A tumor microenvironment-responsive LYTAC molecule, its preparation method and application

Abstract

This invention discloses a tumor microenvironment-responsive LYTAC molecule, its preparation method, and its applications. The LYTAC molecule uses a PD-L1 antibody as the target recognition unit and IGF2 as the ligand for the lysosomal targeting receptor IGF2R. IGF2 is fused to the light chain end of the antibody, and the molecule is linked to a "mask" protein via a protease-responsive linker. The "mask" protein masks the binding region of IGF2 and IGF2R, making it difficult for the molecule to effectively bind to IGF2R in normal tissue environments. In the tumor microenvironment, after cleavage by relevant proteases, the IGF2 functional region is exposed, restoring its binding ability to IGF2R. This, in turn, mediates the internalization of PD-L1 on the tumor cell surface into lysosomes for degradation. This invention improves the tumor selectivity of PD-L1 degradation, reduces the off-target risk in normal tissues, and has a well-defined structure, is easy to prepare, and has promising application prospects.

Description

Technical Field

[0001] This invention relates to a tumor microenvironment-responsive LYTAC molecule that degrades PD-L1 on the surface of tumor cells and its preparation method, as well as its use in the preparation of anti-tumor drugs, PD-L1-targeted degradation inducers for treating tumor cells, tumor cell proliferation inhibitors, or tumor cell migration inhibitors. Background Technology

[0002] Cancer poses a serious threat to human life and health. Immune checkpoint blockade therapy, especially strategies targeting programmed death-ligand 1 (PD-L1) or programmed death receptor 1 (PD-1), has become an important means of cancer treatment and has shown good clinical benefits in some patients. However, the overall response rate of existing immune checkpoint inhibitors remains limited, and PD-L1 is expressed not only on the surface of tumor cells but also in normal tissues such as the heart, lungs, and skeletal muscle, potentially triggering immune-related adverse reactions during treatment. Furthermore, PD-L1 mediates immune escape not only through its extracellular domain binding to PD-1 on the surface of T cells, but its intracellular domain can also participate in the transduction of pro-growth signals within tumor cells. Therefore, simply blocking PD-L1 function is insufficient to simultaneously eliminate its extracellular immunosuppressive effects and intracellular tumor-promoting effects. Developing new strategies that can directly clear PD-L1 protein is of great significance.

[0003] In recent years, lysosomal pathway-based targeted protein degradation technology has provided a new approach for clearing cell membrane and extracellular proteins. Lysosomal targeted chimeras (LYTACs) utilize cation-independent mannose-6-phosphate receptors (CI-M6PR / IGF2R) and other receptors to mediate the endocytosis of target proteins into lysosomes for degradation, thereby achieving the clearance of membrane proteins that are difficult to handle by traditional proteasome degradation technologies. Although existing LYTAC molecules have been shown to effectively degrade cell surface proteins such as EGFR, their clinical application is limited by problems such as complex molecular structure, high difficulty in preparation, insufficient in vivo stability, and insufficient targeting due to the wide distribution of lysosomal receptors. Especially for targets like PD-L1, which are expressed in both tumor tissues and some normal tissues, how to selectively activate LYTAC molecules in the tumor microenvironment through structural optimization, thereby achieving precise degradation of PD-L1 in tumor cells while ensuring efficacy and safety, remains a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0004] Objectives of this invention: Addressing the limitations of existing PD-L1 immune checkpoint inhibitors, which only block the binding of PD-L1 to PD-1 and fail to eliminate the intracellular pro-tumor signaling effects of PD-L1, as well as the problems of existing LYTAC technology such as complex structure, insufficient tumor targeting, and easy off-target degradation in normal tissues, one objective of this invention is to provide a tumor microenvironment-responsive LYTAC molecule. Another objective is to provide a method for preparing this LYTAC molecule. A final objective is to provide multiple applications of this LYTAC molecule in the preparation of anti-tumor drugs, the preparation of PD-L1-targeted degradation inducers for treating tumor cells, and the preparation of tumor cell proliferation inhibitors or tumor cell migration inhibitors. The LYTAC molecule is used to specifically degrade PD-L1 on the surface of tumor cells, thereby improving the safety and efficacy of anti-tumor therapy.

[0005] Technical solution: The present invention provides a tumor microenvironment-responsive LYTAC molecule, wherein the heavy chain Mi-LYTAC-H of the LYTAC molecule has the sequence shown in SEQ ID NO.1, and the light chain Mi-LYTAC-L has the sequence shown in SEQ ID NO.4.

[0006] The method for preparing the tumor microenvironment-responsive LYTAC molecule of the present invention includes: using PD-L1 antibody as the target recognition unit, using IGF2 as the ligand of lysosomal target receptor IGF2R, fusing IGF2 at the light chain end of the antibody, and then linking the "Mask" protein through a proteolytic enzyme-responsive linker peptide.

[0007] The "Mask" protein is derived from the CD loop region of IGF2R protein domain 11, and its sequence is shown in SEQ ID NO.5. The proteolytic enzyme is matrix metalloproteinase MMP2 or matrix metalloproteinase MMP9.

[0008] The present invention also includes a method for inducing the degradation of PD-L1 protein in tumor cells for non-disease diagnosis or treatment purposes, characterized in that the tumor cells are treated with the tumor microenvironment-responsive LYTAC molecule as described in claim 1.

[0009] The concentration of LYTAC molecules was above 100 nM, and the treatment time was above 24 hours.

[0010] The application of the tumor microenvironment-responsive LYTAC molecule described in this invention in the preparation of tumor therapeutic agents.

[0011] The application of the tumor microenvironment-responsive LYTAC molecule described in this invention in the preparation of PD-L1 targeted degradation inducers, tumor cell proliferation inhibitors, and / or tumor cell migration inhibitors for the treatment of tumor cells.

[0012] Among the applications mentioned above, tumor cells, such as breast cancer cells (e.g., MDA-MB-231 cells), are used. LYTAC molecules have the effect of masking IGF2.

[0013] The tumor microenvironment-responsive LYTAC molecule of this invention is based on the lysosomal targeting receptor IGF2R, using IGF2 as the ligand for IGF2R, i.e., using the IGF2 protein as an inducer, and the PD-L1 antibody atezolizumab (Atz) recognizing the target protein, i.e., using the PD-L1 antibody as the target recognition unit. On this basis, IGF2 is fused to the light chain end of the antibody, and a "mask" protein is linked to it via a protease-responsive linker peptide, constructing a tumor microenvironment-responsive LYTAC molecule, abbreviated as Mi-LYTAC. The "mask" protein is used to mask the binding region of IGF2 and IGF2R, making it difficult for the molecule to effectively bind IGF2R in the normal tissue environment. In the tumor microenvironment, after being cleaved by relevant proteases, the IGF2 functional region is exposed, restoring its binding ability to IGF2R, thereby mediating the internalization of PD-L1 on the tumor cell surface into the lysosome for degradation. In normal tissue environments, the IGF2 receptor-binding region of this molecule is masked, making it difficult to efficiently bind to IGF2R and induce PD-L1 degradation. However, in the tumor microenvironment, the masking structure can be cleaved or inactivated by MMP2 or MMP9 proteases, thereby exposing the IGF2 functional region and restoring its ability to bind to IGF2R and mediate PD-L1 endocytosis into lysosomes for degradation. This allows for selective clearance of PD-L1 from the surface of tumor cells, rather than simple functional blockade. This molecule can be obtained through expression and purification in eukaryotic cells, improving the tumor selectivity of PD-L1 degradation and reducing the off-target risk in normal tissues. It has a well-defined structure, is easy to prepare, and shows promising application prospects.

[0014] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0015] (1) This invention removes PD-L1 by protein degradation rather than simple blocking, which is expected to weaken its extracellular immunosuppressive effect and intracellular tumor-promoting effect at the same time;

[0016] (2) The Mi-LYTAC provided by this invention is a tumor microenvironment-responsive masking design that can improve the activation specificity of LYTAC molecules at the tumor site and reduce the off-target risk caused by the widespread expression of IGF2R and PD-L1 in normal tissues.

[0017] (3) The present invention uses a fusion protein strategy for molecular construction. Compared with the polysaccharide-coupled LYTAC system, the structure is more defined, the design is simpler, and it is easier to express, purify and transform. Attached Figure Description

[0018] Figure 1 The diagram below shows the simplified molecular structure of MiLYTAC in Example 1. In this diagram, MiYTAC① is a uPA enzyme-responsive peptide linking the "Mask" protein to IGF2; MiYTAC② is a Matriptase A-responsive peptide linking the "Mask" protein to IGF2; and MiYTAC③ is a matrix metalloproteinase (MMP2 or MMP9)-responsive peptide linking the "Mask" protein to IGF2.

[0019] Figure 2 The above are SDS-PAGE images of Mi-LYTAC①, Mi-LYTAC②, and Mi-LYTAC③ in Example 1.

[0020] Figure 3 The graph shows the flow cytometry results of the affinity activities of Mi-LYTAC①, Mi-LYTAC②, Mi-LYTAC③ and the positive control LYTAC with cell surface IGF2R and PD-L1, respectively, in Example 1. In the graph, a represents PD-L1 and b represents IGF2R.

[0021] Figure 4 The image shows the expression levels of PD-L1 in MDA-MB-231 cells after 24 hours of treatment with 100 nM Mi-LYTAC, LYTAC, and PD-L1 monoclonal antibodies by Western blotting in Example 1. In the image, a is the Western blotting result and b is the quantitative result of Western blotting.

[0022] Figure 5 The images show the SDS-PAGE and WB analysis results of MDA-MB-231 cells and / or 100 nM Mi-LYTAC and 200 ng / mL MMP2 or MMP9 after co-treatment at 37°C for 12 h in Example 1. In the images, a is the SDS-PAGE image of Mi-LYTAC digested by MMP2 and MMP9, and b is the WB analysis image of PD-L1 expression in the cells. Detailed Implementation

[0023] Example 1

[0024] (1) Mi-LYTAC molecular structure design

[0025] Based on the PD-L1 antibody atezolizumab (Atz), mature IGF2 was fused to the N-terminus of the antibody light chain. Then, peptides responding to different proteases were used to link IGF2 to a "mask" protein, resulting in the Mi-LYTAC molecule. The coding sequence was synthesized by Suzhou Genewiz Biotechnology Co., Ltd. and inserted into the pCDNA3.4 vector for subsequent suspension cell expression. The "mask" protein, used to mask the IGF2 receptor-binding region, is from the CD loop region of IGF2R protein domain 11: GENENCPPGVGACFGQTRISVGKANK (SEQ ID NO. 5), which has been proven to be one of the key domains capable of binding IGF2. The different proteases are: urokinase (uPA), membrane-type serine protease 1 (Matriptase A), and matrix metalloproteinases (MMP2 or MMP9). These three proteases are proteins highly expressed in tumors, endowing Mi-LYTAC with tumor-targeting protein degradation properties. Inserting different proteolytic peptide segments can alter the structure of Mi-LYTAC, potentially affecting the recognition and binding of substrates, PD-L1, or target proteins. The next step will be to screen for conformations that can successfully mask IGF2 to achieve protease-responsive PD-L1 degradation.

[0026] like Figure 1 As shown, three types of proteolytic enzyme-responsive Mi-LYTAC molecules were constructed. These three Mi-LYTAC molecules consist of a heavy chain Mi-LYTAC-H (sequence shown in SEQ ID NO.1) and a light chain (Mi-LYTAC-L) (sequences shown in SEQ ID NO.2-SEQ ID NO.4, respectively). The Mi-LYTAC molecules are denoted as Mi-LYTAC. (Mi-LYTAC linker: uPA response), Mi-LYTAC (Mi-LYTAC linker: Matriptase A response) and Mi-LYTAC (LYTAC linker: MMP2 / 9 response).

[0027] SEQ1: Mi-LYTAC-H (SEQ ID NO.1)

[0028] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVCPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGK;

[0029] SEQ2:Mi-LYTAC-L-uPA(SEQ ID NO.2)

[0030] GENENCPPGVGACFGQTRISVGKANKGGGGSGGGGSLSGRSDNHGGGSGGGSAYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSESGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPCDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0031] SEQ3:Mi-LYTAC-L-Matriptase A(SEQ ID NO.3)

[0032] GENENCPPGVGACFGQTRISVGKANKGGGGSGGGGSKSTSRQARGGGSGGGSAYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSESGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPCDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0033] SEQ4: Mi-LYTAC-L-MMP2 / 9 (SEQ ID NO.4)

[0034] GENENCPPGVGACFGQTRISVGKANKGGGGSGGGGSGILGVPGILGVPGILGVPGGGGSAYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSESGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPCDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0035] (2) Expression and purification of Mi-LYTAC protein

[0036] Expi 293F suspension cells (Thermo Scientific, USA) were cultured in serum-free medium (OPMbiosciences, China) at 37°C and 5% CO2 in a shaker at 150 rpm. The mi-LYTAC cDNA sequence was constructed in pCDNA3.4 and transiently transfected into Expi 293F suspension cells using PEI. On day 6 post-transfection, the cell suspension was collected, centrifuged at 6000 rpm for 20 minutes, and the supernatant was collected. The supernatant was filtered through a 0.45 μm filter and purified using a protein G affinity column (NanoMicro, China). The purity and molecular weight of the purified protein were preliminarily identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results are as follows: Figure 2 As shown, the SDS-PAGE electrophoresis results indicate that the purity of the three Mi-LYTAC molecules is high (≥85%), and the molecular weights of the heavy chain (H) and the light chain (L) containing the "mask" are in line with the expected size.

[0037] (3) Assay of the binding activity of Mi-LYTAC to target proteins

[0038] MDA-MB-231 cells (PD-L1 positive cells) and SK-Hep-1 cells (PD-L1 negative) were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin to the logarithmic growth phase. LYTAC molecules (100 nM) without "Mask" protein modification were added as a control, and Mi-LYTAC① (100 nM), Mi-LYTA② (100 nM), and Mi-LYTA③ (100 nM) were added to MDA-MB-231 cells and SK-Hep-1 cells, respectively. The cells were incubated at 4°C for 30 minutes, washed with 2% FBS-PBS, and then anti-human IgG (H+L) fluorescent antibody (1:2000 dilution) was added. The cells were incubated at 4°C for 20 minutes, washed with 2% FBS-PBS, and resuspended. The affinity activity of the fusion antibody with the cells was measured using a flow cytometer (CytoFLEX, Beckman, USA). The results are shown below. Figure 3 As shown. Figure 3 As shown in Figure a, the affinity activities of the three Mi-LYTACs for PD-L1 are close to those of the control LYTAC molecule, indicating that they can recognize PD-L1 normally. However, the affinity of Mi-LYTACs for cell surface IGF2R has changed (see Figure a). Figure 3 (b). Among them, Mi-LYTAC③ could not be calculated to obtain an effective affinity value, indicating that its affinity with cells is reduced.

[0039] (4) Determination of the protein degradation capacity of Mi-LYTAC

[0040] Following step (3), MDA-MB-231 cells were cultured to the logarithmic growth phase and then inoculated with 2×10⁻⁶ cells. 5 Cells were seeded per well in 12-well plates and cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin at 37°C for 24 hours. Then, 100 nM Mi-LYTAC and control LYTAC were added, respectively. After 24 hours, cells were collected, and 100 μL of RIPA lysis buffer containing 1 mM protease inhibitor was added to each well. Cells were incubated on ice for 30 minutes, followed by high-speed centrifugation to collect the cell lysate supernatant. The supernatant was subjected to SDS-PAGE electrophoresis for 1 hour, then transferred to a PVDF membrane at constant current for 90 minutes. The membrane was blocked with 5% skim milk powder for 1 hour, followed by overnight incubation at 4°C with anti-human PD-L1 primary antibody (1:1000). Finally, the membrane was incubated at room temperature for 1 hour with anti-rabbit HRP secondary antibody (1:5000) before imaging. Results are as follows: Figure 4 As shown, the configurations of Mi-LYTAC① and Mi-LYTAC② do not affect the degradation of PD-L1, but the ability of Mi-LYTAC③ to degrade PD-L1 is significantly reduced, indicating that the configuration design of Mi-LYTAC③ can play a role in shielding IGF2R, resulting in its inability to bind effectively with IGF2R.

[0041] Furthermore, after incubating 100 nM Mi-LYTAC③ with 200 ng / mL MMP2 or MMP9 protease at 37°C for 12 hours, the molecular weight of the light chain decreased, and the "mask" protein on the surface light chain was effectively cleaved by MMP2 or MMP9 protease (see...). Figure 5 (a), and its ability to degrade PD-L1 was restored after cleavage (see a). Figure 5 (b)

Claims

1. A tumor microenvironment-responsive LYTAC molecule, characterized in that, The sequence of the heavy chain Mi-LYTAC-H of the LYTAC molecule is shown in SEQ ID NO.1, and the sequence of the light chain Mi-LYTAC-L is shown in SEQ ID NO.

4.

2. The method for preparing the tumor microenvironment-responsive LYTAC molecule according to claim 1, characterized in that, include: Using PD-L1 antibody as the target recognition unit and IGF2 as the ligand for the lysosomal target receptor IGF2R, IGF2 is fused to the light chain end of the antibody, and then the "Mask" protein is linked by a proteolytic enzyme-responsive linker peptide.

3. The preparation method according to claim 2, characterized in that, The "Mask" protein is derived from the CD loop region of IGF2R protein domain 11, and its sequence is shown in SEQ ID NO.

5.

4. The preparation method according to claim 2, characterized in that, The proteolytic enzyme is matrix metalloproteinase MMP2 or matrix metalloproteinase MMP9.

5. A method for inducing the degradation of PD-L1 protein in tumor cells for non-disease diagnosis or treatment purposes, characterized in that, Tumor cells were treated with the tumor microenvironment-responsive LYTAC molecule as described in claim 1.

6. The method according to claim 5, characterized in that, The concentration of LYTAC molecules was above 100 nM, and the treatment time was above 24 hours.

7. The use of the tumor microenvironment-responsive LYTAC molecule according to claim 1 in the preparation of antitumor agents.

8. The use of the tumor microenvironment-responsive LYTAC molecule of claim 1 in the preparation of a PD-L1-targeted degradation inducer, a tumor cell proliferation inhibitor, and / or a tumor cell migration inhibitor for the treatment of tumor cells.

9. The application according to claim 7 or 8, characterized in that, Tumor cells, breast cancer cells.

10. The application according to claim 7 or 8, characterized in that, LYTAC molecules have the effect of blocking IGF2.

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