IgG (immunoglobulin G) binding type immune activator protein as well as preparation method and application thereof

Abstract

The invention provides an IgG (immunoglobulin G) binding type immune activator protein as well as a preparation method and application thereof, and belongs to the technical field of medicines. It is found for the first time that after three Fab binding peptides CFab and N215 are fused, the N215 can be endowed with the IgG antibody binding capacity, the obtained fusion protein N215-CFab3 and the IgG antibody can form a stable compound, and the compound retains the functions of the N215 and the antibody, can mediate binding of the N215 to target cells, remarkably prolongs the half-life period of the N215, promotes proliferation of T and NK cells, and has the advantages of being capable of achieving the purpose of improving the immunogenicity of the N215 and improving the immunogenicity of the N215. The remarkably enhanced in-vivo anti-tumor effect is exerted. A compound formed by the fusion protein N215-CFab3 and an IgG antibody can be used as an immune activator, and has a wide application prospect in immunotherapy of diseases such as tumors and infection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to an IgG binding type immune activation protein as well as a preparation method and application thereof. BACKGROUND

[0002] T cells and natural killer (NK) cells are the main immune cells of the body to fight against serious diseases such as tumors and infections. The receptor molecules IL-2Rβγ expressed by the two cells transmit proliferation signals after binding with agonists, so that the T cells and NK cells proliferate in large quantities, thereby generating a strong immune response to fight against diseases such as tumors and infections.

[0003] Neo-2 / 15 (abbreviated as N215) is a de novo synthesized protein, which can activate IL-2Rβγ to promote the proliferation of T cells and NK cells and enhance the anti-tumor immune response, and is a very potential candidate drug for tumor immunotherapy (Silva et al., Nature, 2019; 565(7738): 186-191). However, the clinical application of N215 is limited by various factors. On the one hand, N215 has a small molecular weight, a short half-life in vivo, and a short duration of efficacy; on the other hand, IL-2Rβγ is widely expressed in various cells, and N215 is widely distributed in the body after entering the body, and the amount of N215 combined to T cells and NK cells is not enough, so that T cells and NK cells cannot be activated efficiently. Therefore, direct use of N215 often requires multiple large-dose administrations to ensure efficacy. However, large-dose administration is prone to induce adverse immune side reactions. In order to promote the clinical application of N215, it is urgent to prolong the half-life of N215 while enhancing the targeting of T cells and NK cells, so as to reduce the drug dosage and improve the safety of N215. SUMMARY

[0004] A first object of the present application is to provide an IgG binding type immune activation protein as well as a preparation method and application thereof, and a second object of the present application is to provide a complex of the aforementioned immune activation protein and IgG type antibodies as well as a preparation method and application thereof.

[0005] The present application provides a recombinant protein, the amino acid sequence of which is shown as SEQ ID NO. 13.

[0006] The present application also provides a gene encoding the aforementioned recombinant protein, the nucleotide sequence of which is shown as SEQ ID NO. 14.

[0007] The present application also provides a recombinant expression plasmid, which is a recombinant expression plasmid comprising the aforementioned gene.

[0008] The present application also provides a recombinant bacterium, which contains the aforementioned recombinant expression plasmid.

[0009] The present application also provides a method for preparing the aforementioned recombinant protein, comprising the following steps:

[0010] (1) constructing a recombinant expression plasmid comprising the aforementioned gene;

[0011] (2) transforming the recombinant expression plasmid into a host bacterium, culturing, and inducing expression of the recombinant protein.

[0012] Further, the host bacterium is Escherichia coli, and the inducing agent used for inducing expression is isopropyl-β-D-thiogalactopyranoside.

[0013] Further, the method further comprises the following purification steps: harvesting the bacterial solution, lysing, centrifuging, and taking the supernatant for separation and purification by Ni-NTA agarose gel.

[0014] The present application also provides a complex, which is a complex of the aforementioned recombinant protein and an IgG antibody.

[0015] Further, the IgG antibody is an anti-PD-1 antibody or an anti-NKG2A antibody; and / or, the molar ratio of the recombinant protein to the IgG antibody is 1:(0.5-2), preferably 1:1.

[0016] The present application also provides a method for preparing the aforementioned complex, comprising the following steps: mixing the recombinant protein and the IgG antibody in an aqueous solution to obtain the complex.

[0017] Further, the aqueous solution is water or a buffer.

[0018] The present application also provides the use of the aforementioned complex in the preparation of an immunotherapeutic drug.

[0019] Further, the immunotherapeutic drug is a drug for treating tumors or anti-infection.

[0020] Further, the tumor is melanoma.

[0021] The present application finds that the fusion of Fc binding peptide Z Fc to N215 can endow N215 with the ability to bind antibodies, but the binding of IgG antibodies will cause precipitation, which cannot be utilized. The present application also finds that the fusion of a single Fab binding peptide C Fab to N215 cannot enhance the binding ability of N215 to IgG antibodies, and cannot form stable complexes. However, the fusion of three Fab binding peptides C Fab to N215 can endow N215 with the ability to bind IgG antibodies, and the obtained fusion protein N215-C Fab3can form stable complexes with IgG class antibodies (including anti-PD-1 antibodies and anti-NKG2A antibodies), which retain the functions of N215 and the antibodies, can mediate the binding of N215 to target cells, significantly prolong the half-life of N215, promote T and NK cell proliferation, and exert significantly enhanced anti-tumor effects in vivo.

[0022] In addition to anti-PD-1 antibodies and anti-NKG2A antibodies, N215-C Fab 3may also form complexes with other IgG class antibodies.

[0023] The fusion protein N215-C Fab 3formed with IgG class antibodies can be used as an immune activator and has broad application prospects in the immunotherapy of diseases such as tumors and infections.

[0024] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and common practice in the art, other various forms of modifications, substitutions or changes can be made without departing from the above basic technical ideas of the present application.

[0025] The above content of the present application will be further described in detail through the specific embodiments in the form of examples. However, this should not be understood as limiting the scope of the above subject matter of the present application to the following examples. Any technology realized based on the above content of the present application belongs to the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Preparation and characterization of fusion protein N215-Z Fc A: molecular design schematic diagram; B: polyacrylamide gel electrophoresis diagram of the protein after purification.

[0027] Figure 2 Preparation and characterization of N215-C Fab and N215-C Fab 3. A: molecular design schematic diagram; B: polyacrylamide gel electrophoresis diagram of the protein after purification.

[0028] Figure 3 Preparation and characterization of fusion protein N215-Z Fc Turbidity analysis after mixing with IgG class antibodies.

[0029] Figure 4 Gel filtration analysis of N215-C Fab and N215-C Fab 3binding to IgG class antibodies.

[0030] Figure 5 Preparation and characterization of N215-C Fab3 Cell binding ability to form complex with αPD-1 or αNKG2A.

[0031] Figure 6 N215-C Fab 3 Pharmacokinetic analysis of complex formed with αNKG2A.

[0032] Figure 7 N215-C Fab 3 Effect of complex formed with IgG class antibody on cell proliferation activity of T cells (A) and NK cells (B).

[0033] Figure 8 N215-C Fab 3 Anti-tumor effect in vivo of complex formed with IgG class antibody. A: N215-C Fab 3 Complex formed with αPD-1; B: N215-C Fab 3 Complex formed with αNKG2A. DETAILED DESCRIPTION

[0034] The raw materials and equipment used in the present application are known products, which are obtained by purchasing commercially available products.

[0035] In the present application, room temperature refers to 25±5℃.

[0036] Example 1: Preparation of fusion protein N215-C Fab 3

[0037] The three Fab binding peptides (referred to as C Fab , the amino acid sequence is shown as SEQ ID NO. 9) are designed to be connected in series, and then fused to the C-terminal of N215 through a (G4S)3 linker (the amino acid sequence is shown as SEQ ID NO. 5) to prepare the fusion protein N215-C Fab 3 (the amino acid sequence is shown as SEQ ID NO. 13). The specific preparation method of the fusion protein N215-C Fab 3 is as follows:

[0038] (1) Synthesis of N215-C Fab 3 encoding gene (the nucleotide sequence is shown as SEQ ID NO. 14), the N215-C Fab 3 encoding gene is cloned into the pQE30 vector through BamHI and SalI to obtain a recombinant expression plasmid.

[0039] (2) The recombinant expression plasmid was transformed into E. coli M15, and the bacteria were cultured in LB medium to the logarithmic growth phase, and then induced with 1 mM isopropyl-β-D-thiogalactoside overnight. The bacteria were harvested, lysed, centrifuged, and the supernatant was separated and purified by commercial Ni-NTA agarose gel according to the provided method to obtain the fusion protein N215-C. Fab 3 (the amino acid sequence is shown in SEQ ID NO. 13).

[0040] The purified fusion protein N215-C Fab 3 was dialyzed against phosphate buffered saline (PBS; 137 mM NaCl, 2.68 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) overnight for standby use.

[0041] Example 2: Preparation of complex αPD-1-N215

[0042] The fusion protein N215-C Fab 3 was mixed with the anti-PD-1 antibody αPD-1 at a molar ratio of 1:1 in PBS (137 mM NaCl, 2.68 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) and incubated at room temperature for 30 minutes to obtain the complex αPD-1-N215.

[0043] The above-mentioned anti-PD-1 antibody is an anti-human PD-1 antibody or an anti-mouse PD-1 antibody.

[0044] Example 3: Preparation of complex αNKG2A-N215

[0045] The fusion protein N215-C Fab 3 was mixed with the anti-NKG2A antibody αNKG2A at a molar ratio of 1:1 in PBS (137 mM NaCl, 2.68 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) and incubated at room temperature for 30 minutes to obtain the complex αNKG2A-N215.

[0046] The above-mentioned anti-NKG2A antibody is an anti-human NKG2A antibody or an anti-mouse NKG2A antibody.

[0047] The following is the preparation method of the control sample.

[0048] Example 1: Preparation of N215 protein

[0049] The N215-encoding gene (nucleotide sequence as shown in SEQ ID NO. 2) was synthesized. The N215-encoding gene was cloned into the pQE30 vector through BamHI and Sail according to the method of Example 1 to obtain a recombinant expression plasmid. The recombinant was expressed, the bacterial solution was harvested, lysed, centrifuged, and the supernatant was separated and purified through a commercial Ni column to obtain the protein N215 (amino acid sequence as shown in SEQ ID NO. 1).

[0050] Example 2: Preparation of fusion protein N215-Z Fc

[0051] It was designed to fuse the Z peptide (abbreviated as Z Fc , amino acid sequence as shown in SEQ ID NO. 3) capable of binding IgG antibody through the Fc segment at the C-terminal end of N215 through a (G4S)3 linker (amino acid sequence as shown in SEQ ID NO. 5) to prepare the fusion protein N215-Z Fc (amino acid sequence as shown in SEQ ID NO. 7). The fusion protein N215-Z Fc was prepared according to the following specific method:

[0052] The N215-Z Fc -encoding gene (nucleotide sequence as shown in SEQ ID NO. 8) was synthesized. The N215-Z Fc -encoding gene was cloned into the pQE30 vector through BamHI and Sail according to the method of Example 1 to obtain a recombinant expression plasmid. The recombinant was expressed, the bacterial solution was harvested, lysed, centrifuged, and the supernatant was separated and purified through a commercial Ni column to obtain the fusion protein N215-Z Fc (amino acid sequence as shown in SEQ ID NO. 7).

[0053] Example 3: Preparation of fusion protein N215-C Fab

[0054] It was designed to fuse the Fab binding peptide (abbreviated as C Fab , amino acid sequence as shown in SEQ ID NO. 9) at the C-terminal end of N215 through a (G4S)3 linker (amino acid sequence as shown in SEQ ID NO. 5) to prepare the fusion protein N215-C Fab (amino acid sequence as shown in SEQ ID NO. 11). The fusion protein N215-C Fab was prepared according to the following specific method:

[0055] The N215-C Fab -encoding gene (nucleotide sequence as shown in SEQ ID NO. 12) was synthesized. The N215-C FabThe encoding gene was cloned into the pQE30 vector using BamHI and SalI to obtain a recombinant expression plasmid. After recombinant expression, the bacterial culture was harvested, lysed, centrifuged, and the supernatant was purified using a commercially available Ni column to obtain the fusion protein N215-C. Fab (Amino acid sequence as shown in SEQ ID NO.11).

[0056] The following experimental examples demonstrate the beneficial effects of the present invention.

[0057] Experimental Example 1: Gel Electrophoresis Test

[0058] 1. Experimental Samples

[0059] Comparative Examples 1, 2, and 3: Recombinant proteins prepared in Example 1.

[0060] 2. Experimental Methods

[0061] Polyacrylamide gel electrophoresis.

[0062] 3. Experimental Results

[0063] like Figure 1 , Figure 2 As shown, the recombinant expression of N215 and N215-Z Fc N215-C Fab and N215-C Fab 3. After purification by Ni column, N215 and N215-Z were observed as a single band on polyacrylamide gel electrophoresis, with the molecular weight consistent with expectations, indicating that the above method yielded N215 and N215-Z. Fc N215-C Fab and N215-C Fab 3. Pure protein.

[0064] Experimental Example 2: Protein-induced Antibody Aggregation and Precipitation Test

[0065] 1. Experimental Samples

[0066] Comparative Examples 1, 2, and 3: Recombinant proteins prepared in Example 1.

[0067] 2. Experimental Methods

[0068] When proteins containing IgBD bind to antibodies, the antibodies may aggregate and precipitate, leading to loss of function. To determine whether the binding of recombinant proteins to IgG antibodies causes antibody aggregation and precipitation, this experiment tested the recombinant proteins with IgG antibodies.

[0069] (1) First, dilute the anti-human PD-1 antibody (αhPD-1) with PBS to 1 mg / ml, and then add different concentrations (0.25-1 mg / ml) of recombinant protein N215 and N215-Z to it.Fc After mixing, the solution was left at room temperature for 30 minutes, and the absorbance value was read at 340 nm. The precipitated rate of the antibody caused by the protein was calculated as 100% using the precipitated αhPD-1 antibody with 10% trichloroacetic acid.

[0070] (2) N215, N215-C Fab , N215-C Fab 3 The three recombinant proteins were mixed with different IgG class antibodies (anti-PD-1 antibody, anti-NKG2A antibody), and then the turbidity was measured at 340 nm.

[0071] 3. Experimental results

[0072] As shown in Figure 3 , the addition of N215 to the αhPD-1 solution did not increase the turbidity of the solution, indicating that the added N215 did not cause the precipitation of αhPD-1. However, the addition of N215-Z Fc protein to the αhPD-1 antibody solution increased the turbidity of the solution with the increase of the amount of N215-Z Fc protein, indicating that N215-Z Fc caused the precipitation of the αhPD-1 antibody.

[0073] N215, N215-C Fab , N215-C Fab 3 The three recombinant proteins did not cause precipitation reaction after being mixed with αhPD-1.

[0074] The above experimental results show that N215-Z Fc produced after the fusion of Fc binding peptide Z Fc can cause the aggregation and precipitation of IgG class antibodies and cannot be utilized, while N215-C Fab , N215-C Fab 3 do not cause the aggregation and precipitation of IgG class antibodies.

[0075] Experimental Example 3: Verification of complex formation

[0076] 1. Experimental samples

[0077] Recombinant proteins prepared in Control Example 1, Control Example 3, and Example 1.

[0078] 2. Experimental method

[0079] It has been proven in the foregoing that N215, N215-C Fab , N215-C Fab 3 do not cause the aggregation and precipitation of IgG class antibodies, and in order to further determine N215, N215-C Fab , N215-C Fab3 whether it can bind to IgG class antibodies to form a complex, in this experiment, N215, N215-C Fab , N215-C Fab 3 were mixed with IgG class antibodies at a 1:1 molar ratio in PBS and incubated for 30 minutes. Then the protein components in the mixture were detected by high performance liquid chromatography using a gel filtration column (Superdex™ 200 10 / 300 GL) with PBS as the mobile phase, and compared with the components before mixing.

[0080] 3. Experimental results

[0081] The results are shown in Figure 4 N215 mixed with anti-mouse PD-1 antibody αmPD-1 showed two protein components on the gel filtration column corresponding to the molecular weights of αmPD-1 and N215 alone. Similarly, N215-C Fab mixed with αmPD-1 also showed two protein peaks corresponding to the molecular weights of αmPD-1 and N215-C Fab alone. These results show that N215 itself does not bind to IgG class antibodies; the fusion of a C Fab at its C-terminus also does not allow N215 to bind to IgG antibodies. However, N215-C Fab 3 mixed with αmPD-1 showed a new protein component on the gel filtration column, which was larger than both N215-C Fab 3 and αmPD-1, indicating that N215-C Fab 3 binds to αmPD-1 to form a complex. Analysis under the same conditions found that N215-C Fab 3 can also bind to anti-mouse NKG2A antibody (αmNKG2A), anti-human PD-1 antibody (αhPD-1) and anti-human NKG2A antibody (αhNKG2A) to form a complex.

[0082] The above experimental results show that the fusion protein N215-C Fab 3 obtained by coupling three C Fab in series with N215 can bind to IgG class antibodies to form a complex without causing antibody precipitation.

[0083] Whether the complex formed by N215-C Fab 3 and IgG class antibodies can play the expected role depends mainly on whether the functions of N215 and antibodies in the complex are retained. The following experiments were performed to test this.

[0084] Example 4: Antigen recognition properties of the complex formed by N215-C Fab 3 and IgG class antibodies

[0085] 1. Experimental method

[0086] Antigen recognition is one of the important functions of antibodies, which determines the cell targeting mediated by antibodies. To determine whether the complex has the antigen recognition specificity of the original antibody, the present experiment first labeled N215-C Fab 3 with FAM, then mixed it with anti-mouse PD-1 antibody amPD-1 or anti-mouse NKG2A antibody amNKG2A at a molar ratio of 1:1 in PBS to form a complex at room temperature for 30 minutes, and then incubated with T or NK cells with high expression of amPD-1 and amNKG2A at room temperature for 30 minutes. Finally, the complex bound to the cells was detected by conventional flow cytometry. At the same time, FAM-labeled N215 was used as a control for comparison. Since T and NK cells also express IL-2Rβγ, to avoid the involvement of N215 in the complex in cell binding, T and NK cells were first blocked with an excess of unlabeled N215.

[0087] 2. Experimental results

[0088] The results, as shown in Figure 5 , the complex amPD-1-N215 formed by N215-C Fab 3 and anti-mouse PD-1 antibody amPD-1 and the complex amNKG2A-N215 formed by it and anti-mouse NKG2A antibody amNKG2A can both bind to T and NK cells, and the binding rate is higher than that of N215, indicating that the antibody in the complex retains the original antigen recognition characteristics and can mediate the binding of N215 to target cells.

[0089] Experimental Example 5: Half-life of the complex formed by N215-C Fab 3 and IgG class antibodies

[0090] 1. Experimental method

[0091] To determine the half-life of the complex, the present experiment first labeled N215-C Fab 3 with biotin (Biotin), and then mixed it with amPD-1 or amNKG2A antibody at a molar ratio of 1:1 in PBS to form a complex at room temperature for 30 minutes. The complex was injected into mice through the tail vein, and blood was taken at regular intervals. The content of N215-C Fab 3 in the plasma was determined by streptavidin (Streptavidin)-mediated enzyme-linked immunosorbent assay, and the pharmacokinetic curve was drawn to calculate its half-life. Biotin-labeled N215 was used as a control for comparison.

[0092] 2. Experimental results

[0093] The results, as shown in Figure 6N215-C Fab 3 and its complex with amPD-1, amPD-1-N215, and its complex with amNKG2A, amNKG2A-N215, N215-C Fab 3 is significantly slower than N215, indicating that by fusing C Fab 3, N215 can form a complex with IgG antibodies, which can significantly prolong the half-life of N215.

[0094] Experimental Example 6: Functional analysis of N215 in the complex of N215-C Fab 3 with IgG class antibodies

[0095] 1. Experimental method

[0096] The main function of N215 is to stimulate T cell and NK cell proliferation. N215-C Fab3 Whether its T cell and NK cell proliferation ability is retained after forming a complex with aPD-1 or aNKG2A antibody will determine whether the complex has immune activation activity. To determine whether the complex still has the ability to promote T cell and NK cell proliferation, in this experiment, N215-C Fab 3 was mixed with aPD-1 (amPD-1, ahPD-1) or aNKG2A (amNKG2A, ahNKG2A) antibody at a molar ratio of 1:1 in PBS at room temperature for 30 minutes to form a complex, and then diluted to different concentrations and added to T cells (CTLL2) or NK cells (NK92). In 96-well plates, continue to culture for 72 hours, and then use CCK-8 reagent to determine the number of surviving cells. Compared with the control group without adding protein, it is determined whether the complex of N215-C Fab 3 and aPD-1 or aNKG2A antibody still retains the cell proliferation ability of N215.

[0097] 2. Experimental results

[0098] The results are shown in Figure 7 N215-C Fab 3 and aPD-1 (amPD-1-N215, ahPD-1-N215) or aNKG2A (amNKG2A-N215, ahNKG2A-N215) antibody complex showed concentration-dependent cell proliferation activity. It is indicated that the function of N215 in promoting T and NK cell proliferation is retained in the complex.

[0099] The above experimental results show that N215-C Fab3 The complex formed with IgG class antibody retains the function of N215 and antibody, can mediate the binding of N215 to target cells, significantly prolongs the half-life of N215, and can promote the proliferation of T and NK cells.

[0100] Experimental Example 7: N215-C Fab 3 In vivo anti-tumor effect of the complex formed with IgG class antibody

[0101] 1. Experimental sample

[0102] The complex prepared in Example 2 and Example 3.

[0103] 2. Experimental method

[0104] To test the in vivo anti-tumor activity of N215-C Fab 3 In vivo anti-tumor activity of the complex amPD-1-N215 formed by N215-C 5 3 and the antibody against mouse PD-1 amPD-1, melanoma cells B16 / F1 were inoculated subcutaneously on the flank of mice at a dose of 5x10 3 6.5mg / kg, respectively. Mice were randomly divided into groups of 8, and when the average tumor volume reached 150mm Fab 3, the complex amPD-1-N215 formed by N215-C 2 3 and amPD-1 or the mixture amPD-1+N215 of N215 and amPD-1. Untreated mice were given the same volume of PBS. Drug administration was performed every other day, for a total of three times. During this period, the length (L) and width (W) of the tumor were measured daily, and the tumor volume was calculated according to the formula V=LxW Fab / 2, and the tumor growth curve was plotted.

[0105] To test the in vivo anti-tumor activity of N215-C Fab 3 and the antibody against mouse NKG2A amNKG2A, melanoma cells B16 / F1 were inoculated subcutaneously on the flank of mice at a dose of 5x10 Fab 6.5mg / kg, respectively. Mice were randomly divided into groups of 8, and when the average tumor volume reached 150mm Fab 3, the complex amPD-1-N215 formed by N215-C Fab 3 and amPD-1 or the mixture amPD-1+N215 of N215 and amPD-1. Untreated mice were given the same volume of PBS. Drug administration was performed every other day, for a total of three times. During this period, the length (L) and width (W) of the tumor were measured daily, and the tumor volume was calculated according to the formula V=LxW Fab / 2, and the tumor growth curve was plotted.

[0106] 3. Experimental results

[0107] The results showed that, compared with the control group treated with PBS, the mixture of N215 and αmPD-1 (αmPD-1+N215) did not show a significant growth inhibitory effect on melanoma; however, the tumor growth rate was significantly slower in the group treated with the same dose of the αmPD-1-N215 complex. At the end of the observation period, the mean tumor volume in the PBS, αmPD-1+N215, and αmPD-1-N215 groups was 1840 mmHg. 3 1699mm 3 and 551mm 3 ( Figure 8 A). Similarly, compared with the control group treated with PBS, the mixture of N215 and αmNKG2A (αmNKG2A+N215) did not show a significant growth inhibitory effect on melanoma; however, the same dose of the complex αmNKG2A-N215 significantly slowed tumor growth. By the end of the observation period, the average tumor volume in the PBS, αmNKG2A+N215, and αmNKG2A-N215 groups was 1840 mm². 3 1718mm 3 and 553mm 3 ( Figure 8 B).

[0108] The above experimental results show that N215-C Fab The antitumor effects of complexes formed with αmPD-1 or αmNKG2A (αmPD-1-N215, αmNKG2A-N215) were significantly better than those of mixtures of N215 with αmPD-1 or αmNKG2A (αmPD-1+N215α, αmNKG2A+N215). This is mainly due to N215-C Fab 3 can bind to IgG antibodies, while N215 cannot. N215-C Fab The complex formed by 3 and IgG antibodies exerted a significantly enhanced in vivo antitumor effect.

[0109] The sequence information involved in this invention is as follows:

[0110] 1) N215 amino acid sequence (SEQ ID NO.1):

[0111] NH2-PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS-COOH.

[0112] N215 nucleotide sequence (SEQ ID NO.2):

[0113] 5-cctaaaaagaaaatccagctgcacgcagaacacgcactgtacgacgcactgatgatcctgaatattgtcaaaaccaacagcccgccggcagaagaaaaactggaagactacgcattcaactttgaactgattctggaagaaattgcgcgtctgtttgaaagcggtgatcagaaagacgaagcagaaaaagcaaaacgcatgaaagaatggatgaaacgtattaaaaccaccgcaagcgaagatgaacaggaagaaatggcaaatgcaattattaccatcctgcagagctggatttttagt-3.

[0114] 2) Z Fc Amino acid sequence (SEQ ID NO. 3)

[0115] NH2-VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK-COOH.

[0116] Z Fc Nucleotide sequence (SEQ ID NO. 4):

[0117] 5-gtggataacaaattcaacaaagaacaacaaaatgctttctatgaaatcttacatttacctaacttaaacgaagaacaacgcaatgctttcatccaaagcctaaaagatgacccaagccaaagcgctaaccttttagcagaagctaaaaagctaaatgatgctcaagcaccaaaa-3.

[0118] 3) (G4S)3 Amino acid sequence (SEQ ID NO. 5):

[0119] NH2-GGGGSGGGGSGGGGS-COOH.

[0120] (G4S)3 Nucleotide sequence (SEQ ID NO. 6):

[0121] 5-ggcggaggcggttcaggcggaggtggctctggcggtggcggctcc-3.

[0122] 4) N215-ZFc Amino acid sequence (SEQ ID NO. 7):

[0123] NH2-PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFSGGGGSGGGGSGGGGSVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK-COOH.

[0124] N215-Z Fc Nucleotide sequence (SEQ ID NO. 8):

[0125] 5- cctaaaaagaaaatccagctgcacgcagaacacgcactgtacgacgcactgatgatcctgaatattgtcaaaaccaacagcccgccggcagaagaaaaactggaagactacgcattcaactttgaactgattctggaagaaattgcgcgtctgtttgaaagcggtgatcagaaagacgaagcagaaaaagcaaaacgcatgaaagaatggatgaaacgtattaaaaccaccgcaagcgaagatgaacaggaagaaatggcaaatgcaattattaccatcctgcagagctggatttttagtggcggaggcggttcaggcggaggtggctctggcggtggcggctccgtggataacaaattcaacaaagaacaacaaaatgctttctatgaaatcttacatttacctaacttaaacgaagaacaacgcaatgctttcatccaaagcctaaaagatgacccaagccaaagcgctaaccttttagcagaagctaaaaagctaaatgatgctcaagcaccaaaa-3.

[0126] 5) C Fab Amino acid sequence (SEQ ID NO. 9):

[0127] NH2-TTYKLVINGKTLKGETTTKAVDAETAAAAFAQYARRNGVDGVWTYDDATKTFTVTE-COOH.

[0128] C Fab Amino acid sequence (SEQ ID NO. 11):

[0129] 5 -accacctacaagctggtgatcaacggcaagaccctgaaaggcgagaccaccaccaaagcggttgacgcggagaccgcggcggcggcgttcgcgcagtacgcgcgtcgcaacggtgtggatggcgtttggacctatgacgatgcgaccaagacctttaccgtgaccgaa-3.

[0130] 6) N215-C Fab Amino acid sequence (SEQ ID NO. 11):

[0131] NH2-TTYKLVINGKTLKGETTTKAVDAETAAAAFAQYARRNGVDGVWTYDDATKTFTVTE-COOH.

[0132] N215-C Fab Amino acid sequence (SEQ ID NO. 11):

[0133] 5- cctaaaaagaaaatccagctgcacgcagaacacgcactgtacgacgcactgatgatcctgaatattgtcaaaaccaacagcccgccggcagaagaaaaactggaagactacgcattcaactttgaactgattctggaagaaattgcgcgtctgtttgaaagcggtgatcagaaagacgaagcagaaaaagcaaaacgcatgaaagaatggatgaaacgtattaaaaccaccgcaagcgaagatgaacaggaagaaatggcaaatgcaattattaccatcctgcagagctggatttttagtggcggtggcggatcgggcggaggcggttcaggcggaggtggctctaccacctacaagctggtgatcaacggcaagaccctgaaaggcgagaccaccaccaaagcggttgacgcggagaccgcggcggcggcgttcgcgcagtacgcgcgtcgcaacggtgtggatggcgtttggacctatgacgatgcgaccaagacctttaccgtgaccgaa-3.

[0134] 7) N215-C Fab 3 Amino acid sequence (SEQ ID NO. 13):

[0135] NH2-PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFSGGGGSGGGGSGGGGSTTYKLVINGKTLKGETTTKAVDAETAAAAFAQYARRNGVDGVWTYDDATKTFTVTEGGGGSGGGGSGGGGSTTYKLVINGKTLKGETTTKAVDAETAAAAFAQYARRNGVDGVWTYDDATKTFTVTEGGGGSGGGGSGGGGSTTYKLVINGKTLKGETTTKAVDAETAAAAFAQYARRNGVDGVWTYDDATKTFTVTE-COOH.

[0136] N215-C Fab 3 Nucleotide sequence (SEQ ID NO. 14):

[0137] 5- CTTAAAAAGAAAATCCAGCTGCACGCAGAACACGCAC TGTACGACGC ACTGATGATCCTGAA TATTGTCAA AACCAACAGCCC GCCGGCAGA AGAAAACTGGAAGACTACGCATTCAACTTTGA ACTGATTCTGGAAGAAATTGC GC GTCTGTTTGAAAGCGGTGATCAGAAAGACGAAGC AGAAAAAGCAA AACGCATGAAAGAATGGATGAAACGTATTAAAACCACCGCAAGCGAAGATGAACAGGAAGAAATGGCAAATGCAATTAT TACC ATCCTGCAGAGCTGGATTTTTAGTGGCGGTGGCGGATCGGGCGGAGGCGGTT CAGGC GGAGGTGGCTCTACCACCTACAAGCTG GTGATCAACGGCAAGAC CCTGAAAGGC GAGACCACCACCAAAGCGGTTGACGC GGA GACC GC GGC GGC GGC GTTCGC GC AGTACGC GC GT CGCAACGGTGTGGATGGCGTTTGGACCTATGACGATGC GACCAAGACCTTTACC GTGACC GAAACCACCTACAAGCTGGTGATCAACGGCAAGACCTTGAAAGGC GAGACCACCACCAAAGCGGTTGACGC GGA GACC GC GGC GGC GGC GTTCGC GC AGTACGC GC GT CGCAACGGTGTGGATGGCGTTTGGACCTATGACGATGC GACCAAGACCTTTACC GTGACC GAAACCACCTACAAGCTGGTGATCAACGGCAAGACCTTGAAAGGC GAGACCACCACCAAAGCGGTTGACGC GGA GACC GC GGC GGC GGC GTTCGC GC AGTACGC GC GT CGCAACGGTGTGGATGGCGTTTGGACCTATGACGATGC GACCAAGACCTTTACC GTGACC GAAACCACCTACAAGCTGGTGATCAACGGCAAGACCTTGAAAGGC GAGACCACCACCAAAGCGGTTGACGC GGA GACC GC GGC GGC GGC GTTCGC GC AGTACGC GC GT CGCAACGGTGTGGATGGCGTTTGGACCTATGACGATGC GACCAAGACCTTTACC GTGACC GAAACCACCTACAAGCTGGTGATCAACGGCAAGACCTTGAAAGGC GAGACCACCACCAAAGCGGTTGACGC GGA GACC GC GGC GGC GGC GTTCGC GC AGTACGC GC GT CGCAACGGTGTGGATGGCGTTTGGACCTATGACGATGC GACCAAGACCTTTACC GTGACC GAAACCACCTACAAGCTGGTGATCAACGGCAAGACCTTGAAAGGC GAGACCACCACCAAAGCGGTTGACGC GGA GACC GC GGC GGC GGC GTTCGC GC AGTACGC GC GT CGCAACGGTGTGGATGGCGTTTGGACCTATGACGATGC GACCAAGACCTTTACC GTGACC GAAACCACCTACAAGCTGGTGATCAACGGCAAGACCTTGAAAGGC GAGACCACCACCAAAGCGGTTGACGC GGA GACC GC GGC GGC GGC GTTCGC GC AGTACGC GC GT CGCAACGGTGTGGATGGCGTTTGGACCTATGACGATGC GACCAAGACCTTTACC GTGACC GAAACCACCTACAAGCTGGTGATCAACGGCAAGACCTTGAAAGGC GAGACCACCACCAAAGCGGTTGACGC GGA GACC GC GGC GGC GGC GTTCGC GC AGTACGC GC GT CGCA

Claims

1. A recombinant protein, characterized in that: The amino acid sequence of the protein is shown as SEQ ID NO.

13.

2. A gene encoding the recombinant protein of claim 1, characterized in that: The nucleotide sequence of the gene is shown as SEQ ID NO.

14.

3. A recombinant expression plasmid, characterized by: It is a recombinant expression plasmid comprising the gene of claim 2.

4. A recombinant bacterium, characterized by: It contains the recombinant expression plasmid of claim 3.

5. A method for preparing the recombinant protein of claim 1, characterized in that: The method comprises the following steps: (1) constructing a recombinant expression plasmid comprising the gene of claim 2; (2) transforming the recombinant expression plasmid into a host cell, culturing, and inducing expression of the recombinant protein.

6. A composite, characterized in that, It is a complex of the recombinant protein of claim 1 and an IgG class antibody.

7. The composite of claim 6, wherein, The IgG class antibody is an anti-PD-1 antibody or an anti-NKG2A antibody; and / or, the molar ratio of the recombinant protein to the IgG class antibody is 1: (0.5-2), preferably 1:

1.

8. A method of preparing the complex of any one of claims 6-7, characterized in that, The method comprises the following steps: mixing the recombinant protein with the IgG class antibody in an aqueous solution to obtain a complex.

9. Use of the complex of any one of claims 6-7 in the preparation of an immunotherapeutic drug.

10. Use according to claim 9, characterized in that, The immunotherapeutic drug is a drug for treating tumors or anti-infection.

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