Use of CD55 in enhancing nk cell anti-tumor function and engineered nk cells overexpressing cd55

Abstract

The application discloses application of CD55 in enhancing NK cell anti-tumor function and engineered NK cells overexpressing CD55. It is verified by the application that CD55 can trigger membrane lipid raft aggregation and LCK kinase activation of NK cells by trans-combining with CD97 on the surface of tumor cells, and further improve killing activity, cytokine secretion level and proliferation capacity of the NK cells. Further research shows that the expression level of CD55 of the NK cells in a tumor microenvironment presents a progressive downward trend, and further causes the anti-tumor function of the NK cells to be impaired. Based on the above-mentioned mechanism, the application provides a drug screening method taking CD55 as a target, and further provides use of engineered NK cells or CAR-NK cells overexpressing CD55 in preparation of an anti-tumor drug. Overexpression of CD55 can restore and enhance the effect function and survival stability of the NK cells in the tumor microenvironment, and can provide a brand-new strategy for tumor immunotherapy.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and tumor immunotherapy, specifically to novel uses of CD55 in enhancing the anti-tumor function of natural killer (NK) cells, and engineered NK cells based on CD55 overexpression and their application in tumor therapy. Background Technology

[0002] Natural killer (NK) cells are core effector cells of the innate immune system, playing a crucial role in anti-tumor immune surveillance. Adoptive cell therapy based on NK cells has shown promising clinical application prospects in the treatment of hematologic malignancies and some solid tumors. However, the efficacy of this therapy is still limited by several factors, one of the core challenges being that adopted NK cells rapidly undergo functional exhaustion after entering the tumor microenvironment (TME), specifically manifested as reduced killing activity, decreased cytokine secretion, and impaired proliferation. This phenomenon significantly limits the clinical benefits of NK cell therapy.

[0003] Current understanding of NK cell dysfunction primarily focuses on the upregulation of inhibitory receptors or the role of immunosuppressive cytokines. However, the key molecular events regulating the sustained activation of NK cells at the tumor recognition interface remain unclear. CD55, also known as decay accelerator factor (DAF), is traditionally classified as a complement regulatory protein, which can protect autologous cells from attack by inhibiting the complement cascade. However, its non-complement regulatory function in NK cells, particularly its role in anti-tumor immunity, has not been previously explored. Summary of the Invention

[0004] This invention aims to overcome the shortcomings of existing NK cell therapies in terms of the easy depletion of cell function, and provides a method and product for enhancing the anti-tumor function of NK cells based on CD55 overexpression.

[0005] This invention, based on in-depth and systematic research, elucidates for the first time a novel, non-classical role of CD55 in the anti-tumor function of NK cells. Contrary to conventional understanding, CD55 interacts with CD97 molecules on the surface of tumor cells, initiating an independent activation signaling axis within NK cells. This process plays a crucial role in maintaining the effector function and persistence of NK cells. This invention is based on this groundbreaking research.

[0006] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a method for enhancing the anti-tumor function of NK cells, the method comprising promoting or maintaining the expression and / or function of CD55 on the surface of NK cells.

[0007] Furthermore, the step of promoting or maintaining CD55 expression on the surface of NK cells is to overexpress CD55 protein in NK cells.

[0008] Furthermore, the ways to promote or maintain the function of CD55 on the surface of NK cells include: promoting the binding of CD55 to its ligand CD97, promoting the aggregation of lipid rafts on the NK cell membrane, or promoting the activation of LCK kinase.

[0009] Secondly, the present invention provides an engineered NK cell, wherein the NK cell is modified to achieve CD55 overexpression, thereby enhancing its anti-tumor activity.

[0010] Furthermore, the NK cells also express chimeric antigen receptors (CARs) or T-cell receptors (TCRs).

[0011] Preferably, the CAR targets tumor-associated antigens.

[0012] More preferably, it targets CD19, CD22, BCMA, HER2, or mesothelin.

[0013] Furthermore, the NK cells are primary human NK cells, NK92 cell lines, or NK cells derived from umbilical cord blood.

[0014] Thirdly, the present invention provides a pharmaceutical composition comprising engineered NK cells as described in any one of the above-mentioned claims, and a pharmaceutically acceptable carrier or adjuvant.

[0015] Fourthly, the present invention provides the use of CD55 or nucleic acid encoding CD55 in the preparation of reagents or drugs that enhance the anti-tumor function of NK cells.

[0016] Fifthly, the present invention provides a method for screening candidate drugs that enhance the anti-tumor function of NK cells, the method comprising the following steps: a. Introduce the candidate substance into NK cells expressing CD55; b. Detect the activity of CD55-mediated signaling pathways, which include the CD97-CD55-lipid raft-LCK signal axis; c. If the candidate substance can enhance the activity of the signaling pathway, for example, by promoting the binding of CD55 to its ligand CD97, promoting lipid raft aggregation, or promoting LCK phosphorylation, then the candidate substance is determined to be a candidate drug that can enhance the anti-tumor function of NK cells. The present invention has the following beneficial effects: 1. Corrected the cognitive bias of existing technologies: For the first time, it was clarified that CD55 has non-complement regulatory functions in NK cells, that is, CD55 can play a positive regulatory role on the anti-tumor activity of NK cells through the "CD97-CD55-lipid raft-LCK" signaling axis, breaking the traditional understanding in the industry that it was only identified as a complement inhibitory molecule.

[0017] 2. A novel treatment strategy is provided: Addressing the functional exhaustion of NK cells in the tumor microenvironment caused by the downregulation of CD55 expression, a novel strategy is proposed to enhance NK cell function by forcibly expressing CD55 through genetic engineering. This strategy is applicable not only to conventional NK cells but also to CAR-NK cells, significantly improving the anti-tumor efficacy and in vivo survival capacity of these cells.

[0018] 3. Identified novel drug screening targets: This invention establishes that CD55 and its downstream signaling pathways can serve as targets for screening NK cell enhancers, pointing the way for the development of novel immunotherapy drugs. Attached Figure Description

[0019] Figure 1CD55 is crucial for the antitumor effector function of NK cells. (A) CD55 expression was detected in expanded human NK cells (CD55cRNP vs. control TRACcRNP) and in spleen NK cells isolated from Cd55flox / flox and Ncr1-Cd55Δ mice using non-viral CRISPR-Cas9RNP technology. (B) Cell viability (CYTOX ⇌ Annexin V ⇌ percentage) of CD55 knockout or wild-type (WT) human NK cells (n=3 donors) and mouse NK cells (n=5 mice) was analyzed by flow cytometry. (C) Calcein release assays were used to evaluate the cytotoxicity of CD55 knockout or WT human NK cells (n=3 donors, E:T=2:1, cultured for 4 hours) and mouse NK cells (n=5 mice, E:T=5:1) against K562 and YAC-1 target cells. (DF) Flow cytometry analysis of the proportions of CD107a+ (D), IFN-γ+ (E), and Ki-67+ (F) NK cells 16 hours after K562 stimulation (E:T=1:1, n=3 donors). (G) Representative images (left) and quantitative results (right) of Cd55flox / flox and Ncr1-Cd55Δ lung metastases in the B16F0 model (n=7 mice). (H) Subcutaneous B16F0 (n=5) and RMA-S (n=7) tumor volumes in the two groups of mice. (IJ) Quantitative analysis of the percentage of surviving NK cells (CYTOX¯AnnexinV¯, I) and the number of tumor-infiltrating NK cells (J) in the two groups of RMA-S tumors (n=7 mice). (K) Flow cytometry analysis of CD107a+, IFN-γ+, and Ki-67+ cells in tumor-infiltrating lymphocytes (TIL-NK cells) of tumor-bearing Cd55flox / flox and Ncr1-Cd55△ mice (n=7 mice). Paired t-tests (BF) and unpaired t-tests (GK) were used for comparison. Data are expressed as mean ± standard deviation (B, DK) and mean ± standard error (C). Ns indicates p>0.05; **p<0.01; ***p<0.001; ****p<0.0001. Figure 2CD55-CD97 interaction triggers lipid raft-LCK signaling to enhance NK cell antitumor activity. (A) Immunofluorescence shows the interaction between CD55 and LCK in primary human NK cells (red: CD55, green: LCK, blue: Hoechst, scale bar = 10 μM). (B) CD55 expression was detected by anti-Flag immunoprecipitation and Western blot analysis after expressing Flag-tagged LCK or an empty vector in primary human NK cells. (C) Immunofluorescence analysis was performed on lipid rafts (red) and LCK (green) in wild-type (TRACcRNP) or CD55 knockout (CD55cRNP) human NK cells after stimulation for 30 minutes on culture plates coated with IgG, α-CD55 activating antibody (α-CD55ago), or CD97 protein (CD97pro). Images represent results from multiple independent experiments. Scale bar is 2 μM. (D) CD55 activation recruits an LCK-centric kinase signaling network. (Top) Immunoprecipitation procedure used for subsequent mass spectrometry analysis validated by Western blot. (Bottom) Protein-protein interaction network of LCK-specific chaperone binding after CD55 binding. This network is visualized by margins scaled according to mass spectrometry interaction scores, revealing the activation process of downstream kinase cascades (such as ZAP70 and SYK) crucial for NK cell activation. (EF) p-LCK-positive and Ki-67-positive NK cells (n=3 donors) after 48 hours of stimulation with IgG, α-CD55, or CD97 pre-coated culture plates. (G)(E) Cytotoxicity assay of NK cells against K562 cells (4 hours of culture, E:T=5:1, n=3 donors). (H) Human NK cells pretreated with IgG or CD55 neutralizing antibody (α-CD55blk) and then co-cultured with K562 tumor cells pretreated with IgG or CD97 neutralizing antibody (α-CD97blk). Data were obtained from n=3 donors (E:T=5:1, co-cultured for 1 hour). (I) p-LCK (Y394) MFI of Cd55flox / flox or Ncr1-Cd55△ mouse spleen NK cells after co-culturing with YAC-1 cells for 1 hour (E:T=20:1, n=5 mice). (JK)(H) Ki67 expression (J) and cytotoxicity (K) of human NK cells after co-culturing with K562 cells for 16 hours (E:T=5:1, n=3 donors). (L) Human primary NK cells were pretreated with an LCK inhibitor (LCKi) for 30 minutes, followed by co-culturing with K562 cells for 1 hour. Flow cytometry was used to detect phosphorylated LCK at the Y394 site (n=3 donors).(M) The responses of human NK cells amplified by TRACcRNP and CD55cRNP and K562 cells after co-culturing for 4 hours (n=3 donors, E:T=2:1) ​​and pretreated with LCKi or DMSO for 30 minutes were analyzed. Paired t-tests (I, L) and Holm-Sidak one-way ANOVA were used for multiple comparisons (EH, JK, M). Data are expressed as mean ± standard deviation (EF, HI, J, L) and mean ± standard error (SEM) (G, K, M). Figure 3 Sustained CD55 expression can enhance the efficacy of NK cell immunotherapy. (A) CD55 expression in primary NK cells transfected with empty vector (EV-NK) or CD55 overexpression vector (OE-NK) (n=4 donors). (B) CD55 MFI of EV-NK and OE-NK after 0-3 days of K562 cell stimulation (E:T=5:1, n=3 donors). (C) Cytotoxicity of EV-NK and OE-NK to K562 cells after 2 or 3 days of K562 cell stimulation (stimulation duration 4 hours, E:T=5:1, n=3 donors). (D) Cytotoxicity of CD19-CAR (CARNK92) and CD19-CAR-CD55NK92 (CAR-CD55NK92) to Raji cells (stimulation 4 hours, E:T=2.5:1, n=3 independent experiments). (EG) NCG mice carrying Raji tumors (day 0) were injected with CARNK92 or CAR-CD55NK92 cells via tail vein on day 1. Peripheral blood was collected on day 7 post-injection to detect CD55 expression (E), LCK, CD107a, and IFN-γ expression (F) in circulating CARNK92 cells (n=4 mice per group); 50µl peripheral blood samples were collected on days 0, 1, 3, and 5 to quantify human CD56. +Absolute cell count (G). (H) NCG mice injected with Raji-luc tumor cells (day 0) treated with saline, CARNK92, or CAR-CD55NK92 on days 1, 8, and 15, respectively. (I) Bioluminescence imaging results on days 5, 12, and 19; summary chart (n=5 mice). (J) Survival curves of tumor-bearing mice treated with saline, CARNK92, or CAR-CD55NK92 (n=9 mice per group). Treatment began on day 1 and continued weekly until death. Comparisons were performed using paired t-tests (C, D), unpaired t-tests (F), one-way ANOVA (B, I), and two-way ANOVA (A, G). Survival analyses were performed using the log-rank test (J). Data are expressed as mean ± standard deviation (A–B, F–G, I–J) and mean ± standard error (SEM) (C, D). Ns represents p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Detailed Implementation

[0020] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0021] For experiments not specifically described in the examples, follow standard experimental procedures in the field or the manufacturer's instructions. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0022] All procedures involving human or animal operations in this invention have been approved by the ethics committee.

[0023] Example 1: Verification of the necessity of CD55 in the anti-tumor function of NK cells The purpose of this study is to verify the key role of CD55 in NK cell killing of tumor cells.

[0024] Experimental methods: Gene knockout experiment: Using non-viral CRISPR-Cas9 ribonucleoprotein (cRNP) technology, CD55 gene knockout was performed on in vitro expanded human primary NK cells (denoted as CD55^cRNP^), and cells targeting the TRAC gene were selected as controls (denoted as TRAC^cRNP^). NK cell-specific Cd55 conditional knockout mice (denoted as Cd55^CKO^) were simultaneously constructed, and Cd55^fl / fl^ mice were selected as controls.

[0025] Functional assays: Under complement inactivation conditions (using heat-inactivated autologous serum, i.e., HI-AS), the cytotoxic activity of NK cells against tumor cells (human K562 cells and mouse YAC-1 cells) was detected by calcein release assay. Flow cytometry was used to detect the degranulation level (CD107a), cytokine (IFN-γ) production capacity, and proliferation capacity (Ki-67) of NK cells after co-culturing with tumor cells.

[0026] In vivo models: In the B16F0 lung metastasis model, B16 subcutaneous xenograft model, and RMA-S subcutaneous xenograft model, the differences in tumor burden, number of metastatic lesions, and function of tumor-infiltrating NK cells (TIL-NK) between Cd55^CKO^ mice and Cd55^fl / fl^ mice were compared.

[0027] Experimental results: Flow cytometry analysis confirmed that highly efficient CD55-specific knockout was achieved in both human and mouse NK cells. Figure 1 a).

[0028] Under complement inactivation conditions, the killing activity of CD55^cRNP^ human NK cells against K562 cells was significantly lower than that of the control group, and the killing activity of mouse Cd55^CKO^ NK cells against YAC-1 cells was also significantly reduced. Figure 1 c). Simultaneously, after tumor stimulation, the positive rates of CD107a, IFN-γ, and Ki-67 in CD55-deficient human NK cells all decreased significantly. Figure 1 df).

[0029] The number of lung metastatic nodules in Cd55^CKO^ mice was significantly higher than that in the control group. Figure 1 g), and the subcutaneous tumor growth rate was also significantly faster than that of the control group ( Figure 1 h). Analysis of intratumoral TIL-NK cells showed that although CD55 deficiency did not affect NK cell survival, the absolute number of NK cells, CD107a expression level, IFN-γ expression level, and Ki-67 expression level were all significantly decreased. Figure 1 ik).

[0030] Conclusion: CD55 is an essential molecule for maintaining the effector function and proliferative capacity of NK cells under tumor stimulation. CD55 deficiency severely impairs the in vivo and in vitro antitumor activity of NK cells, and this effect is independent of the complement regulatory function of CD55.

[0031] Example 2: Study on the mechanism by which CD55 enhances NK cell function The purpose of this study is to elucidate the molecular mechanism by which CD55 enhances NK cell function.

[0032] Experimental methods: Immunoprecipitation and immunofluorescence detection: Immunoprecipitation (Co-IP) and immunofluorescence staining techniques were used to verify the physical interaction and co-localization of CD55 and LCK on the NK cell membrane.

[0033] Signaling pathway activation detection: NK cells were stimulated with CD55 agonist antibody or recombinant CD97 protein, and the phosphorylation level of LCK was detected by Western blotting and flow cytometry; cells were treated with lipid raft inhibitor MβCD, and the effect of this treatment on signal activation was observed.

[0034] Blocking experiment: In the co-culture system, the interaction between CD55 and CD97 was blocked using CD55 neutralizing antibody (α-CD55 blk) and CD97 neutralizing antibody (α-CD97 blk), respectively. The effect of this operation on LCK phosphorylation level, NK cell killing ability and proliferation ability was detected.

[0035] Mass spectrometry analysis: Immunoprecipitation-mass spectrometry (Co-IP-MS) was used to identify the protein complex components that bind to LCK after CD55 activation.

[0036] Experimental results: Immunofluorescence and immunoprecipitation assays confirmed that CD55 and LCK co-localize and physically interact on the NK cell membrane. Figure 2 ab).

[0037] Stimulation with CD55 agonist antibodies or CD97 protein significantly induced lipid raft aggregation in NK cell membranes, promoted LCK recruitment to lipid rafts, and upregulated phosphorylation at the LCK Y394 site, thereby promoting NK cell proliferation and cytotoxic function; the lipid raft inhibitor MβCD completely blocked the above effects. Figure 2 cg).

[0038] Blocking the interaction between CD55 and CD97 (treatment with α-CD55 blk or α-CD97 blk) significantly reduced LCK phosphorylation levels, NK cell proliferation, and cytotoxic activity in NK cell-tumor cell co-culture systems. Figure 2 (HK).

[0039] Immunoprecipitation-mass spectrometry analysis showed that after CD55 activation, key downstream kinases such as ZAP70 and SYK were enriched in the LCK immune complex. Figure 2 d).

[0040] Conclusion: CD55 binds to CD97 on the surface of tumor cells, triggering lipid raft aggregation in NK cell membranes, inducing LCK kinase recruitment and activation, forming an independent "CD97-CD55-lipid raft-LCK" signaling axis, ultimately driving NK cells to exert anti-tumor functions.

[0041] Example 3: Overexpression of CD55 can enhance the anti-tumor efficacy of primary NK cells and CAR-NK cells. This embodiment can directly verify the core strategy of the present invention, namely, that overexpression of CD55 can effectively enhance the anti-tumor ability of NK cells.

[0042] Experimental methods: Construction of overexpression cells: CD55 overexpression plasmids were constructed and CD55 was stably overexpressed in primary human NK cells and CD19 CAR-NK cells (including primary cells and NK92 cell lines) by electroporation or lentiviral transduction technology. These were denoted as OE-NK and CD19CAR-CD55, respectively. An empty vector control group was set up and denoted as EV-NK and CD19CAR.

[0043] In vitro continuous stimulation experiment: OE-NK was co-cultured with EV-NK and K562 tumor cells (culture period of 0-3 days), and the expression maintenance level of CD55 and the killing ability of OE-NK and EV-NK when exposed to tumor cells again after 2 days and 3 days of co-culture were detected.

[0044] In vivo anti-tumor model: In the Raji-luc lymphoma model of NCG mice, saline, CD19CAR-NK cells and CD19CAR-CD55-NK cells (primary cells or NK92 cells) were injected via the tail vein. The tumor burden was monitored by in vivo imaging technology, and the survival status, functional level and in vivo persistence of CAR-NK cells in peripheral blood were detected.

[0045] Experimental results: Primary NK cells and CD19 CAR-NK cells that stably overexpress CD55 were successfully constructed and obtained. Figure 3 a).

[0046] Without tumor stimulation, there was no significant difference in cellular function between OE-NK and EV-NK; however, after long-term co-culture with tumor cells, OE-NK maintained a higher level of CD55 expression; in the re-killing challenge experiment conducted after 2 or 3 days of co-culture, OE-NK exhibited significantly stronger and more durable killing activity than EV-NK. Figure 3 c).

[0047] In the Raji tumor-bearing mouse model, compared with the CD19CAR control group, the CD19CAR-CD55 primary NK cell therapy group showed: 1) CD55 maintained a higher expression level in vivo ( Figure 3 e); 2) CAR-NK cells in peripheral blood exhibit higher levels of LCK phosphorylation, degranulation activity (CD107a), and IFN-γ production capacity ( Figure 3 f); 3) The persistence (absolute cell number) of CAR-NK cells in mice was significantly improved ( Figure 3 g); 4) Better tumor control effect and significantly prolonged mouse survival ( Figure 3 Repeated experiments conducted in the NK92 cell line yielded consistent results.

[0048] Conclusion: By forcibly overexpressing CD55 in primary NK cells or CAR-NK cells through genetic engineering, the loss of CD55 expression and cell function exhaustion induced by the tumor microenvironment can be resisted, and the anti-tumor effect, in vivo persistence and therapeutic efficacy of NK cells or CAR-NK cells can be significantly enhanced.

[0049] In summary, this invention confirms that CD55 is a key target for enhancing the anti-tumor function of NK cells. Overexpressing CD55 to modify NK cells or CAR-NK cells is an effective and promising new strategy for tumor immunotherapy.

Claims

1. A method for enhancing the anti-tumor function of NK cells, characterized in that, The method includes promoting or maintaining the expression and / or function of CD55 on the surface of NK cells.

2. The method for enhancing the anti-tumor function of NK cells according to claim 1, characterized in that, The step of promoting or maintaining CD55 expression on the surface of NK cells is to overexpress CD55 protein in NK cells.

3. The method for enhancing the anti-tumor function of NK cells according to claim 1, characterized in that, The ways to promote or maintain the function of CD55 on the surface of NK cells include: promoting the binding of CD55 to its ligand CD97, promoting the aggregation of lipid rafts on the NK cell membrane, or promoting the activation of LCK kinase.

4. An engineered NK cell, characterized in that, The NK cells were modified to achieve CD55 overexpression.

5. The engineered NK cell according to claim 4, characterized in that, The NK cells also express chimeric antigen receptors or T-cell receptors.

6. The engineered NK cells according to claim 4 or claim 5, characterized in that, The NK cells are primary human NK cells, NK92 cell lines, or NK cells derived from umbilical cord blood.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises engineered NK cells as described in any one of claims 4-6, and a pharmaceutically acceptable carrier or adjuvant.

8. Application of CD55 or nucleic acids encoding CD55 in the preparation of reagents or drugs that enhance the anti-tumor function of NK cells.

9. A method for screening candidate drugs that enhance the anti-tumor function of NK cells, characterized in that, The method includes the following steps: a. Introduce the candidate substance into NK cells expressing CD55; b. Detect the activity of CD55-mediated signaling pathways, which include the CD97-CD55-lipid raft-LCK signal axis; c. If a candidate substance can enhance the activity of the signaling pathway, such as promoting the binding of CD55 to its ligand CD97, promoting lipid raft aggregation, or promoting LCK phosphorylation, then the candidate substance is determined to be a candidate drug that can enhance the anti-tumor function of NK cells.

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