8226 by concentration-dependent PBMCs, respectively, and the results show that DF00 can activate T-cell-dominated immunocytes of PBMCs to kill tumor cells by approximately 60% of NCI-H929 and 40% of RPMI8226 cells, respectively, and has significantly better cytotoxicity than F00; the results show that DF00 exerts the drug effect mainly through the core action of the T cells for killing the tumor cells, recruits a large amount of activated T cells to the periphery of the tumor cells and further enhances the killing function.

Fig. 7 is an experiment of DF 00-mediated cytotoxicity of gradient effect targets versus myeloma cells expressing different BCMAs. Fig. 7A depicts the cytotoxic effect of DF 00-mediated gradient effect targets versus RPMI8226 and R-BCMA, fig. 7B is the statistical analysis result of fig. 7A; FIG. 7C depicts the cytotoxic effect of DF 00-mediated gradient effect target versus NCI-H929 and NCI-H929-shBCMA, FIG. 7D is the statistical analysis of FIG. 7C; the result shows that DF00 can obviously enhance the killing effect of PBMCs on myeloma cells, and the killing effect is also obviously enhanced under the condition of higher effective target ratio, and meanwhile, the killing effect also depends on the expression of BCMA on the surface of the myeloma cells, and the higher the BCMA expression is, the killing effect is correspondingly enhanced.

Detailed Description

Example 1 construction of bispecific antibody DF 00.

Firstly, a BCMA-targeted single-chain antibody 2A9 and a PD-1-targeted antibody D00 which are obtained by screening in a laboratory through a phage display technology and optimized by a company are taken as template design primers to call heavy and light chain variable region genes, and the IgG4 constant region (Fc segment is mutated through nanobs-int-holes, Fab segment is transformed through Crossover) genes are taken as template design primersThe heavy and light chain constant region gene is adjusted, and the variable region and the constant region of the heavy and light chain are respectively connected by overlap PCR technology to construct the heavy and light chain gene of the bispecific antibody DF 00; EcoRI and NotI double enzyme digestion is carried out on the obtained PCR product and a pCMV3 vector respectively, a target fragment and the double enzyme digestion vector are recovered, and then T4 DNA ligase is used for connecting overnight at 16 ℃, so as to obtain four groups of recombinant plasmids pCMV3-F00-H/L-Chain (knob end) and pCMV3-D00-H/L-Chain (hole end). CaCl₂The method comprises the steps of transforming the enzyme-linked product into escherichia coli DH5 alpha competent cells, coating a plate, selecting positive monoclonals for sequencing comparison, selecting bacteria with correct sequencing results, amplifying and storing.

Example 2 expression purification and characterization of bispecific antibody DF 00.

Firstly, four recombinant plasmids pCMV3-F00-H/L-Chain (knob end) and pCMV3-D00-H/L-Chain (hole end) are transfected into HEK293 cells by PEI transfection reagent according to a certain proportion, the cells are changed and passaged the next day, the cell fermentation culture scale is gradually enlarged, the cell culture solution is collected, samples are filtered by a 0.22 mu m filter membrane and then subjected to protein A column affinity chromatography purification, and finally a large amount of target proteins are obtained. And respectively carrying out 8% non-reduction and 12% reduction SDS-PAGE electrophoresis, identifying the molecular weight and the assembly condition by Western blotting, and carrying out preliminary verification on the target protein. The results are shown in fig. 2, F00 was successfully expressed with DF00 and assembled correctly.

Example 3 binding affinity of DF00 to BCMA and PD-1 was verified by ELISA analysis.

The antigens BCMA and PD-1 were first treated with 50mM NaHCO₃Diluting the buffer solution to 1 mu g/ml, and coating the buffer solution on an activated enzyme label plate at 4 ℃ overnight; washing with PBS to remove antigen not bound to the ELISA plate, sealing 5% skimmed milk at 37 deg.C for 2 hr; after washing the plate again with PBST, a series of dilutions of the antibody (500nM-0.244nM/250nM-0.122nM) were incubated at 37 ℃ for 2 h; then PBST washing plate again, washing away the antibody not combined with the antigen, using goat anti-human IgG (H + L) with HRP label to incubate, 37 ℃, 2H; finally, after PBST washing the plate, TMB color developing solution is used for developing color, when obvious color gradient appears, 2M dilute sulphuric acid is used for stopping reaction, and an enzyme-labeling instrument is used for detecting OD₄₅₀-OD₆₃₀As the final result. The results are showninFigure 3, dual anti-DF 00 was comparable in affinity to the two parent antibodies at the molecular level.

Example 4 flow validation of targeting affinity of DF00 at cellular level.

Separately constructed BCMA and PD-1 overexpression stable HEK293-BCMA/PD-1 cell strains, in the experiment, 2 x 10 is firstly added⁵Individual HEK293, HEK293-BCMA and HEK293-PD-1 cells were resuspended in 250. mu.l PBS to prepare single cell suspensions. An equal volume of DF00 at a concentration of 250nM was then added to each cell suspension for co-incubation for 1 h. Washing with PBS to remove unbound DF00, incubating tumor cells with an anti-human IgG (H + L) antibody with a FITC label, detecting the proportion of cells bound with DF00 on the surface of the tumor cells by a flow cytometer after washing with PBS, and calculating the binding rate of DF00 and HEK293-BCMA/PD-1 cells. The results are shown in fig. 4, and the double anti-DF 00 also shows better binding affinity to BCMA, PD-1 at the cellular level.

Example 5 flow cytometry was used to test the binding capacity of the bispecific antibody DF00 to different BCMA-expressing human-derived multiple myeloma cell lines.

The human myeloma cell line NCI-H929 with high BCMA expression and RPMI-8226 with low BCMA expression are selected as research objects, and the Raji cell with negative BCMA is selected as a control. In this experiment, 2 x 10 was first introduced⁵Individual NCI-H929, RPMI8226 and Raji cells were resuspended in 250. mu.l PBS to prepare single cell suspensions. An equal volume of F00 at a concentration of 250nM and DF00 were then added to each cell suspension for 1h of incubation. After unbound F00 and DF00 are removed by PBS washing, an anti-human IgG (H + L) antibody with a FITC label is used for incubating with tumor cells, the ratio of cells bound with F00 and DF00 on the surface of the tumor cells is detected by a flow cytometer after PBS washing, and the binding rate of DF00 with NCI-H929, RPMI8226 and Raji cells is calculated. The results are shown in fig. 5, the higher the binding rate of DF00 to myeloma cells with higher BCMA expression, and the binding of F00 to myeloma cells was not significantly reduced compared to DF 00.

Example 6 LDH lactate dehydrogenase cytotoxicity assay the killing of myeloma cells NCI-H929 and RPMI8226 by DF 00-mediated concentration-dependent PBMCs as effector cells and NCI-H929 and RPMI8226 as target cells was examined.Firstly, adding 1000U/mL IL-2 into the culture supernatant of PBMCs 24h in advance to stimulate effector cells, diluting the culture supernatant by concentration times and adding DF00 (from 1000nM to 7.8125nM) under the condition of an effective target ratio of 50:1, and detecting the killing effect of DF00 on T cell-dominant immune cells in the PBMCs on target cells under the condition of different concentrations of DF 00. Wherein a target cell spontaneous release group is set: target cells + culture medium; effector cell spontaneous release group: effector cells + culture medium; background blank control group: a cell-free medium; volume corrected control group: LDH lysate + culture medium; target cell maximum release group: adding 10% LDH cell lysate into the target cells and the culture medium 1h before the culture time is finished, and repeatedly blowing, beating and uniformly mixing; each group was set with three parallel secondary wells, incubated in a 5% CO2 incubator at 37 ℃ for a period of time, centrifuged at 400g for 5min, 60. mu.l of culture supernatant was aspirated from each well to a new 96-well plate, LDH working solution was prepared, 30. mu.l of working solution was added to each well and mixed well, incubated at room temperature in the dark for 30min, OD was measured₄₉₀-OD₆₃₀The absorbance of the background blank control group (where the absorbance of the target cell maximum release group is subtracted from the absorbance of the volume correction group) is subtracted from the absorbance of each group, and the lysis rate is calculated as (experimental group-effector cell spontaneous release group-target cell spontaneous release group OD value)/(target cell maximum release group-target cell spontaneous release group OD value) × 100%. The results are shown in fig. 6, DF00 mediated killing of myeloma cells is concentration dependent, and the mediated cytotoxicity is significantly better than F00.

Example 7 LDH lactate dehydrogenase cytotoxicity assay to detect killing of myeloma cells expressing different BCMA by DF 00-mediated gradient effect target versus PBMCs as effector cells and RPMI8226/R-BCMA, NCI-H929/NCI-H929-shBCMA as target cells. Adding 1000U/mL IL-2 into the culture supernatant of the PBMCs 24h in advance to stimulate effector cells, and detecting the killing effect of immune cells mainly comprising T cells in the PBMCs activated by DF00 on target cells under the conditions of a certain concentration of 500nM DF00 and different effect-target ratios (5:1/50:1/500: 1). The result is shown in fig. 7, DF00 can significantly enhance the killing effect of PBMCs on myeloma cells compared to their parental antibodies, and the killing effect is also significantly enhanced under the condition of higher effective target ratio, and the killing effect is also dependent on the expression of BCMA on the surface of myeloma cells, and the higher the expression of BCMA, the higher the killing effect is.

Sequence listing

<110> university of Chinese pharmacy

<120> bispecific antibody for multiple myeloma and application thereof

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<212> PRT

<213> DF00 knob terminal heavy chain amino acid sequence (2 Ambystoma laterale x Ambystoma jeffersonanum)

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Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

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Leu Arg Gly Ala Arg Cys Met Ala Glu Val Gln Leu Gln Gln Ser Gly

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Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr Ala

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Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn Gly

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Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr Ala

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Asp Thr Ser Ser Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr Ser

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Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Trp Val Tyr Trp Gly Gln

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Gly Thr Thr Leu Thr Val Ser Thr Ala Ser Thr Lys Gly Pro Ser Val

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Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

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Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

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Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

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Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

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Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

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Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

225 230 235 240

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

245 250 255

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

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Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

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Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

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Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

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Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

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Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

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Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

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Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

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His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

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<213> DF00 knob terminal heavy chain nucleotide sequence (2 Ambystoma laterale x Ambystoma jeffersonanum)

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atggacatga gagtgccagc acagctgctg ggactgctgc tgctgtggct gaggggagca 60

agatgcatgg cagaggtgca gctgcagcag agcggagcag agctggtgaa gcctggagcc 120

tccgtgaagc tgtcttgtac cgccagcggc ttcaacatca aggatacata catgcactgg 180

gtgaagcaga ggccagagca gggactggag tggatcggca gaatcgaccc agccaacggc 240

aataccaagt acgatcccaa gtttcagggc aaggccacca tcacagccga caccagctcc 300

aatacagcct atctgcagct gtctagcctg acctccgagg atacagccgt gtactattgc 360

gcaagatggg tgtactgggg acagggaacc acactgaccg tgtccacagc tagcaccaag 420

ggccccagcg tgttccccct ggccccttgc agcagaagca ccagcgagag cacagccgcc 480

ctgggctgcc tggtgaagga ctacttcccc gagcccgtga ccgtgtcctg gaacagcggc 540

gctctgacca gcggcgtgca taccttcccc gccgtgctcc agagcagcgg actgtactcc 600

ctgagcagcg tggtgaccgt gccttccagc agcctgggca ccaagaccta cacctgcaac 660

gtggaccaca agcccagcaa caccaaggtg gacaagagag tggagagcaa gtacggccct 720

ccctgccccc cttgccctgc ccccgagttc ctgggcggac ctagcgtgtt cctgttcccc 780

cccaagccca aggacaccct gatgatcagc agaacccccg aggtgacctg cgtggtggtg 840

gacgtgtccc aggaggaccc cgaggtccag tttaattggt acgtggacgg cgtggaagtg 900

cataacgcca agaccaagcc cagagaggag cagttcaaca gcacctacag agtggtgtcc 960

gtgctgaccg tgctgcacca ggactggctg aacggcaagg aatacaagtg caaggtctcc 1020

aacaagggcc tgcctagcag catcgagaag accatcagca aggccaaggg ccagccacgg 1080

gagccccagg tctacaccct gccaccttgc caagaggaga tgaccaagaa ccaggtgtcc 1140

ctgtggtgtc tggtgaaagg cttctatccc agcgatatcg ccgtggagtg ggagagcaac 1200

ggccagcccg agaacaacta caagaccacc ccccctgtgc tggacagcga cggcagcttc 1260

ttcctgtact ccaagctgac cgtggacaag tccagatggc aggagggcaa cgtcttcagc 1320

tgctccgtga tgcacgaggc cctgcacaac cactacaccc agaagtccct gagcctgagc 1380

ctgggcaagt ga 1392

<210> 3

<211> 237

<212> PRT

<213> DF00 knob terminal light chain amino acid sequence (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 3

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Arg Gly Ala Arg Cys Asp Val Val Met Thr Gln Ser Pro Ser Ser

20 25 30

Met Tyr Ala Ser Leu Gly Glu Arg Val Thr Ile Thr Cys Lys Ala Ser

35 40 45

Gln Asp Ile Asn Ser Tyr Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys

50 55 60

Ser Pro Lys Thr Leu Ile Tyr Arg Ala Asn Arg Leu Val Asp Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr

85 90 95

Ile Ser Ser Leu Glu Tyr Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln

100 105 110

Tyr Asp Glu Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

115 120 125

Lys Arg Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser

130 135 140

Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn

145 150 155 160

Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala

165 170 175

Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys

180 185 190

Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp

195 200 205

Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu

210 215 220

Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 4

<211> 714

<212> DNA

<213> DF00 knob terminal light chain nucleotide sequence (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 4

atggacatga gagtgccagc acagctgctg ggactgctgc tgctgtggct gaggggagca 60

agatgcgacg tggtcatgac ccagagcccc tcctctatgt atgcctccct gggcgagcgc 120

gtgaccatca cctgtaaggc ctcccaggat atcaactctt acctgagctg gttccagcag 180

aagcccggca agtctcctaa gaccctgatc tatagggcaa ataggctggt ggacggagtg 240

ccatctcggt tttctggcag cggctccggc caggattaca gcctgacaat cagctccctg 300

gagtatgagg acatgggcat ctactattgc ctgcagtacg atgagttccc ttataccttt 360

ggcggcggca caaagctgga gatcaagcgg cgaactgtgg ctgcaccatc tgtcttcatc 420

ttcccgccat ctgatgagca gttgaaatct ggaactgcct ctgttgtgtg cctgctgaat 480

aacttctatc ccagagaggc caaagtacag tggaaggtgg ataacgccct ccaatcgggt 540

aactcccagg agagtgtcac agagcaggac agcaaggaca gcacctacag cctcagcagc 600

accctgacgc tgagcaaagc agactacgag aaacacaaag tctacgcctg cgaagtcacc 660

catcagggcc tgagctcgcc cgtcacaaag agcttcaaca ggggagagtg ttag 714

<210> 5

<211> 483

<212> PRT

<213> DF00 hole end heavy chain amino acid sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 5

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Arg Gly Ala Arg Cys Gln Gly Gln Leu Val Gln Ser Gly Ala Glu

20 25 30

Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly

35 40 45

Tyr Thr Phe Thr Asp Tyr Glu Met His Trp Val Arg Gln Ala Pro Ile

50 55 60

His Gly Leu Glu Trp Ile Gly Val Ile Glu Ser Glu Thr Gly Gly Thr

65 70 75 80

Ala Tyr Asn Gln Lys Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Lys

85 90 95

Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp

100 105 110

Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Ile Thr Thr Val Ala Thr

115 120 125

Thr Tyr Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr

130 135 140

Val Ser Ser Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

145 150 155 160

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

165 170 175

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn

180 185 190

Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser

195 200 205

Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala

210 215 220

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly

225 230 235 240

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Glu Ser

245 250 255

Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly

260 265 270

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

275 280 285

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln

290 295 300

Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val

305 310 315 320

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr

325 330 335

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

340 345 350

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile

355 360 365

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

370 375 380

Cys Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser

385 390 395 400

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

405 410 415

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

420 425 430

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

435 440 445

Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

450 455 460

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

465 470 475 480

Leu Gly Lys

<210> 6

<211> 1452

<212> DNA

<213> DF00 hole end heavy chain nucleotide sequence (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 6

atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct gagaggtgcc 60

agatgtcagg gccagctggt gcagagcggc gccgaggtga agaagcccgg cgccagcgtg 120

aaggtgagct gcaaggccag cggctacacc ttcaccgact acgagatgca ctgggtgaga 180

caggccccca tccacggcct ggagtggatc ggcgtgatcg agagcgagac cggcggcacc 240

gcctacaacc agaagttcaa gggcagagtg accatcaccg ccgacaagag caccagcacc 300

gcctacatgg agctgagcag cctgagaagc gaggacaccg ccgtgtacta ctgcgccaga 360

gagggcatca ccaccgtggc caccacctac tactggtact tcgacgtgtg gggccagggc 420

accaccgtga ccgtgagcag ccgaactgtg gctgcaccat ctgtcttcat cttcccgcca 480

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 540

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 600

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 660

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 720

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gtgagagcaa gtacggccct 780

ccctgccccc cttgccctgc ccccgagttc ctgggcggac ctagcgtgtt cctgttcccc 840

cccaagccca aggacaccct gatgatcagc agaacccccg aggtgacctg cgtggtggtg 900

gacgtgtccc aggaggaccc cgaggtccag tttaattggt acgtggacgg cgtggaagtg 960

cataacgcca agaccaagcc cagagaggag cagttcaaca gcacctacag agtggtgtcc 1020

gtgctgaccg tgctgcacca ggactggctg aacggcaagg aatacaagtg caaggtctcc 1080

aacaagggcc tgcctagcag catcgagaag accatcagca aggccaaggg ccagccacgg 1140

gagccccagg tctgcaccct gccacctagc caagaggaga tgaccaagaa ccaggtgtcc 1200

ctgagctgtg ccgtgaaagg cttctatccc agcgatatcg ccgtggagtg ggagagcaac 1260

ggccagcccg agaacaacta caagaccacc ccccctgtgc tggacagcga cggcagcttc 1320

ttcctggtgt ccaagctgac cgtggacaag tccagatggc aggagggcaa cgtcttcagc 1380

tgctccgtga tgcacgaggc cctgcacaac cactacaccc agaagtccct gagcctgagc 1440

ctgggcaagt ga 1452

<210> 7

<211> 232

<212> PRT

<213> DF00 hole end light chain amino acid sequence (2 Ambystoma laterale x Ambystoma jeffersonanium)

<400> 7

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Arg Gly Ala Arg Cys Asp Val Val Met Thr Gln Ser Pro Leu Ser

20 25 30

Leu Pro Val Thr Leu Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser

35 40 45

Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu

50 55 60

Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn

65 70 75 80

Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

85 90 95

Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val

100 105 110

Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Leu Thr Phe Gly Gln Gly

115 120 125

Thr Lys Leu Glu Ile Lys Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

130 135 140

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

145 150 155 160

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

165 170 175

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

180 185 190

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

195 200 205

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

210 215 220

Asn Thr Lys Val Asp Lys Arg Val

225 230

<210> 8

<211> 699

<212> DNA

<213> DF00 hole end light chain nucleotide sequence (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 8

atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct gagaggtgcc 60

agatgtgatg tggtgatgac ccagagcccg ctgagcctgc cggtgaccct gggccagccg 120

gcgagcatta gctgccgcag cagccagagc attgtgcata gcaacggcaa cacctatctg 180

gaatggtatc tgcagaaacc gggccagagc ccgcagctgc tgatttataa agtgagcaac 240

cgctttagcg gcgtgccgga tcgctttagc ggcagcggca gcggcaccga ttttaccctg 300

aaaattagcc gcgtggaagc ggaagatgtg ggcgtgtatt attgctttca gggcagccat 360

gtgccgctga cctttggcca gggcaccaaa ctggaaatta aagctagcac caagggcccc 420

agcgtgttcc ccctggcccc ttgcagcaga agcaccagcg agagcacagc cgccctgggc 480

tgcctggtga aggactactt ccccgagccc gtgaccgtgt cctggaacag cggcgctctg 540

accagcggcg tgcatacctt ccccgccgtg ctccagagca gcggactgta ctccctgagc 600

agcgtggtga ccgtgccttc cagcagcctg ggcaccaaga cctacacctg caacgtggac 660

cacaagccca gcaacaccaa ggtggacaag agagtgtag 699

Claims (6)

Translated fromChinese

1.一种双特异性抗体，其特征在于，该双特异性抗体包括抗人 BCMA 与抗人PD-1两个亲本全长抗体的可变区序列；所述抗人BCMA亲本抗体的重链的氨基酸序列如SEQ ID NO.1所示，抗人BCMA亲本抗体的轻链的氨基酸序列如SEQ ID NO.3所示；所述抗人PD-1亲本抗体的重链的氨基酸序列如SEQ ID NO.5所示，所述抗人PD-1亲本抗体的轻链的氨基酸序列如SEQ ID NO.7所示。1. a bispecific antibody, it is characterized in that, this bispecific antibody comprises the variable region sequence of two parental full-length antibodies of anti-human BCMA and anti-human PD-1; The heavy chain of described anti-human BCMA parental antibody The amino acid sequence of the anti-human BCMA parental antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain of the anti-human BCMA parental antibody is shown in SEQ ID NO.3; the amino acid sequence of the heavy chain of the anti-human PD-1 parental antibody is shown in SEQ ID NO.3. As shown in NO.5, the amino acid sequence of the light chain of the anti-human PD-1 parental antibody is shown in SEQ ID NO.7.2.编码权利要求1所述的双特异性抗体的核酸分子，其特征在于，所述抗人BCMA亲本抗体的重链的核苷酸序列如SEQ ID NO.2所示，所述抗人BCMA亲本抗体的轻链的核苷酸序列如SEQ ID NO.4所示；所述抗人PD-1亲本抗体的重链的核苷酸序列如SEQ ID NO.6所示，所述抗人PD-1亲本抗体的轻链的核苷酸序列如SEQ ID NO.8所示。2. The nucleic acid molecule encoding the bispecific antibody of claim 1, wherein the nucleotide sequence of the heavy chain of the anti-human BCMA parental antibody is shown in SEQ ID NO.2, and the anti-human BCMA The nucleotide sequence of the light chain of the parent antibody is shown in SEQ ID NO. 4; the nucleotide sequence of the heavy chain of the anti-human PD-1 parent antibody is shown in SEQ ID NO. 6, the anti-human PD The nucleotide sequence of the light chain of the -1 parent antibody is shown in SEQ ID NO.8.3.一种表达载体，其特征在于，包含权利要求2所述的核酸分子。3. An expression vector, comprising the nucleic acid molecule of claim 2.4.一种宿主细胞，其特征在于，包含权利要求3所述的表达载体。4. A host cell comprising the expression vector of claim 3.5.根据权利要求1所述抗体、权利要求3所述的表达载体或权利要求4所述的宿主细胞在制备治疗肿瘤药物中的应用。5 . The application of the antibody according to claim 1 , the expression vector according to claim 3 or the host cell according to claim 4 in the preparation of a drug for treating tumors. 6 .6.根据权利要求5所述的应用，其特征在于所述的肿瘤为多发性骨髓瘤、白血病或淋巴瘤。6. The application according to claim 5, wherein the tumor is multiple myeloma, leukemia or lymphoma.

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