A protein scaffold, its preparation method and application

Abstract

This application discloses a protein scaffold, its preparation method, and its applications, belonging to the field of biomedical technology. The protein scaffold of this application is a hexamer composed of six identical fusion proteins, wherein the fusion protein contains a modified IgGFc and two Fc receptors; the modified IgGFc contains an IgGFc mutant and an IgM tail, with the IgM tail fused to the C-terminus of the IgGFc mutant; the IgGFc mutant possesses the amino acid sequence shown in SEQ ID NO.1. This protein scaffold can not only directly achieve antibody polymerization, greatly improving the effector function and titer of therapeutic antibodies, but also non-covalently bind to the Fc portion of the antibody through Fc receptors, maximizing the preservation of the antibody's structure and function.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, and in particular relates to a protein scaffold, its preparation method and application. Background Technology

[0002] Monoclonal antibody drugs have shown great promise in cancer treatment due to their high specificity, efficacy, and safety. However, numerous clinical trials have revealed significant differences in the therapeutic effects of antibody drugs among different cancer patients, with an overall low response rate. Therefore, there is an urgent need to develop effective strategies to enhance the anti-tumor efficacy of antibodies.

[0003] In related technologies, anti-tumor effects can be enhanced by targeting TNFRSF antibodies and immune checkpoint antibodies. Developing TNFRSF-targeting antibodies requires activating downstream TNFRSF signaling pathways. There are two strategies to promote this activation: 1) Inducing cross-linking and aggregation of TNFRSF-targeting antibodies. Specifically, this involves designing and modifying the Fc fragment of the TNFRSF-targeting antibody, then utilizing Fc receptors on the surface of immune effector cells to mediate aggregation and cross-linking, thereby promoting the aggregation of TNFRSF target receptors to form receptor superclusters, resulting in stronger downstream signal transduction; 2) Preparing polyprotein drugs by tandemly expressing TNFRSF-targeting antibody fragments or ligand molecules. However, the two strategies for activating the downstream signaling pathway of TNFRSF have the following problems in clinical anti-tumor treatment: First, the design of immune effector cells expressing Fc receptors to induce cross-linking aggregation of antibodies targeting TNFRSF is greatly limited in clinical anti-tumor treatment because the tumor microenvironment usually lacks the required immune effector cells. Second, achieving high-valence aggregation usually requires the tandem expression of multiple proteins. Since the tandem expression of multiple proteins affects the binding of protein drugs to target receptors, and the resulting polypeptide drugs do not have spatial directionality, the protein drugs are randomly arranged after tandem expression, making it difficult to induce TNFRSF target receptors to aggregate more effectively in the same direction.

[0004] Developing immune checkpoint antibodies involves using chemical conjugation or DNA recombination techniques to create multivalent antibodies that promote binding to target molecules. However, the preparation of multivalent antibodies typically requires the polymerization of chemical materials or proteins not naturally occurring in vivo, posing potential risks such as biocompatibility and immunogenicity. Furthermore, the preparation of each multivalent antibody drug targeting a specific target requires de novo design, making the process cumbersome and hindering the high-throughput preparation of various multivalent antibodies. Summary of the Invention

[0005] This application addresses two problems: firstly, by providing a protein scaffold, its preparation method, and its application, it solves the problems arising from the development of TNFRSF-targeting antibodies to activate downstream signaling pathways of TNFRSF; and secondly, it addresses the problems arising from the development of immune checkpoint antibodies.

[0006] To achieve the above objectives, the technical solution of this application is:

[0007] The first aspect of this application provides a protein scaffold. The protein scaffold of this application is a hexamer composed of six identical fusion proteins, wherein the fusion proteins contain a modified IgG Fc and two Fc receptors; the modified IgG Fc contains an IgG Fc mutant and an IgM tail, the IgM tail being fused to the C-terminus of the IgG Fc mutant; the IgG Fc mutant has the amino acid sequence shown in SEQ ID NO.1.

[0008] Preferably, in conjunction with the first aspect, the type of the Fc receptor is FcγRI.

[0009] Preferably, in conjunction with the first aspect, the Fc receptor contains a full-length or partial amino acid sequence; and / or, the modified IgG Fc contains a full-length or partial amino acid sequence.

[0010] Preferably, in conjunction with the first aspect, the amino acid sequence of the full-length fragment is increased or decreased by one or more natural amino acid sequences; and / or, the amino acid sequence of the partial fragment is increased or decreased by one or more natural amino acid sequences.

[0011] In conjunction with the first aspect, preferably, the monomeric fusion protein forming the protein scaffold has the amino acid sequence shown in SEQ ID NO. 2.

[0012] Preferably, in conjunction with the first aspect, the average particle size of the protein scaffold is 50-200 nm.

[0013] The second aspect of this application provides a method for preparing the protein scaffold described in the first aspect, comprising the following steps:

[0014] S10: Construct the amino acid sequence shown in SEQ ID NO.2 into a lentiviral vector plasmid to obtain a lentivirus carrying the amino acid sequence;

[0015] S20: After transducing the lentivirus into cells expressing the protein, resistance-pressure screening and limiting dilution culture are performed to obtain a monoclonal cell line expressing the protein scaffold.

[0016] S30: After collecting the culture supernatant of the monoclonal cell line, perform Protein G affinity chromatography separation and purification to obtain the protein scaffold.

[0017] The third aspect of this application provides the use of the protein scaffold described in the first aspect in binding monoclonal antibodies and achieving antibody polymerization.

[0018] In conjunction with the third aspect, preferably, the monoclonal antibody contains an antibody targeting TNFRSF and an immune checkpoint antibody.

[0019] Preferably, in conjunction with the third aspect, the antibody targeting TNFRSF contains antibodies targeting OX40, CD40, 4-1BB, CD27, GITR, DR5, and Fas.

[0020] In conjunction with the third aspect, preferably, the protein scaffold is used to bind at least one of the antibodies targeting TNFRSF to form a polyantibody.

[0021] In conjunction with the third aspect, preferably, the polyantibody is used in the preparation of drugs that enhance the anti-tumor effect of T cells and induce tumor cell apoptosis.

[0022] Preferably, in conjunction with the third aspect, the binding immune checkpoint antibody contains antibodies targeting PD-L1, PD-1, CTLA-4, LAG-3, TIM-3, TIGHT, and VISTA.

[0023] In conjunction with the third aspect, preferably, the protein scaffold is used to bind at least one of the immune checkpoint antibodies to form a multivalent antibody.

[0024] In conjunction with the third aspect, preferably, the multivalent antibody is used in the preparation of drugs that enhance antibody titer and improve anti-tumor efficacy.

[0025] Compared with the prior art, the advantages or beneficial effects of the embodiments of this application include at least the following:

[0026] The protein scaffold of this application is a hexamer formed by the fusion expression of Fc receptor, IgG Fc mutant, and IgM tail. Firstly, it can directly induce TNFRSF target receptor aggregation and promote downstream signal activation in the presence of immune cells independent of Fc receptor, enhancing the efficacy of tumor immunotherapy and greatly promoting the application of TNFRSF-targeting antibodies in clinical anti-tumor treatment. Secondly, based on a simulated IgM aggregation process, the resulting aggregated structure forms a three-dimensional umbrella-like structure similar to IgM, possessing spatial orientation, which can better induce TNFRSF target receptors to aggregate in the same direction. Thirdly, it can be used to bind existing immune checkpoint antibodies to improve their titer and enhance their anti-tumor effects. Fourthly, this protein scaffold can directly bind existing commercial immune checkpoint antibodies and be used to prepare multivalent antibodies, eliminating the need for de novo design and expression, saving cumbersome processes and steps. Specifically:

[0027] First, the protein scaffold of this application can directly achieve antibody polymerization, especially by simple physical mixing, which can conveniently, efficiently and controllably achieve antibody polymerization, greatly promoting the effector function and potency of therapeutic antibodies.

[0028] Second, the hexamer structure of the protein scaffold in this application has good stability and does not require the introduction of non-in vivo natural proteins, thereby avoiding dependence on chemical materials, resulting in low immunogenicity and excellent biocompatibility.

[0029] Third, the protein scaffold of this application can bind to the Fc part of the antibody through the Fc receptor, and the binding between the two is non-covalent rather than chemically bonded, thus preserving the structure and function of the antibody to the greatest extent.

[0030] Fourth, the protein scaffold of this application can be used to bind antibodies targeting TNFRSF, thereby inducing TNFRSF polymerization and enhancing the tumor immunotherapy effect of TNFRSF antibodies. Simultaneously, the protein scaffold of this application can also be used to bind immune checkpoint antibodies, increasing the titer of immune checkpoint antibodies and promoting their anti-tumor effects. Attached Figure Description

[0031] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1A schematic diagram of the protein structure of the protein scaffold (MV-FcR) provided in the embodiments of this application;

[0033] Figure 2 This is a schematic diagram of the sequence structure of the fusion protein (FcR-mut IgG Fc-TP) provided in the embodiments of this application;

[0034] Figure 3 An expression plasmid map of an embodiment of this application that can express and assemble to form a protein scaffold (MV-FcR);

[0035] Figure 4 A graph showing the characterization results of the molecular weight range of the protein scaffold (MV-FcR) provided in the embodiments of this application;

[0036] Figure 5 This is a graph showing the characterization results of the antibody affinity of the protein scaffold (MV-FcR) provided in the embodiments of this application;

[0037] Figure 6 This is a schematic diagram of the process for preparing the polyantibody MV-αOX40 provided in the embodiments of this application;

[0038] Figure 7 The graph shows the detection results of the polyantibody MV-αOX40 and αOX40 titer provided in the embodiments of this application;

[0039] Figure 8 This is a diagram showing the results of the in vitro binding of the polyantibody MV-αOX40 to T cells provided in the embodiments of this application;

[0040] Figure 9 This is a diagram showing the results of the in vitro promotion of T cell proliferation by the polyantibody MV-αOX40 provided in the embodiments of this application;

[0041] Figure 10 This is a diagram showing the results of the in vitro promotion of T cell activation by the polyantibody MV-αOX40 provided in the embodiments of this application;

[0042] Figure 11 This is a diagram showing the results of the polyantibody MV-αOX40 promoting cytokine expression in T cells in vitro, as provided in the embodiments of this application.

[0043] Figure 12 This is a diagram showing the results of the polyantibody MV-αOX40 enhancing T cell function in vivo, as provided in the embodiments of this application.

[0044] Figure 13 This is a diagram showing the results of the polyantibody MV-αOX40 inhibiting tumor growth in a mouse colorectal cancer model, as provided in the embodiments of this application.

[0045] Figure 14This is a diagram showing the results of the polyantibody MV-αOX40 prolonging the survival of a mouse colorectal cancer model provided in the embodiments of this application;

[0046] Figure 15 This is a schematic diagram of the process for preparing the polyantibody MV-αPD-L1 provided in the embodiments of this application;

[0047] Figure 16 This is a diagram showing the results of the polyantibody MV-αPD-L1 enhancing T cell proliferation and activation in vivo, as provided in the embodiments of this application.

[0048] Figure 17 This is a diagram showing the results of the polyantibody MV-αPD-L1 inhibiting tumor growth in a mouse melanoma model, as provided in the embodiments of this application.

[0049] Figure 18 This is a diagram showing the results of the polyantibody MV-αPD-L1 prolonging the survival of a mouse melanoma model provided in the embodiments of this application;

[0050] Figure 19 This is a graph showing the changes in cytokines in tumors after treatment with the polyantibody MV-αPD-L1 provided in the embodiments of this application. Detailed Implementation

[0051] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0052] In the following description of this embodiment, the term "and / or" is used to describe the association relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, B existing alone, and A and B existing simultaneously. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0053] In the following description of this embodiment, the term "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0054] Those skilled in the art should understand that in the following description of the embodiments of this application, the sequence of numbers does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0055] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms "a" and "the" as used in the embodiments of this application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0056] In a first aspect, embodiments of this application provide a protein scaffold (MV-FcR).

[0057] Please see Figure 1 As shown, the protein scaffold (MV-FcR) of this application embodiment is a hexamer composed of six identical fusion proteins (FcR-mut IgG Fc-TP). Each fusion protein (FcR-mut IgG Fc-TP) consists of a modified IgG Fc (mut IgG Fc-TP) and two Fc receptors (FcR). The modified IgG Fc (mut IgG Fc-TP) contains an IgG Fc mutant and an IgM tail, with the IgM tail fused to the C-terminus of the IgG Fc mutant. The IgG Fc mutant possesses the amino acid sequence shown in SEQ ID NO. 1.

[0058] The amino acid sequences of the Fc receptor (FcR) and IgM tailpiece (TP) can be directly obtained from the NCBI database, and will not be specifically described in this application embodiment. The IgG Fc mutant (mut IgG Fc) can be constructed according to the genetic engineering techniques known in the art. For example, firstly, the original amino acid sequence of IgG Fc (specifically shown in SEQ ID NO.3) is obtained from the NCBI database. Then, using the amino acid sequence shown in SEQ ID NO.3 as a template, the leucine at position 309 is mutated to cysteine, and the histidine sequence at position 310 is mutated to leucine, thus obtaining the amino acid sequence shown in SEQ ID NO.1, which is the IgG Fc mutant (mut IgG Fc).

[0059] It should be noted that the Fc receptor can be a naturally occurring FcR present in vivo, such as a human or mouse FcR. The protein scaffold (MV-FcR) of this application embodiment can directly bind to and polymerize existing antibodies against TNFRSF, thereby inducing TNFRSF target aggregation. This process does not require de novo design to express polymerized antibodies, nor does it rely on the presence of immune cells that can express receptors for cross-linked polymerized antibodies at the target site, greatly promoting practical applications. At the same time, this application embodiment can achieve self-assembly during the fusion expression of IgM tail (TP) and IgG Fc mutant (mut IgG Fc), thereby enabling the polymerization of IgG Fc fragments to form disulfide bonds between the fusion proteins (FcR-mut IgG Fc-TP), further stabilizing the formation of a hexamer structure.

[0060] Those skilled in the art should understand that the embodiments of this application do not impose special limitations on the basic structure of FcR, and it may contain / or not contain a hinge region. Furthermore, the embodiments of this application do not specifically limit the connection method between the sequences. Therefore, FcR and the IgG Fc mutant (mut IgG Fc) may or may not contain a peptide linker, and the IgG Fc mutant (mut IgG Fc) and the IgM tail (TP) may or may not contain a peptide linker, as long as stable connections can be achieved between FcR and the IgG Fc mutant (mut IgG Fc), and between the IgG Fc mutant (mut IgG Fc) and the IgM tail (TP).

[0061] In a specific embodiment, the type of Fc receptor is preferably FcγRI, which can specifically bind to various IgG type monoclonal antibodies.

[0062] In specific embodiments, the Fc receptor contains a full-length or partial amino acid sequence, and / or the modified IgG Fc contains a full-length or partial amino acid sequence. Wherein, when the Fc receptor is a full-length amino acid sequence, the modified IgG Fc can be either a full-length or partial amino acid sequence; similarly, when the Fc receptor is a partial amino acid sequence, the modified IgG Fc can be either a full-length or partial amino acid sequence. This application does not impose any particular limitation on this, as long as the fusion expression can form a hexamer structure with the corresponding function.

[0063] In specific embodiments, the amino acid sequence of the full-length fragment may have at least one natural amino acid sequence added or removed, and / or the amino acid sequence of the partial fragment may have one or more natural amino acid sequences added or removed. The amino acid sequence of the full-length fragment may have one or more natural amino acid sequences added or removed; similarly, the amino acid sequence of the partial fragment may have one or more natural amino acid sequences added or removed. This application does not impose any particular limitations on this, as long as the fusion expression can form a hexamer structure with the corresponding function.

[0064] In a specific embodiment, based on the above-mentioned sequences, all of which are mouse-derived sequences, the monomeric fusion protein forming the protein scaffold has the amino acid sequence shown in SEQ ID NO.2.

[0065] In a specific embodiment, the average particle size of the protein scaffold (MV-FcR) is preferably 50-200 nm.

[0066] Secondly, embodiments of this application provide a method for preparing the protein scaffold (MV-FcR) described in the first aspect, wherein the method is formed by self-assembly when the fusion protein (FcR-mut IgG Fc-TP) is expressed, specifically including steps S10-S30.

[0067] S10: The amino acid sequence shown in SEQ ID NO.2 is constructed into a lentiviral vector plasmid to obtain a lentivirus carrying the amino acid sequence, specifically:

[0068] S101: Construct the sequence structure of the fusion protein (FcR-mut IgG Fc-TP), wherein the sequence structure of the fusion protein (FcR-mut IgG Fc-TP) is as follows: Figure 2 As shown in the diagram, the sequence structure consists of the IL-2 secretion signal peptide sequence, the Fc receptor sequence, the IgG Fc mutant sequence, and the IgM TP sequence, from the 5' end to the 3' end.

[0069] S102: After codon optimization of the fusion protein (FcR-mut IgG Fc-TP) sequence structure, it was sent to Shanghai Sangon Biotech Co., Ltd. for sequence synthesis, and then inserted into the expression plasmid (Addgene, 72266) of the lentiviral vector pCDH-EF1 to obtain an expression plasmid capable of expressing and assembling to form a protein scaffold (MV-FcR). The map of this expression plasmid capable of expressing and assembling to form a protein scaffold (MV-FcR) is as follows: Figure 3 As shown.

[0070] S20: After transducing the lentivirus into cells expressing the protein scaffold, resistance-controlled selection and limiting dilution culture were performed to obtain monoclonal cell lines capable of expressing the protein scaffold (MV-FcR), specifically:

[0071] S201: Lentiviral vector plasmid strains capable of expressing protein scaffolds (MV-FcR) were constructed using shake flasks, along with psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259) plasmid strains for lentivirus packaging. These were then transferred to ampicillin-resistant LB medium and cultured at 37°C for 220 rpm for 20 h in a constant temperature shaker. The MV-FcR expression plasmid and lentivirus packaging plasmid were extracted using an endotoxin-free plasmid extraction kit.

[0072] S202: The cultured 293T cells were digested, neutralized, centrifuged, and counted sequentially, and then seeded in 100mm diameter culture dishes at a density of 5×10⁶ cells / year. 6 Cells were cultured at 37°C and 5% CO2 for 12 h. 250 μL of serum-free DMEM medium was added to an enzyme-free EP tube, followed by the addition of 1.64 pmol of a lentiviral vector plasmid capable of expressing and assembling a protein scaffold (MV-FcR), 1.30 pmol of psPAX2, and 0.72 pmol of pMD2.G lentiviral packaging plasmid, and mixed thoroughly. Another 250 μL of serum-free DMEM medium was added to a different enzyme-free EP tube, followed by the addition of 1 mg / mL PEI solution to a final concentration with a PEI:DNA ratio of 3:1. The liquids in both EP tubes were mixed and incubated at room temperature for 20 min. The cultured 293T cells were then replaced with fresh serum-free DMEM medium, and the mixture from the EP tubes was added and shaken well. The cells were then transfected and cultured at 37°C and 5% CO2 for 12 h. After transfection culture, the culture medium in the 293T cells was replaced with DMEM complete medium containing 10% FBS for continued culture to package lentiviruses expressing the MV-FcR sequence. After 48 hours of culture, the culture supernatant containing the packaged lentivirus was collected. Cells and debris in the collected supernatant were removed by filtration using a 0.45 μm sterile filter. The lentivirus in the collected supernatant was then concentrated using a viral concentrator.

[0073] S203: Count the cultured CHO-K1 cells and seed 1×10⁶ cells into each well of a 24-well plate. 5After the cells were cultured, they were incubated in a constant temperature incubator at 37℃ and 5% CO2 for 12 hours. Then, concentrated lentivirus containing the FcR-mut IgG Fc-TP sequence was added to transduce CHO-K1 cells for 12 hours. After that, the CHO-K1 cell culture medium was replaced with fresh F-12K complete medium and cultured for 36 hours. Antibiotic pressure screening was used, and multiple monoclonal cell lines expressing MV-FcR were obtained by limiting dilution. From these, monoclonal cell lines with high expression levels were selected for expansion culture and preservation.

[0074] S30: The culture supernatant of CHO-K1 cell line expressing protein scaffold (MV-FcR) was separated and purified by Protein G affinity chromatography, and concentrated by ultrafiltration to obtain the protein scaffold (MV-FcR).

[0075] To confirm the successful synthesis of the protein scaffold (MV-FcR) described above, this application embodiment characterized the molecular weight range of the protein scaffold (MV-FcR) by detecting size exclusion chromatography. The specific method is as follows:

[0076] First, prepare the Marker stock solution and working solution: Turn on the AKTApure protein purification instrument, connect the size exclusion column to the purification instrument, and equilibrate two column volumes using filtered and degassed 20% ethanol, ultrapure water, and mobile phase buffer (0.15M NaCl, 20mM Na2HPO4, pH=7.4). Then, add 0.5mL of sterile deionized water to the size exclusion Marker powder (18mg / vial) to obtain the Marker stock solution. Take 50μL of the Marker stock solution and dilute it to 3mg / mL as the working solution. Seal the remaining Marker stock solution and store it in a refrigerator at 4℃.

[0077] Next, the diluted size exclusion marker working solution was injected into the column using a 500 μL loading loop. The flow rate of the mobile phase buffer was 0.5 mL / min, and the peak chromatogram of the MV-FcR protein was recorded and saved.

[0078] Next, after loading the marker, wash the column with the mobile phase for two column volumes. Then, adjust the protein scaffold drug sample to 0.5 mg / mL, and use a 500 μL loading loop to inject the protein sample into the column. The flow rate of the mobile phase is 0.5 mL / min, and the peak chromatogram of the protein marker is recorded and saved.

[0079] Finally, the two peak spectra recorded above are superimposed to obtain... Figure 4 The graph shows the molecular weight range characterization results of the protein scaffold (MV-FcR). The molecular weight range of the protein scaffold can be obtained by comparing two peak spectra.

[0080] according to Figure 4 It can be seen that the MV-FcR protein sample has only one large and relatively obvious main peak at a position greater than 670kDa, while the calculated molecular weight of the protein scaffold MV-FcR is 825.6kDa, which is consistent with the expected result.

[0081] Thirdly, embodiments of this application provide the application of the protein scaffold (MV-FcR) described above in binding antibodies and mediating antibody polymerization, the method comprising:

[0082] First, the OX40 antigen stock solution was diluted to 5 μg / mL with sterile PBS, and 50 μL was added to each well of a MaxiSorp flat-bottom protein binding plate. The plate was incubated overnight at 4°C. The supernatant was discarded, and after washing with PBS, 200 μL of 5% BSA was added to each well to block the remaining protein binding sites. The plate was then incubated on a shaker at room temperature for 2 h. The plate was then washed twice with PBS to obtain the αOX40 antibody.

[0083] Next, add 50 μL of 20 μg / mL αOX40 antibody to each well, incubate at room temperature for 2 h, wash the plate twice with PBS and add protein scaffolds (MV-FcR) of different concentrations in sequence.

[0084] Finally, the ability of the antibody to bind was determined by detecting the binding of MV-FcR on the plate, and the results were as follows: Figure 5 As shown. Among them, Figure 5 The figure shows the results of antibody affinity characterization for the protein scaffold (MV-FcR).

[0085] according to Figure 5 By performing fitting calculations, the dissociation constant KD value of MV-FcR after binding with αOX40 antibody was found to be 32.93 nM, indicating that the protein scaffold (MV-FcR) can effectively bind with αOX40 antibody and that the affinity between the two is strong.

[0086] To detect the ability of MV-FcR to mediate antibody polymerization and increase antibody titer, the embodiments of this application firstly based on Figure 6 The process shown is used to prepare MV-αOX40, wherein, Figure 6 A schematic diagram of the process for preparing the polyantibody MV-αOX40, specifically including:

[0087] After mixing MV-FcR and αOX40 at a mass ratio of 1:1, incubate at 4°C for 2 hours to obtain MV-αOX40.

[0088] Next, different concentrations of αOX40 or MV-αOX40, as described above, were added sequentially to the plates. After incubation at room temperature for 2 hours, the binding of αOX40 or MV-αOX40 on the plates was detected, as well as the change in the amount of antigen bound after MV-FcR-mediated αOX40 polymerization to MV-αOX40 was detected. The results were as follows: Figure 7 As shown. Among them, Figure 7 The image shows the detection results of the polyantibody MV-αOX40 and the αOX40 titer.

[0089] according to Figure 7 It is evident that the MV-αOX40 complex prepared in the embodiments of this application has a higher titer and can bind more antigens compared to αOX40, indicating that MV-FcR-mediated antibody polymerization can improve antibody titer.

[0090] Fourthly, embodiments of this application provide an in vitro binding experiment of the polyantibody MV-αOX40 to T cells as described above, the method including:

[0091] First, mouse T cells were sorted using CD3 magnetic beads and cultured for 72 h in T cell culture medium containing 2 μg / mL mouse CD3 activating antibody.

[0092] Secondly, MV-αOX40 labeled with Cy5 fluorescent molecules Cy5 Add to cultured activated T cells and incubate for 2 hours;

[0093] Finally, after incubation, the T cells were washed with PBS by centrifugation, then added to a glass slide. Laser confocal microscopy was used to image the binding of MV-αOX40 to the T cells. The results were as follows: Figure 8 As shown. Among them, Figure 8 This is a diagram showing the results of the polyantibody MV-αOX40 binding to T cells in vitro.

[0094] according to Figure 8 It can be seen that T cells have obvious MV-αOX40 on their surface. Cy5 The combination indicates that the polyantibody MV-αOX40 can bind to T cells in vitro.

[0095] Fifthly, embodiments of this application provide an in vitro T cell proliferation and activation experiment using the aforementioned polyantibody MV-αOX40, the method of which includes:

[0096] First, mouse T cells were sorted by CD3 magnetic beads and then cultured in T cell culture medium containing 2 μg / mL mouse CD3 activating antibody and 5 μg / mL MV-αOX40 for 72 h.

[0097] Next, after incubation, T cells were washed with PBS, collected by centrifugation, counted, and then blocked with anti-CD16 / 32 antibody. Flow cytometry antibodies anti-CD45, anti-CD3, and anti-CD69 were used to label cell surface markers. For labeling Ki-67 in the cell nucleus, the cells were treated with a nuclear membrane penetration kit and then labeled with anti-Ki-67 antibody. After sample processing, the samples were filtered through a 200-mesh nylon mesh and transferred to flow cytometry tubes for detection using a flow cytometer. The results were as follows: Figures 9 to 10 .in, Figure 9 This is a diagram showing the results of the polyantibody MV-αOX40 promoting T cell proliferation in vitro. Figure 10 This is a diagram showing the results of T cell activation promoted in vitro by the polyantibody MV-αOX40.

[0098] according to Figure 9 It can be seen that MV-αOX40 can effectively promote the proliferation of T cells, and compared with the use of αOX40 antibody alone, the T cell proliferation rate increased from 56.7% to 74.1%.

[0099] according to Figure 10 It can be seen that MV-αOX40 further promoted T cell activation, and the expression level of CD69 molecules increased from 50.5% to 56.3% compared with the use of αOX40 antibody.

[0100] Sixthly, embodiments of this application provide the above-described in vitro T cell function enhancement experiment using the polyantibody MV-αOX40, the method of which includes:

[0101] First, mouse T cells were sorted by CD3 magnetic beads and then cultured for 72 h in T cell culture medium containing 2 μg / mL mouse CD3 activating antibody and 5 μg / mL MV-αOX40, respectively.

[0102] Secondly, after incubating the T cells, they were washed and collected by centrifugation with PBS, and then the total RNA of the cells was extracted using RNAiso plus and reverse transcribed.

[0103] Finally, qRT-PCR was used to detect changes in the mRNA expression levels of IL-2 and IFN-γ in T cells. The results were as follows: Figure 11 As shown. Among them, Figure 11 This figure shows the results of the polyantibody MV-αOX40 promoting cytokine expression in T cells in vitro.

[0104] according to Figure 11It was found that, compared with the PBS group, the mRNA expression level of IL-2 in T cells increased by 4.6 times after αOX40 treatment, and further increased by 6.8 times after MV-αOX40 treatment; at the same time, the expression level of IFN-γ in T cells was also significantly increased, indicating that MV-αOX40 further promoted the secretion of IL-2 by T cells to enhance T cell proliferation and survival, as well as the expression of IFN-γ cytokines to enhance tumor killing.

[0105] Seventhly, embodiments of this application provide an in vivo T cell function enhancement experiment using the polyantibody MV-αOX40 described above, to detect the effect of MV-αOX40 on T cell function in vivo, the method comprising:

[0106] A mouse colorectal cancer model was established. After expanding the culture of mouse CT26 cells, the tumor cell culture medium was replaced with serum-free medium, and the tumor cells were starved for 4 hours. The starved tumor cells were collected and centrifuged at 500g for 3 minutes at 4°C. The cell pellet was resuspended in PBS, counted, and then matrix gel was added to a final concentration of 10%, adjusting the cell density to 1×10⁻⁶ cells / mL. 7 Cells / mL, stored on ice. A CT26 colorectal cancer model was established using BALB / c mice. 1×10⁶ cells / mL were subcutaneously implanted in the right back of the mice. 6 Cells. In mice, tumor volume approached 100 mm. 3 At that time, all mice were randomly divided into groups and intraperitoneally injected with 5 mg / kg of MV-αOX40, administered every other day for three times.

[0107] After treatment, mouse tumors were removed, minced, and placed on ice. Tumor digestion solution (serum-free DMEM medium with collagenase to 1 mg / mL, hyaluronidase to 100 μg / mL, and DNase to 100 μg / mL) was added to the minced tumor tissue and incubated at 37°C in a shaker for 30 min. After centrifugation at 500g for 3 min at 4°C to remove the digestion solution, the tumor tissue was washed with PBS and passed through a 200-mesh nylon screen. Cells were counted and blocked with anti-CD16 / 32 antibody. Then, flow cytometry was used to label the cell surface markers with anti-CD45, anti-CD3, anti-CD8, anti-CD4, anti-CD69, anti-CD44, and anti-CD62L antibodies. For labeling intracellular cytokines, a stimulant is first added to further promote cytokine production, while a protein transport inhibitor is added to inhibit secretion. The resulting cytokines are then immobilized in the cells for flow cytometry detection. After treatment with a cell membrane penetration kit, antibodies against IFN-γ, Perforin, Granzyme B, IL-2, and IL-4 are added to label the cytokines. After sample processing, the samples are filtered through a 200-mesh nylon mesh and transferred to flow cytometry tubes for detection. The results are as follows: Figure 12 As shown. Among them, Figure 12 Figure showing the results of the multi-antibody MV-αOX40 enhancing T cell function in vivo.

[0108] according to Figure 12 It can be seen that after treatment, compared with the control group and the αOX40 group, the T cell function in the tumors of mice in the MV-αOX40 group was significantly enhanced. The secretion of anti-tumor functional cytokines IFN-γ, cytotoxic effector factors Perforin and Granzyme B, as well as cytokines IL-2 and IL-4 that maintain T cell proliferation and survival, were significantly increased, indicating that MV-αOX40 effectively enhanced T cell activation and improved T cell function.

[0109] Eighthly, embodiments of this application provide an in vivo treatment experiment of the aforementioned polyantibody MV-αOX40 for mouse colorectal cancer, to detect the therapeutic effect of MV-αOX40 on mouse colorectal cancer in vivo, the method comprising:

[0110] A mouse CT26 colorectal cancer model was established. After expanding the culture medium of mouse CT26 cells, the tumor cell culture medium was replaced with serum-free medium, and the tumor cells were starved for 4 hours. The starved tumor cells were collected and centrifuged at 500g for 3 minutes at 4°C. The cell pellet was resuspended in PBS, counted, and then matrix gel was added to a final concentration of 10%, adjusting the cell density to 1×10⁻⁶.7 Cells / mL, stored on ice. A CT26 colorectal cancer model was established using BALB / c mice, with 1×10⁶ cells subcutaneously implanted into the right back of the mouse. Tumor volume in mice approaching 100 mm² was measured. 3 At that time, all mice were randomly divided into groups and intraperitoneally injected with 5 mg / kg of MV-αOX40, administered every other day for three times. Tumor growth and survival changes in mice were monitored, and the results were as follows: Figures 13 to 14 As shown. Among them, Figure 13 The figure shows the results of the polyantibody MV-αOX40 inhibiting tumor growth in a mouse colorectal cancer model. Figure 14 Figure showing the results of prolonging the survival of a mouse colorectal cancer model using the polyantibody MV-αOX40.

[0111] according to Figure 13 It can be seen that after administration of MV-αOX40, the growth of tumors in the mouse CT26 colorectal cancer model was significantly inhibited.

[0112] according to Figure 14 It can be seen that the survival time of the mouse model was significantly prolonged after MV-αOX40 treatment.

[0113] This experiment shows that MV-αOX40 can effectively inhibit tumor growth and prolong the survival time of mice by enhancing the function of T cells in killing tumor cells.

[0114] Ninthly, embodiments of this application provide an in vivo experiment on the polyantibody MV-αPD-L1 described above to relieve T cell inhibition and promote tumor cell killing, in order to detect the effect of MV-αPD-L1 on T cell function in vivo.

[0115] First, according to Figure 15 The procedure shown is for preparing the polyantibody MV-αPD-L1. Among other things, Figure 15 This is a schematic diagram of the process for preparing the polyantibody MV-αPD-L1. The specific methods include:

[0116] MV-FcR and αPD-L1 were mixed at a mass ratio of 1:1 and incubated at 4°C for 2 hours to obtain MV-αPD-L1.

[0117] Next, the B16-F10 cells were expanded. The tumor cell culture medium was replaced with serum-free medium, and the tumor cells were starved for 4 hours. The starved tumor cells were collected and centrifuged at 500g for 3 minutes at 4°C. The cell pellet was resuspended in PBS, counted, and then matrix gel was added to a final concentration of 10%, and the cell density was adjusted to 1×10⁻⁶ cells / year. 7 After collecting 100 cells / mL, place on ice.

[0118] Next, a B16-F10 melanoma model was established using C57BL / 6 mice, and 5×10⁶ cells were implanted subcutaneously on the right side of the mouse back. 5 Cells. In mice, tumor volume approached 100 mm. 3 At that time, all mice were randomly divided into groups and injected intraperitoneally with 10 mg / kg of MV-αPD-L1, administered every other day, for a total of three times.

[0119] Finally, after treatment, mouse tumors were removed, minced, and placed on ice. Tumor digestion solution (serum-free DMEM medium with collagenase to 1 mg / mL, hyaluronidase to 100 μg / mL, and DNase to 100 μg / mL) was added to the minced tumor tissue and incubated at 37°C in a shaker for 30 min. The tumor digestion solution was removed by centrifugation at 500g for 3 min at 4°C. After washing with PBS, the tissue was passed through a 200-mesh nylon screen, counted, and blocked with anti-CD16 / 32 antibody. Simultaneously, flow cytometry was used to label cell surface markers with anti-CD45, anti-CD3, anti-CD8, anti-CD4, anti-CD69, anti-CD44, anti-CD62L, anti-ICOS, and anti-CD25. For labeling intranuclear FOXP3 and Ki-67, the cells were treated with a nuclear membrane penetration kit before adding anti-FOXP3 and anti-Ki-67 antibodies. After sample processing, the sample was filtered through a 200-mesh nylon mesh and transferred to a flow cytometer for analysis. The results were as follows: Figure 16 As shown. Among them, Figure 16 This is a diagram showing the results of the polyantibody MV-αPD-L1 enhancing T cell proliferation and activation in vivo.

[0120] according to Figure 16 It was found that after administration of MV-αPD-L1, the proportions of Ki-67+, CD69+, CD44+, CD8+, and ICOS+ cells in CD3+ T cells increased to 88.7%, 40.4%, 84.4%, 47.1%, and 34.8%, respectively, while the proportion of FOXP3+ cells decreased to 31.2%. Simultaneously, compared to the control group and the use of αPD-L1 antibody alone, administration of MV-αPD-L1 significantly enhanced the proliferation and activation of functional T cells, significantly increased the proportion of cytotoxic T cells, significantly increased the expression of the induced activated T cell co-stimulatory molecule ICOS, and reduced the proportion of immunosuppressive cells.

[0121] Tenthly, the in vivo treatment experiment of the polyantibody MV-αPD-L1 for mouse melanoma described above in the embodiments of this application, to detect the therapeutic effect of MV-αPD-L1 on mouse melanoma in vivo, specifically includes:

[0122] A mouse B16-F10 melanoma model was established. B16-F10 cells were cultured in a large-scale manner. The tumor cell culture medium was replaced with serum-free medium, and the tumor cells were starved for 4 hours. The starved tumor cells were collected and centrifuged at 500g for 3 minutes at 4°C. The cell pellet was resuspended in PBS, counted, and then matrix gel was added to a final concentration of 10%, adjusting the cell density to 1×10⁻⁶ cells / mL. 7 Cells / mL, stored on ice. A B16-F10 melanoma model was established using C57BL / 6 mice, with 5 × 10⁵ cells implanted subcutaneously into the right back of the mouse. Tumor volume in mice approaching 100 mm² was measured. 3 All mice were randomly assigned to groups and intraperitoneally injected with 10 mg / kg MV-αPD-L1, administered every other day for three doses. Tumor growth and survival were monitored. The results were as follows: Figures 17 to 18 As shown. Among them, Figure 17 This is a diagram showing the results of the polyantibody MV-αPD-L1 inhibiting tumor growth in a mouse melanoma model. Figure 18 Figure showing the results of prolonging the survival of a mouse melanoma model using the polyantibody MV-αPD-L1.

[0123] according to Figure 17 It can be seen that after administration of MV-αPD-L1, the growth of tumors in the mouse B16-F10 melanoma model was significantly inhibited, and the inhibitory effect was better than that of the monomeric αPD-L1 antibody.

[0124] according to Figure 18 It can be seen that the survival time of mouse models was prolonged after MV-αPD-L1 treatment.

[0125] Simultaneously, to detect changes in cytokines in mouse melanoma tumors after in vivo treatment with MV-αPD-L1, in this embodiment of the application, after treatment, mouse tumors were harvested, 200 mg of tumor tissue was weighed, and 1 mL of tissue lysis buffer containing protease inhibitors from an ELISA kit was added. The tumor tissue was homogenized using a Dounce tissue homogenizer in an ice-water bath. The homogenate was centrifuged at 10000g for 3 min at 4°C, and the supernatant was collected. Changes in cytokines in the tumors after treatment were detected using mouse IL-6 ELISA kits, mouse IL-12p70 ELISA kits, and mouse TNF-α ELISA kits, respectively. The results were as follows: Figure 19 As shown. Among them, Figure 19 Figure showing the changes in cytokines in tumors after treatment with the multi-antibody MV-αPD-L1.

[0126] according to Figure 19It is known that MV-αPD-L1 treatment significantly enhances the production of inflammatory cytokines in tumors, which can further promote the effective killing of tumors by T cells and improve the anti-tumor efficacy.

[0127] The various embodiments in this specification are described in a progressive manner. The same or similar parts between the various embodiments can be referred to each other. Each embodiment focuses on describing the differences from other embodiments.

[0128] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit this application. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of this application.

Claims

1. A protein scaffold, characterized in that, The protein scaffold is a hexamer composed of six identical fusion proteins, which consist of the amino acid sequence shown in SEQ ID NO.2; The fusion protein consists of a modified IgG Fc and two Fc receptors; The modified IgGFc consists of an IgGFc mutant and an IgM tail fin, wherein the IgM tail fin is fused and expressed at the C-terminus of the IgGFc mutant. The IgGFc mutant consists of the amino acid sequence shown in SEQ ID NO.

1.

2. The protein scaffold according to claim 1, characterized in that, The type of the Fc receptor is FcγRI.

3. The protein scaffold according to claim 1, characterized in that, Its average particle size is 50-200 nm.

4. A method for preparing a protein scaffold according to claim 1, characterized in that, Includes the following steps: S10: Construct the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2 into a lentiviral vector plasmid to obtain a lentivirus carrying the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2; S20: After transducing the lentivirus into cells expressing the protein, resistance-pressure screening and limiting dilution culture are performed to obtain a monoclonal cell line expressing the protein scaffold. S30: After collecting the culture supernatant of the monoclonal cell line, perform Protein G affinity chromatography separation and purification to obtain the protein scaffold.

5. The use of a protein scaffold according to any one of claims 1-3 in binding to OX40 antibody and / or PD-L1 antibody.

6. The application according to claim 5, characterized in that, The protein scaffold is used to bind OX40 antibodies to form polyantibodies.

7. The application according to claim 6, characterized in that, The application of the polymeric antibody in the preparation of drugs for treating colorectal cancer.

8. The application according to claim 5, characterized in that, The protein scaffold is used to bind PD-L1 antibodies to form multivalent antibodies.

9. The application according to claim 8, characterized in that, The application of the multivalent antibody in the preparation of drugs for treating melanoma.

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