Bispecific antibody LILRB4-CD16A as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a bispecific antibody LILRB4-CD16A as well as a preparation method and application thereof. In order to effectively utilize an LILRB4 target spot to treat MM and reduce defects of CAR-T cells or T cell treatment, the invention provides a bispecific antibody LILRB4-CD16A, and the bispecific antibody can link NK cells and LILRB4 positive MM cells with high specificity, so that the NK cells kill tumor cells in a targeted manner. The invention also provides application of the bispecific antibody in preparation of drugs for resisting multiple myeloma, especially application of the bispecific antibody in combination with a cGAS-STING signaling pathway agonist for tumor treatment. The invention provides a new thought for NK cell targeted therapy of recurrent / refractory MM, and has important practical significance.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a bispecific antibody LILRB4-CD16A and a preparation method and use thereof. Background Art

[0002] Multiple myeloma (MM) is a bone marrow plasma cell cancer that accounts for approximately 10% of hematologic malignancies and 1% of all cancers. The disease often leads to hypercalcemia, renal failure caused by monoclonal immunoglobulin free light chains, bone destruction, and pernicious anemia due to bone marrow failure. In the precancerous stage, heterogeneous plasma cells proliferate and develop a monoclonal gammopathy of undetermined significance. Once transformed into MM, only a small percentage of cases can be cured or remain dormant indefinitely, while the majority will relapse.

[0003] Thalidomide and lenalidomide were the first drugs used to alleviate disease. However, these treatments lacked targeted therapy and had significant side effects, including cytopenias, weakness, nausea, and cardiotoxicity. With the development of immunotherapy, monoclonal antibodies have ushered in a new era of immunotherapy for multiple myeloma. First, the FDA has approved the anti-CD38 monoclonal antibodies daratumumab and isatuximab for the treatment of multiple myeloma, as well as the anti-SLAMF7 monoclonal antibody elotuzumab. CD38 is widely expressed in lymphocytes and myeloid subsets, with high expression in normal plasma cells. While mediating tumor cell killing, it also kills healthy plasma cells and immune cells. Elotuzumab, on the other hand, relies primarily on natural killer (NK) cells for its tumor cell killing. Clinically, it has little therapeutic effect when used alone. It is only effective when used in combination with immunomodulatory imide drugs, but this increases the risk of side effects. Newer immunotherapies include chimeric antigen receptor T cells (CARTs) and T cell-binding bispecific antibodies. BCMA-targeting CARTs and BCMA / CD3 bispecific antibodies have shown responses in relapsed / refractory multiple myeloma in clinical trials. A phase II KarMMa trial evaluated the efficacy and safety of BCMA CAR-T cells in relapsed / refractory myeloma. The objective response rate was 73%, and the median progression-free survival was 8.8 months (95% CI, 5.6–11.6 months). Cytokine release syndrome occurred in 84% of patients, neurotoxicity in 23%, anemia in 70%, and thrombocytopenia in 63%. Consequently, chimeric antigen receptor T cells (CARTs) and T cell-binding bispecific antibodies are associated with significant side effects during treatment, making them not reliable cures. Furthermore, BCMA can be genetically lost during treatment, leading to drug resistance. Long-term treatment targeting BCMA can severely deplete normal plasma cells, significantly increasing the risk of infection in patients with relapsed / refractory multiple myeloma. Therefore, it is urgent to develop new and effective targets for the treatment of MM.

[0004] LILRB4 is an immunosuppressive receptor belonging to the LILRB family. It inhibits T cell function and promotes leukemic cell infiltration in monocytic leukemia cells by creating an immunosuppressive microenvironment. Recent studies have found that LILRB4 is highly expressed in multiple myeloma (MM), with even higher expression in extramedullary MM cells, and is significantly negatively correlated with overall survival in MM patients. In vitro and in vivo studies have also demonstrated that LILRB4 knockout or antibody blockade significantly inhibits MM cell proliferation. Furthermore, LILRB4-targeted CAR-T cells have been shown to effectively eliminate MM cells and partially block the MM immunosuppressive tumor microenvironment. Therefore, LILRB4 is a promising target for the treatment of MM. However, therapies based on CAR-T cells or T cell engagers are inherently associated with T cell graft-versus-host disease, are difficult to manufacture, and require long cycles. Therefore, the development of safer and more effective treatments and drugs is crucial.

[0005] The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway represents a central innate immune mechanism with potent anti-tumor activity. As a cytoplasmic DNA sensor, cGAS detects exogenous DNA and generates cGAMP, which binds to and activates STING. Activated STING then binds to IRF3 and NF-κB, driving the production of type I interferons and proinflammatory cytokines. This cascade amplifies the cytotoxic immune response, making cGAS–STING activation crucial for promoting anti-cancer immunity. Studies have demonstrated that agonists of the cGAS-STING pathway can significantly enhance the anti-tumor activity of natural killer (NK) cells. However, to date, no studies have examined the combination of cGAS-STING signaling pathway agonists with specific dual antibodies to enhance the anti-tumor efficacy of NK cells. Summary of the Invention

[0006] In view of this, in order to effectively utilize the LILRB4 target to treat MM and reduce the defects of CAR-T cell or T cell therapy, the present invention aims to provide a bispecific antibody that highly specifically links NK cells with LILRB4-positive MM cells, allowing NK cells to target and kill tumor cells.

[0007] The present invention provides a bispecific antibody LILRB4-CD16A, which comprises: an anti-LILRB4 scFv and an anti-CD16A scFv, wherein the anti-LILRB4 scFv and the anti-CD16A scFv are connected via a human IgG4-Fc segment.

[0008] Furthermore, in the above-mentioned bispecific antibody LILRB4-CD16A, the specific connection mode is CD16A(VLVH)-Gly4Ser-hFc-(Gly4Ser)3-LILRB4(VLVH).

[0009] Furthermore, in the above-mentioned bispecific antibody LILRB4-CD16A, the amino acid sequences of VL and VH of the anti-LILRB4 scFv are shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0010] SEQ ID NO:1 Amino acid sequence of VL of LILRB4 scFv

[0011] DIQMTQSPSSSLSASVGDRVTITTCQASESINSIYLAWYQQKPGKAPKLLIYRASTLASGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYDWGDVENTFGGGTKVEIK.

[0012] SEQ ID NO:2 Amino acid sequence of VH of LILRB4 scFv

[0013] EVQLLESGGGLVQPGGSLRLSCAASGIDFSNHYYIYWVRQAPGKGLEWIGCIFSGDSA STYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGMSTNDWASDLWGQGTL VTVSS.

[0014] Furthermore, in the above-mentioned bispecific antibody LILRB4-CD16A, the amino acid sequences of VL and VH of the anti-CD16A scFv are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0015] SEQ ID NO:3 Amino acid sequence of VL of CD16A scFv

[0016] SYVLTQPSSVSVAPGQTATISCGGHNIGSKNVHWYQQRPGQSPVLVIYQDNKRPSGIPE RFSGSNSGNTATLTISGTQAMDEADYYCQVWDNYSVLFGGGTKLTVL.

[0017] SEQ ID NO:4 Amino acid sequence of VH of CD16A scFv

[0018] QVQLVQSGAEVKKPGESLKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGAIEPMY GSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSAYYYDFADYWGQGTL VTVSS.

[0019] In a second aspect, the present invention further provides a nucleic acid molecule encoding the aforementioned bispecific antibody. The nucleic acid according to an embodiment of the present invention may encode a bispecific antibody that can simultaneously target LILRB4 and CD16A.

[0020] In a third aspect, the present invention provides a vector comprising the aforementioned nucleic acid. The vector constructed using the present invention can effectively express the aforementioned bispecific antibody. Furthermore, the vector is a pVAX vector.

[0021] A fourth aspect of the present invention provides a host cell. The host cell carries the aforementioned nucleic acid or vector, or expresses the aforementioned bispecific antibody. The cell is obtained by transfection or transformation of the vector, and under appropriate conditions, the cell can efficiently express the aforementioned bispecific antibody. Furthermore, the host cell is a CHO cell.

[0022] In a fifth aspect, the present invention provides a use of the aforementioned bispecific antibody, nucleic acid molecule, vector, and host cell in the preparation of an anti-tumor drug. Furthermore, the tumor is multiple myeloma.

[0023] In a sixth aspect, the present invention provides a pharmaceutical composition comprising: the aforementioned bispecific antibody, the aforementioned nucleic acid, the aforementioned vector, or the aforementioned host cell.

[0024] Furthermore, the pharmaceutical composition further comprises a cGAS-STING signaling pathway agonist. Furthermore, the cGAS-STING signaling pathway agonist is SR-717.

[0025] The beneficial effects of the present invention are:

[0026] This study prepared a bispecific antibody, LILRB4-CD16A (BiKE), that can highly specifically link NK cells with LILRB4-positive MM cells, enabling NK cells to target and kill tumor cells. The study also validated the efficacy of the NK cell adapter in killing MM in vitro and in vivo, providing new insights into NK cell-targeted therapy for relapsed / refractory MM. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1Figure 4 shows the expression and activity verification of BiKE. A is a schematic diagram of the BiKE structure. B is a schematic diagram of BiKE-mediated NK cell tumor killing. C is SDS gel electrophoresis of non-reduced and reduced BiKE proteins, stained with Coomassie Brilliant Blue. D is an ELISA test of the affinity of different concentrations of BiKE for the LILRB4 antigen. E is an ELISA test of the affinity of different concentrations of BiKE for the CD16A antigen. F is a flow cytometric test of the binding ability of BiKE to MM1.S, NK, and K562 cells. G is a flow cytometric test of the regulation of NK cell CD69 expression by monoclonal antibodies and bispecific antibodies in the co-culture system after PBMCs were co-cultured with LILRB4+MM1.S cells for 4 hours.

[0028] Figure 2 Figure 1 shows BiKE-mediated NK cell lysis of MM cells. A shows flow cytometric analysis of CD56, CD3, and CD16 expression on purified and expanded NK cells. B shows the cytotoxicity of MM1.S cells when co-cultured with NK cells at different effector-target ratios. C and D show the NK cell-mediated cytotoxicity of tumor cells when co-cultured with MM1.S cells at a 1:1 effector-target ratio. E shows flow cytometric analysis of the percentage of MM1.S cells mediated by BiKE at different concentrations. F shows the co-culture of calcein-labeled NK cells with MM1.S cells stably expressing mCherry. G shows calcein-labeled MM1.S cells, then co-cultured with NK cells at a 1:1 ratio for 4 hours. Cytotoxicity was assessed by measuring the release of free calcein.

[0029] Figure 3 Figure 1 shows the enhancement of BiKE-mediated NK cell cytotoxicity by cGAS-STING signaling agonists. A shows the effect of SR-717 and BiKE on NK cell cytotoxicity in a 1:1 co-culture of NK cells with MM1.S cells. B and C show flow cytometry analysis of CD107a expression on NK cells under different conditions of SR-717 and BiKE co-culture. D shows TNF-α expression. E shows IFN-γ expression. The results were repeated for three donors.

[0030] Figure 4 Figure 1 shows the promotion of NK cell suppression of tumors in vivo by BiKE combined with SR-717. A is a schematic diagram of the NK cell combined with antibody therapy protocol for C-NKG mouse tumors. B is a representative bioluminescence image showing the time course of tumor cells after different treatments, with consistent quantification of tumor flux using Clinx IVScopeEQ Capture software (n = 5 mice per group). C is a comparative analysis of overall survival of tumor-bearing mice using the log-rank test (*P < 0.05 vs. control group). DETAILED DESCRIPTION

[0031] The present invention provides a bispecific antibody LILRB4-CD16A, which comprises: an anti-LILRB4 scFv and an anti-CD16A scFv, wherein the anti-LILRB4 scFv and the anti-CD16A scFv are connected via a human IgG4-Fc segment.

[0032] The present invention also provides the use of the bispecific antibody LILRB4-CD16A in the preparation of an anti-tumor drug. Furthermore, the tumor is multiple myeloma.

[0033] We developed a bispecific antibody, LILRB4-CD16A (BiKE), that highly specifically links NK cells to LILRB4-positive MM cells, enabling NK cells to target and kill tumor cells. We validated the efficacy of this NK cell engager in treating MM in vitro and in vivo, providing new insights into NK cell-targeted therapy for relapsed / refractory MM.

[0034] The bispecific antibody BiKE we prepared has an affinity comparable to that of a monoclonal antibody, and its binding activity to antigen targets on MM1.S and NK cells is also comparable to that of existing monoclonal antibodies. However, the bispecific antibody of the present invention can play a positive role in mediating NK cell killing of tumors by NK cells, thereby improving the effect of NK cells killing tumors and further preparing drugs against multiple myeloma.

[0035] Furthermore, the present invention's research also found that cGAS-STING signaling pathway agonists, especially SR-717, can synergize with the bispecific antibody BiKE of the present invention, significantly promoting NK cells to kill tumor cells and improving the anti-tumor effect.

[0036] Although the use of proteasome inhibitors, immunomodulators, and CD38 monoclonal antibodies has significantly improved patient outcomes, relapse and drug resistance remain major challenges. A study of 77.6% of patients with refractory triple-grade MM found that treatment with a BCMA x CD3 bispecific antibody resulted in an overall response rate of 63%. Another phase 2 trial targeting the same target achieved an objective response rate (ORR) of 61%. GPRC5D is highly expressed on malignant plasma cells, and patients receiving bispecific antibodies targeting this target have achieved an overall response rate of 64-70%, depending on the route of administration. These studies have demonstrated promising efficacy with bispecific antibodies, but both share the common drawbacks of T-cell therapy. A recent study compared the efficacy and safety of CAR-T and bispecific antibodies in the treatment of MM. While CAR-T is more effective than bispecific antibody therapy alone, it is associated with a higher rate of adverse events. Another study demonstrated that, compared with combined T-cell and T-cell engager therapy, combined NK cell and NK cell engager therapy has a stronger anti-tumor effect and produces fewer pro-inflammatory cytokines.

[0037] Due to the special immunosuppressive microenvironment in MM patients, the number of NK and NKT cells in many advanced MM patients decreases, accompanied by impaired effector function. Therefore, direct use of bispecific antibodies for treatment may not be ideal. Existing clinical trial data show that CAR-NK therapy does not have the risk of GVHD. In addition to CAR, there are natural receptors that can target tumors, the risk of hematopoiesis is lower, and antigen downregulation caused by CAR-T treatment is unlikely. However, due to the natural immune pattern of NK cells, the viral transduction efficiency of NK cells is low, and the preparation and application cost of CAR-NK is higher. Therefore, the use of bispecific antibodies combined with NK cell therapy is a good solution to fully utilize the inherent advantages of NK cells.

[0038] With previous treatments, relapses were common even after complete remission. Even after achieving complete remission with highly effective BCMA / GPRC5D CAR-T therapy, relapses can occur even when the tumor is negative for microresidual tumors, and antigen expression is downregulated. Therefore, the selection of the tumor cell target antigen is particularly important. LILRB4 is considered an immune checkpoint on myeloid cells. A study of LILRB4 CAR-T cells in acute monocytic leukemia demonstrated that anti-LILRB4 CAR-T cells were not toxic to normal CD34+ hematopoietic progenitor cells, confirming the safety of this target. Recent studies have shown that RRMM expresses elevated levels of LILRB4, a biomarker for aggressive multiple myeloma. Furthermore, LILRB4+ MM cells themselves promote the production of more monocyte-derived myeloid suppressor cells, which also express high levels of LILRB4. STAR-T cells targeting LILRB4 can dually target both tumor cells and myeloid-derived suppressor cells, enhancing therapeutic efficacy. The IgG-like bispecific antibodies we constructed have a longer half-life than small molecule antibodies without an Fc region, and do not significantly cross the blood-brain barrier. Furthermore, because IgG4 has high affinity only for FcγRI and weak affinity for all other FcγRs, it has no detectable Fc-mediated effector function, and thus activated NK cells will not be eliminated by ADCC.

[0039] cGAS-Sting is a key signaling pathway in innate cancer immunity. Studies have shown that cancer cells with downregulated cGAS-Sting can resist immune cytotoxicity. Active cGAS-Sting in immune cells also plays a crucial role in tumor control. In this study, we co-cultured tumor cells with PBMCs. NK cell CD107a, TNF-α, and IFN-γ levels were significantly higher in the SR-717 combined with BiKE treatment group than in the BiKE alone group, with consistent trends across all three donors. In vivo experiments also significantly prolonged mouse survival. To explore the mechanism by which BiKE combined with a STING agonist promotes NK cell anti-tumor function, we closely mimicked the in vivo integrated response system by co-culturing tumor cells with PBMCs. NK cells were then magnetically sorted for RNA-seq. We found significant upregulation of the NF-κB and TNF signaling pathways in NK cells, with SR-treated cells showing higher upregulation of cytotoxicity molecules such as TNF and IFNB1 than those treated with BiKE alone.

[0040] In this study, we demonstrated the ability of BiKE bispecific antibodies to mediate allogeneic NK cell killing of MM internally and externally, as well as the synergistic effect of STING agonists in this process. However, single-cell analysis revealed that LILRB4 is not highly expressed in all MM cells, but rather in a subset of high-risk / relapsed / refractory MM cells. Therefore, the killing effect in patients undergoing first-line treatment may not be as good as that of widely expressed targets such as BCMA / GPRC5D. However, MM is a highly heterogeneous and recurrent disease, and targeted killing of relapsed / refractory subpopulations of cells is particularly important. Therefore, combining LILRB4 with other targets widely expressed on tumor cells appears to be a promising new therapeutic approach. This is also the subject of our further research, aiming to provide more theoretical approaches for the treatment of relapsed MM.

[0041] The specific embodiments of the present invention will be further explained below through examples, but it is not intended to limit the scope of protection of the present invention to the scope described in the examples.

[0042] MM1.S, RMPI8226, and K562 used in the examples were purchased from the American Type Culture Collection (ATCC). PBMCs were isolated from the peripheral blood of healthy donors. All cell lines were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum, while PBMCs were cultured in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum. Cell culture was maintained at 37°C in a 5% CO2 atmosphere.

[0043] Female NSG mice, 6–8 weeks old, were obtained from Saiye Biotechnology Co., Ltd. All mice were maintained in a pathogen-free vivarium with free access to water and food. All animal experimental procedures were approved by the Medical Ethics Committee of Chengdu Blood Center.

[0044] Example 1 Evaluation of LILRB4 Expression in MM

[0045] We performed dimensionality reduction cluster analysis on the Gene Expression Omnibus (GEO) GSE223060 dataset and divided myeloma cells into 7 subpopulations. We found that LILRB4 was significantly upregulated in the 6th subpopulation and almost not expressed in other subpopulations of myeloma cells. We then performed copy number variation (CNV) analysis on these tumor cells and found that LILRB4 +MM cells have elevated CNV scores. Metabolic pathway enrichment analysis of differentially expressed genes in these tumor cell subpopulations revealed that LILRB4+ MM cells are highly correlated with nitrogen metabolism, which may be related to the high production of immunoglobulins in MM patients. Furthermore, cell communication analysis revealed that this population of cells interacts with B cells via the CXCL12 / CXCR4 pathway. The TCGA database shows that elevated LILRB4 expression is associated with shorter disease-free survival. Subsequently, we evaluated LILRB4 expression in the MM cell lines MM1.S and RPMI-8226. Western blot revealed high LILRB4 expression in MM1.S, consistent with flow cytometry results. These findings highlight LILRB4 as a potential target for killing highly malignant MM cells.

[0046] Example 2 Expression and Activity Verification of Bispecific Antibodies

[0047] In order to obtain a structural diagram such as Figure 1 For the bispecific antibody shown in A, we constructed a pVAX expression vector using anti-LILRB4 and anti-CD16A scFv sequences coupled to the human IgG4 fragment crystallizable Fc domain (hFc). The amino acid sequences of the anti-LILRB4 scFv and anti-CD16A scFv (including VL and VH) are shown in SEQ ID NOs: 1 and 2, and SEQ ID NOs: 3 and 4, respectively. Within the pVAX vector, the VLVH within the CD16A scFv is linked by a (Gly2Ser)7 peptide, while the VLVH within the LILRB4 scFv is linked by a (Gly4Ser)3 peptide. The CD16A scFv and LILRB4 scFv are linked by a human IgG4-Fc domain (CD16A(VLVH)-Gly4Ser-hFc-(Gly4Ser)3-LILRB4(VLVH)). A hexa-histidine (His6) tag was fused to the C-terminus of the BiKE.

[0048] BiKE is expressed in CHO cells. In short, the vector plasmid is first transfected into CHO cells using electroporation, and then cultured on a shaker for 10 days. The cell culture supernatant is collected and purified using human Fc segment high affinity protion A+G (Biyuntian), and then the antibody is concentrated using a cutoff column. The purified antibody is subjected to reducing and non-reducing SDS gel electrophoresis. The reduced single-chain antibody is about 80KDa and the size is correct (such as Figure 1 C). To test the affinity of BiKE to antigens, we simultaneously constructed monoclonal antibodies against CD16A and LILRB4. After antigen coating, the affinity of the antibodies was tested using ELISA (as shown in Figure 1 D and 1E), the results showed that: compared with the monoclonal antibody, the affinity of BiKE is similar to that of the monoclonal antibody, and its structure has no obvious effect on the activity of BiKE. Subsequently, in order to clarify the binding of BiKE to target labeled cells, we used indirect labeling flow cytometry to detect the results. Figure 1 As shown in F, the results showed that compared with monoclonal antibodies, BiKE had no significant difference in binding activity to antigen targets on MM1.S and NK cells, and did not bind to K562 cells without targets. Subsequently, in order to verify whether BiKE plays a positive role in mediating NK cell killing of tumors, we compared human peripheral blood PBMC with LILRB4 + MM1.S cell co-culture (process diagram as shown Figure 1 B), the results are as follows Figure 1 As shown in G, the results showed that compared with the monoclonal antibody and other control groups, the BiKE group had the highest upregulation of CD69 on NK cells, indicating that the degree of NK cell activation was the greatest.

[0049] Example 3 BiKE promotes NK cells to kill MM

[0050] To verify the function of BiKE-mediated NK cell killing of tumor cells, we first isolated PBMC from healthy human peripheral blood and sorted out NK cells using CD56 and CD3 magnetic beads antibodies. After in vitro activation and expansion culture, we found that almost all the expanded NK cells were CD16 positive ( Figure 2 A). Before testing the function of BiKE, in order to avoid masking the BiKE function due to an excessively high effector-target ratio, we screened for a suitable effector-target ratio and selected 1:1 for subsequent experiments (e.g. Figure 2 B). We then co-cultured NK cells with MM1.S cells at a ratio of 1:1 and found that MM1.S cells in the 5μg / ml BiKE group clearly aggregated ( Figure 2 C). Flow cytometry was performed after live-dead staining, and it was found that the high-concentration BiKE group significantly promoted NK cell killing of MM1.S (as shown in Figure 2 D, 2E). Calcein labeling was used to assess cytotoxicity, which showed consistent results (e.g. Figure 2 F and 2G).

[0051] The above experimental results collectively demonstrate that the BiKE constructed in the present invention can promote NK cells to kill MM, providing a new idea for the treatment of MM.

[0052] The relevant experimental process involved in this embodiment is as follows:

[0053] Human Natural Killer Cell Isolation and Ex Vivo Expansion:

[0054] According to a previously reported method, PBMCs were isolated from human peripheral blood using a density gradient using Ficoll (DAKEWE, China), followed by negative selection using CD3 magnetic beads coupled with antibodies, and positive selection using CD56 magnetic beads coupled with antibodies (Miltenyi Biotec, Germany). They were cultured in NK culture medium (Miltenyi Biotec, Germany) containing 5% human AB serum (GeminiBio, USA) and 500 IU / mL IL-2 (Miltenyi Biotec, Germany), and activated for 6 days using an NK cell activation / expansion kit (Miltenyi Biotec, Germany). They were then expanded in a 5% CO2, 37°C incubator using culture medium without the activation magnetic beads. NK cells were initially cultured at a density of 1×10 6 / mL(range:0.8–1.3×10 6 ) cells were seeded at a density of 1-1.5×10 6 The cells were inoculated into T25 culture flasks at a density of / mL, and the cells were counted and cultured in separate flasks every 2-3 days, and complete culture medium was added.

[0055] Enzyme-linked immunosorbent assay (ELISA)

[0056] For antibody affinity testing, LILRB4 (hFc tag) and CD16A (6His tag) recombinant proteins were coated at 50 ng / well in a 96-well plate at 4°C overnight. Blocking was performed with 2% BSA at 37°C for 2 h. Then, graded antibody concentrations were added and incubated at 37°C for 1.5 h. The plates were washed five times with PBST and patted dry. Then, goat anti-human IgG (H+L) or goat anti-6His tag secondary antibodies (Miyoshi Biosciences, China) were added. The plates were incubated at 37°C for 1 h. After washing five times with PBST and patting dry, 100 μL of TMB (Solarbio, China) was added for color development, followed by 50 μL of concentrated sulfuric acid. Finally, absorbance was measured at 450 nm on a microplate reader.

[0057] Flow cytometry

[0058] The cell suspension was collected, centrifuged to remove the culture medium, and washed once with PBS. NK cell-associated markers (CD56-APC / BV-421, CD3-FITC, and CD16-PE) were detected using a direct labeling method. Antibodies were incubated for 30 minutes at room temperature, washed twice with PBS, and then analyzed using a Novocyte flow cytometer. Tumor cell markers were detected using an indirect labeling method. Antibodies (rabbit anti-human LILRB4, 1:1000, Mitaka Biotechnology, China) were incubated for 1 hour at room temperature, washed twice with PBS, and then incubated with a FITC-conjugated goat anti-rabbit IgG (H+L) secondary antibody (1:1000, Mitaka Biotechnology, China) for 20 minutes at room temperature. After washing twice with PBS, the cells were analyzed using a Novocyte flow cytometer. BiKE binding assays used the same indirect labeling method. The secondary antibody used was a FITC-conjugated goat anti-human IgG (H+L) secondary antibody (1:1000, Mitaka Biotechnology, China).

[0059] Determination of NK cell cytotoxicity against tumor cell lines by calcein labeling

[0060] MM1.S cells were washed with PBS and resuspended in 2 μM calcein-AM (Biyuntian, China) solution at a density of 1 × 10 6 / mL. The cells were incubated at 37°C for 30 min. The S cells were washed twice with PBS. The target cells and NK cells were then inoculated in a U-bottom 96-well plate at a ratio of 1:1 and co-cultured at 37°C for 4 hours. The control group contained only target cells (spontaneous release), while the maximum release group was treated with 0.2% Triton X-100. After centrifugation of the cells in the 96-well plate, 100 μL of the supernatant was transferred to a black-walled 96-well plate (Corning, USA). The fluorescence signal was detected using a microplate reader with an excitation wavelength of 490 nm and an emission wavelength of 530 nm. The killing rate was calculated using the following formula: Killing rate = [(co-culture release-spontaneous release) / (maximum release-spontaneous release)] × 100%.

[0061] Example 4 STING agonists promote BiKE-mediated NK cell tumor killing

[0062] The cGAS-STING signaling pathway plays an important role in the innate immune response, mainly by promoting the secretion of type I interferon, thereby directly or indirectly recruiting and activating immune cells to fight tumors. In order to better utilize BiKE-mediated NK cell killing of tumor cells, we tried to use the STING signaling agonist SR-717 while BiKE-mediated NK cell killing of tumor cells. The results showed that the STING signaling agonist SR-717 can indeed significantly promote NK cell killing of tumor cells, and the cells clearly aggregated under the microscope (such as Figure 3 (as shown in A).

[0063] Since all the activated and expanded NK cells in vitro were positive for CD69 and CD107a, in order to better simulate the in vivo immune microenvironment, we co-cultured unstimulated PBMC with MM1.S cells. The results showed that BiKE significantly promoted the expression of CD107a on NK cells, and SR-717 and BiKE had a synergistic effect, further enhancing the expression level of CD107a on NK cells (such as Figure 3 B and 3C). Consistently, the expression levels of TNF-α and IFN-γ in NK cells detected by flow cytometry showed the same trend as CD107a, and the results of the three donors were consistent (as shown in Figure 3 D and 3E), indicating that SR-717 and BiKE synergistically activate NK cell function and enhance tumor killing ability.

[0064] Functional assays measure activation (CD69), degranulation (CD107a), and cytokine production (IFNγ and TNFα)

[0065] NK cell function was assessed by flow cytometry according to previously reported methods. Briefly, PBMCs and target cells were co-cultured for one hour, and the protein transport inhibitor Golgi Stop (BD Biosciences, USA) was added, followed by an additional 3-hour incubation. Cells were stained using a Live / Dead Fixable Aqua staining kit (Thermo Fisher, USA), APC-conjugated anti-CD69, APC-conjugated anti-CD107a, APC- or BV421-conjugated anti-CD56, and FITC-conjugated anti-CD3 (BioLegend, USA). Intracellular cytokines IFNγ and TNFα were surface stained with CD3 and CD56, followed by fixation with 4% paraformaldehyde and permeabilization with 0.1% Triton X-100. Permeabilized cells were stained with BV421-conjugated IFNγ (BioLegend, USA) and APC-conjugated TNFα (BD Biosciences, USA), washed with PBS, and analyzed using a Novocyte flow cytometer.

[0066] Example 5: BiKE combined with SR-717 promotes NK cells to inhibit tumors in vivo

[0067] To evaluate the effect of bispecific antibodies on NK cell-mediated tumor suppression in vivo, we constructed LILRB4+MM1.S cells expressing luciferase, expanded them in vitro, and injected them intravenously into C-NKG mice to construct a disseminated xenograft model (e.g., Figure 4As shown in A). NK cells derived from healthy human peripheral blood were activated and expanded in vitro, and then injected intravenously to evaluate their in vivo killing effect. On the fifth day after tumor inoculation, in vivo tumor growth was detected by in vivo imaging. Treatment was initiated after confirming the successful establishment of the xenogeneic model. Each mouse was injected intravenously with 4×10 6 NK cells, the antibody treatment group received intravenous injection of 1 mg / Kg for 5 consecutive days; SR-717 was intraperitoneally injected with 30 mg / Kg every other day for a total of 3 times. The tumor growth in the mice was then continuously observed by in vivo imaging (e.g. Figure 4 B), the tumor signal decreased significantly on the 5th day after treatment, while the control group showed rapid tumor proliferation. On the 15th day after treatment, the signal increased, indicating tumor growth, but the growth rate of the antibody treatment group was significantly lower than that of the NK cell treatment group alone. All mouse experiments were terminated on the 60th day, and statistical analysis of survival time was performed. It was found that the survival time of the tumor group mice treated with SR-717 combined with BiKE-mediated NK cells was significantly prolonged (as shown in Figure 2). Figure 4 C).

[0068] Methods for establishing animal models:

[0069] The tumor model was established in female C-NKG mice by intravenous injection of 1.5×10 6 LILRB4+MM1.S-luc. Longitudinal tumor growth was monitored by bioluminescence imaging (Qinxiang, China) on day 5. Mice with successfully established tumors were randomly divided into four treatment groups. On the designated days, NK cells isolated and expanded in advance were injected intravenously, followed by five treatments with BiKE (1 mg / kg) or three intraperitoneal injections of SR-717 (30 mg / kg) within a week. The control group was injected with an equal amount of PBS. Mice were monitored twice daily. On the designated days after treatment, bioluminescence imaging was performed under isoflurane anesthesia to detect the therapeutic effect.

[0070] Statistical analysis

[0071] Statistical evaluation was performed using GraphPad Prism 9.0 software. All quantitative data are reported as mean ± standard deviation. One-way ANOVA was used for univariate comparisons of multiple groups, and two-way ANOVA was used for comparisons of multiple groups between two independent variables. Additionally, Brown-Forsythe and Welch ANOVA tests were used for corrected analyses. All experiments were performed with at least three biological replicates, and P < 0.05 was considered statistically significant.

[0072] The above experimental results collectively illustrate that the present invention has constructed a bispecific antibody LILRB4-CD16A, which can highly specifically link NK cells with LILRB4-positive MM cells, allowing NK cells to target and kill tumor cells, providing a new idea for NK cell-targeted therapy for relapsed / refractory MM.

Claims

1. A bispecific antibody LILRB4-CD16A, characterized in that: The bispecific antibody comprises anti-LILRB4 scFv and anti-CD16A scFv, and the anti-LILRB4 scFv and anti-CD16A scFv are connected via a human IgG4-Fc segment.

2. The bispecific antibody LILRB4-CD16A according to claim 1, characterized in that: The specific connection method is CD16A(VLVH)-Gly4Ser-hFc-(Gly4Ser)3-LILRB4(VLVH).

3. The bispecific antibody LILRB4-CD16A according to claim 1, characterized in that: The amino acid sequences of VL and VH of the anti-LILRB4 scFv are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; the amino acid sequences of VL and VH of the anti-CD16A scFv are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

4. A nucleic acid molecule encoding the bispecific antibody LILRB4-CD16A according to any one of claims 1 to 3.

5. A vector comprising the nucleic acid molecule of claim 4.

6. A host cell comprising the nucleic acid molecule of claim 4 or the vector of claim 5.

7. Use of the bispecific antibody LILRB4-CD16A according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the vector according to claim 5, and the host cell according to claim 6 in the preparation of an anti-tumor drug.

8. The use according to claim 7, characterized in that: The tumor is multiple myeloma.

9. A pharmaceutical composition, characterized in that: It comprises the bispecific antibody LILRB4-CD16A according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the vector according to claim 5, or the host cell according to claim 6.

10. The pharmaceutical composition according to claim 9, characterized in that: Also included are cGAS-STING signaling pathway agonists.

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