Pharmaceutical composition and use thereof

Abstract

Disclosed are a pharmaceutical composition and a use thereof. The pharmaceutical composition comprises a recombinant protein and an HIV invasion inhibitor, wherein the recombinant protein comprises: 1) an scFV of an anti-CD3 antibody; and 2) diphtheria toxin.

Description

A pharmaceutical composition and its application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on the Chinese patent application with application number PCT / CN2023 / 117847 and application date September 8, 2023, and claims the priority of the Chinese patent application. The entire content of the Chinese patent application is hereby incorporated into this application by introduction. Technical Field

[0003] The present invention belongs to the field of molecular biology, and in particular, relates to a pharmaceutical composition and its application, and more particularly, relates to an anti-HIV pharmaceutical composition and its application. Background Art

[0004] Acquired Immunodeficiency Syndrome (AIDS), abbreviated as AIDS, is caused by infection with the human immunodeficiency virus (HIV).

[0005] To date, HIV vaccine research has failed. Continuous cocktail therapy (HAART) can maintain a low viral load in HIV-infected individuals, but it cannot eliminate the virus. Patients must take medication long-term, which can cause severe and complex side effects in HIV-infected individuals and AIDS patients. Long-term use of these drugs can also lead to the development of drug-resistant HIV strains. Although broadly neutralizing antibodies (bNAbs) can inhibit infection by multiple HIV strains and have been investigated as drug candidates in preclinical and clinical trials in recent years, their cost is prohibitive, and even if successful, they would be difficult to deploy on a large scale. Currently, most anti-HIV drugs, such as reverse transcriptase inhibitors, protease inhibitors, and integrase inhibitors, are chemical. Biologics offer the advantages of low toxicity and sustained or controlled expression in patients. However, clinically available biologics are extremely scarce and still face challenges such as poor broad-spectrum efficacy, suboptimal inhibitory efficacy, significant side effects, and the presence of resistant strains.

[0006] The virus then invades the target cell through the interaction between HIV envelope protein gp120 or host molecules integrated on the envelope and CD4 on the target cell surface or adsorption receptors such as DC-SIGN, DCIR, Sydecan-3, siglec-1, etc. After the virus invades the target cell, it usually begins to spread throughout the body within 1 to 2 weeks and kills CD4 cells extensively within 8 to 12 weeks. +T cells, establishing systemic latent infection. Once this stage is complete, the virus persists for a long time or even lifelong, making it difficult to eliminate. While developed HIV invasion and fusion inhibitors (EIs / FIs) primarily control HIV infection and transmission by blocking molecular interactions that play a key role in HIV invasion of cells, they effectively inhibit free HIV from invading cells, protecting against HIV at its source. However, they are ineffective against cells already infected with HIV. These infected cells can still produce new HIV virions, leading to subsequent re-infection and proliferation.

[0007] Therefore, there is a need in the art for a product that can clear the HIV viral reservoir, thereby avoiding subsequent repeated infection and amplification.

[0008] Summary of the Invention

[0009] In view of this, in a first aspect, the present invention provides a pharmaceutical composition comprising a recombinant protein and an HIV virus inhibitor, wherein the recombinant protein comprises:

[0010] 1) scFV of anti-CD3 antibody; and

[0011] 2) Diphtheria toxin.

[0012] Furthermore, the recombinant protein heavy chain is 1), 2), or 2), 1) from N-terminus to C-terminus.

[0013] Furthermore, the recombinant protein heavy chain from N-terminus to C-terminus is 2) and 1).

[0014] Furthermore, the recombinant protein also includes linkers of different types and lengths.

[0015] In some specific embodiments, the linker can be PW, (G4S) n ,AADP,(EAAAK) n , KESGSSVSSEQLAQFRSLD, EGKSSGSGSESKST, etc.

[0016] Furthermore, the scFV of the anti-CD3 antibody has the light chain CDR sequences shown in SEQ ID NOs. 1 to 3.

[0017] Furthermore, the scFV of the anti-CD3 antibody has the heavy chain CDR sequences shown in SEQ ID NOs. 4 to 6.

[0018] Furthermore, the scFV of the anti-CD3 antibody has a light chain variable region sequence as shown in SEQ ID NO.7.

[0019] Furthermore, the scFV of the anti-CD3 antibody has a heavy chain variable region sequence as shown in SEQ ID NO.8.

[0020] Furthermore, the scFV of the anti-CD3 antibody has an amino acid sequence as shown in SEQ ID NO.9.

[0021] The pharmaceutical composition of the present invention can be used with CD3 as HIV virus reservoir. + The diphtheria toxin functional group in the recombinant protein enters CD3 under the action of CD3 translocation after binding. + It can cause cell apoptosis inside cells, thus destroying the HIV virus reservoir and avoiding subsequent repeated infection and amplification. At the same time, it can also inhibit HIV.

[0022] In a specific embodiment, the diphtheria toxin has the amino acid sequence shown in SEQ ID NO.10.

[0023] In some specific embodiments, the structure of the recombinant protein is shown in FIG1 , and its sequence is shown in SEQ ID NO.11.

[0024] The use of these recombinant proteins is most effective in destroying the HIV viral reservoir, allowing for the maximum release of HIV and providing conditions for the subsequent elimination of HIV.

[0025] The HIV virus inhibitor refers to an agent that can act on HIV to prevent it from infecting or establishing infection, for example, to prevent HIV from replicating or invading.

[0026] The HIV virus inhibitors include, but are not limited to, drugs used in cocktail therapy, such as HIV reverse transcriptase inhibitors, HIV integrase inhibitors, HIV protease inhibitors, HIV fusion inhibitors, etc.; for example, HIV reverse transcriptase inhibitors include zidovudine, lamivudine, azithromycin, nevirapine, rilpivirine, dolamitinib, etc.; HIV integrase inhibitors include raltegravir, dolutegravir, etc.; HIV protease inhibitors include lopinavir / ritonavir, darunavir / cobicistat, etc.; HIV fusion inhibitors include abovirte, etc.

[0027] The HIV virus inhibitors include but are not limited to HIV virus invasion inhibitors, which are capable of acting on HIV, preventing the virus from binding to cell surface molecules, thereby preventing the virus from entering cells and establishing infection.

[0028] In some specific embodiments, the viral inhibitor may be a neutralizing antibody and / or antibody fragment, or may include a molecule (eg, a fusion protein) that competes with HIV for binding to a cell surface receptor.

[0029] In some specific embodiments, the HIV virus inhibitor is a neutralizing antibody or a fusion protein.

[0030] In some specific embodiments, the neutralizing antibody may be N6, 3BNC117, 10-1074, etc.

[0031] In some specific embodiments, the fusion protein can be VG627-6, VG627-8, VG627-4F, etc.

[0032] In some specific embodiments, one or more of the above-mentioned HIV inhibitors may be used. For example, the pharmaceutical compositions of the present invention may include a recombinant protein and a drug used in cocktail therapy, a recombinant protein and a neutralizing antibody, a recombinant protein and a viral entry inhibitor, or a recombinant protein and a drug used in cocktail therapy and a viral entry inhibitor.

[0033] In some specific embodiments, the neutralizing antibody N6 has a light chain CDR sequence as shown in SEQ ID NOs. 12 to 14; and a heavy chain CDR sequence as shown in SEQ ID NOs. 15 to 17.

[0034] In some specific embodiments, the neutralizing antibody 3BNC117 has a light chain CDR sequence as shown in SEQ ID NOs. 18 to 20; and a heavy chain CDR sequence as shown in SEQ ID NOs. 21 to 23.

[0035] In some specific embodiments, the neutralizing antibody 10-1074 has a light chain CDR sequence as shown in SEQ ID NOs. 24 to 26; and a heavy chain CDR sequence as shown in SEQ ID NOs. 27 to 29.

[0036] In some specific embodiments, the neutralizing antibody N6 has a light chain variable region sequence as shown in SEQ ID NO.30; and a heavy chain variable region sequence as shown in SEQ ID NO.31.

[0037] In some specific embodiments, the 3BNC117 neutralizing antibody has a light chain variable region sequence as shown in SEQ ID NO.32; and a heavy chain variable region sequence as shown in SEQ ID NO.33.

[0038] In some specific embodiments, the neutralizing antibody 10-1074 has a light chain variable region sequence as shown in SEQ ID NO.34; and a heavy chain variable region sequence as shown in SEQ ID NO.35.

[0039] In some specific embodiments, the neutralizing antibody has a light chain sequence N6 as shown in SEQ ID NO.36; and a heavy chain sequence as shown in SEQ ID NO.37.

[0040] In some specific embodiments, the neutralizing antibody 3BNC117 has a light chain sequence as shown in SEQ ID NO.38; and a heavy chain sequence as shown in SEQ ID NO.39.

[0041] In some specific embodiments, the neutralizing antibody 10-1074 has a light chain sequence as shown in SEQ ID NO.40; and a heavy chain sequence as shown in SEQ ID NO.41.

[0042] In some specific embodiments, the fusion protein VG627-6 has the amino acid sequence shown in SEQ ID NO.42.

[0043] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier and / or excipient.

[0044] Furthermore, the HIV is HIV-1 or HIV-2.

[0045] In another aspect, the present invention provides a vector (eg, a cloning vector or an expression vector) comprising the isolated nucleic acid molecule of the present invention.

[0046] In certain preferred embodiments, the vector comprises a nucleotide sequence encoding the pharmaceutical composition of the present invention, specifically, a nucleotide sequence encoding a recombinant protein, a neutralizing antibody, and / or a fusion protein amino acid sequence.

[0047] In certain preferred embodiments, the vector of the present invention is, for example, a plasmid, a cosmid, a phage, a virus, etc. In certain preferred embodiments, the vector is capable of expressing the pharmaceutical composition of the present invention in a subject (eg, a mammal, such as a human).

[0048] In another aspect, the present invention provides a host cell comprising the isolated nucleic acid molecule of the present invention or the vector of the present invention.

[0049] Such host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, eg, mouse cells, human cells, etc.).

[0050] In certain preferred embodiments, the host cell of the present invention is a mammalian cell, such as CHO (eg, CHO-K1, CHO-S, CHO DG44) or HEK293.

[0051] In another aspect, the present invention provides a method for preparing the pharmaceutical composition of the present invention, comprising culturing the host cell of the present invention under conditions allowing expression of the pharmaceutical composition, and recovering the pharmaceutical composition from the cultured host cell.

[0052] In certain preferred embodiments, the method comprises:

[0053] 1) constructing an expression vector comprising a nucleotide sequence encoding the pharmaceutical composition of the present invention;

[0054] 2) transforming the expression vector described in step 1) into a host cell;

[0055] 3) culturing the host cell described in step 2) under conditions that allow expression of the pharmaceutical composition of the present invention; and

[0056] 4) Recovering the pharmaceutical composition from the cultured host cell.

[0057] The pharmaceutical composition of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc.

[0058] The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical composition of the present invention should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such injections can be sterile injection solutions. For example, sterile injection solutions can be prepared by the following method: in an appropriate solvent, the recombinant protein of the present invention is mixed with the necessary dose, and optionally, other desired ingredients (including but not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof) are mixed at the same time, followed by filtration sterilization. In addition, sterile injection solutions can be prepared as sterile lyophilized powders (for example, by vacuum drying or freeze drying) for storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier, such as sterile pyrogen-free water, before use.

[0059] In addition, the recombinant protein of the present invention can be present in a pharmaceutical composition in a unit dosage form, For ease of administration. In certain embodiments, the unit dose is at least 0.1 mg, 1 mg, at least 5 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 45 mg, at least 50 mg, at least 75 mg, or at least 100 mg. In the case where the pharmaceutical composition is a liquid (e.g., injection) dosage form, it can include a concentration of at least 0.1 mg / ml, such as at least 0.25 mg / ml, at least 0.5 mg / ml, at least 1 mg / ml, at least 2.5 mg / ml, at least 5 mg / ml, at least 8 mg / ml, at least 10 mg / ml, at least 15 mg / ml, at least 25 mg / ml, at least 50 mg / ml, at least 75 mg / ml, or at least 100 mg / ml of the recombinant protein of the present invention.

[0060] Recombinant protein of the present invention or pharmaceutical composition can be used by any suitable method known in the art, including but not limited to, oral, oral, sublingual, eyeball, local, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, inguinal, intravesical, local (such as, powder, ointment or drops), or nasal route. But, for many therapeutic uses, preferred route of administration / mode is parenteral administration (such as intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Technicians should understand that route of administration and / or mode will change according to intended purpose. In a preferred embodiment, recombinant protein of the present invention or pharmaceutical composition are given by intravenous infusion or injection.

[0061] In the present invention, the subject may be a mammal, such as a human.

[0062] The pharmaceutical compositions of the present invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of the recombinant protein of the present invention. A "prophylactically effective amount" is an amount sufficient to prevent, prevent, or delay the onset of a disease (e.g., progression to AIDS). A "therapeutically effective amount" is an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount of the recombinant protein of the present invention may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and other treatments administered concurrently.

[0063] Therefore, in another aspect, the present invention provides a kit comprising the pharmaceutical composition of the present invention and instructions, wherein the instructions include instructions for using the pharmaceutical composition of the present invention to treat HIV.

[0064] Furthermore, the instructions include methods for using the pharmaceutical composition of the present invention for treating HIV, as well as dosages of the active ingredients used.

[0065] Furthermore, the instructions include that the dosage of the active ingredient used is not more than 50 μg / kg / person / day.

[0066] Furthermore, the instructions include using a dosage of the active ingredient of no more than 22.5 μg / kg / person / day. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] FIG1 is a schematic diagram of the structure of the recombinant protein of the present invention;

[0068] FIG2 shows the cell status of Jurkat cells after treatment with the recombinant protein of the present invention for 24, 48, and 72 hours;

[0069] FIG3 is a comparison of viral titers in the culture medium of Jurkat cells infected with HIV-1 after treatment with the recombinant protein of the present invention;

[0070] FIG4 shows the neutralization effect of VG627 on HIV-1BaL;

[0071] Figures 5A-D show the neutralization effect of VG627 on free viruses in the culture supernatant of cells treated with VG712;

[0072] Figures 6A-D show the neutralizing effect of neutralizing antibody N6 on free viruses in the culture supernatant of VG712-treated cells;

[0073] Figures 7A-D show the neutralization effect of ART drugs on free viruses in the culture supernatant of VG712-treated cells;

[0074] FIG8 shows the inhibitory effects of different drug combinations on HIV-1. DETAILED DESCRIPTION

[0075] The technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0076] Example 1: Recombinant protein used in the present invention

[0077] The sequence of the recombinant protein of the present invention is shown in Table 1 below.

[0078] Table 1

[0079] The constructed fusion protein is transformed into the working library strain VG712 through conventional strain transformation and preservation, which is the recombinant protein VG712 of the present application.

[0080] Example 2: Expression and purification of the recombinant protein of the present invention

[0081] 1. Fermentation culture

[0082] 1). Materials

[0083] Culture medium: YSG culture medium (Thermo fisher Bacto TM Yeast extract 10g / L, Thermo fisher Difco TM Soytone 20g / L, BD anhydrous glycerin 10g / L)

[0084] Strain: VG712 working library strain

[0085] 2). Operation steps

[0086] A. Add 60 ml of YSG medium to a 250 ml shake flask. Thaw a working bacterial strain at room temperature and inoculate it into the medium. Transfer the inoculated bacterial solution to a shaker and culture for 48 hours.

[0087] B. Add 200 ml of YSG medium to 1 L of shake flask seed, inoculate 20 ml of the superior seed into the medium, and transfer the inoculated bacterial liquid to a shaker for 24 hours.

[0088] C. Remove the secondary culture that has been incubated for 24 ± 1 h, transfer the desired strain to the inoculation bottle, and transfer the strain to the fermenter. Turn on temperature, DO, and pH control.

[0089] D. When the DO peak appears, add 75% glycerol solution.

[0090] E. After a period of cultivation, start feeding methanol and 10% yeast extract and continue feeding until the end of the cultivation.

[0091] F. Use hollow fiber columns to collect the culture supernatant and transfer it to downstream for subsequent protein purification.

[0092] 2. Purification process

[0093] 1)Butyl 650M HIC

[0094] A material

[0095] Buffer: 0.5 mol / L sodium hydroxide solution, TE buffer, 4*BHIC binding buffer, BHIC binding buffer

[0096] TEG buffer, HS packing buffer, LS packing buffer, 20% ethanol solution

[0097] Filler: TOYOPEARL Buty1-650M

[0098] B. Process Description

[0099] Sample dilution: Adjust the conductivity of the clarified harvest solution to 34-42 mS / cm with 4*BHIC banding buffer;

[0100] Chromatography process: Equilibrate the column with BHIC banding buffer and then load the sample. After loading, re-equilibrate with BHIC banding buffer, elute the target protein with TEG buffer, regenerate the column with water for injection, CIP the column with 0.5 mol / L sodium hydroxide solution, and preserve the column with 20% ethanol solution.

[0101] 2)Borate Poros 50HQ AEC

[0102] A material

[0103] Buffer: 0.5mol / L sodium hydroxide solution, TE buffer, HS packing buffer, LS packing buffer, 20% ethanol solution, 0.05mol / L borate in TEG buffer, 0.075mol / L borate in TEG buffer, 0.1mol / L borate in TEG buffer

[0104] Filler: Poros 50HQ

[0105] B. Process Description

[0106] Sample dilution: Dilute the hydrophobic chromatography collection solution 8 times with TE buffer.

[0107] Chromatography process: Equilibrate the column with TE buffer before loading the sample. Re-equilibrate with TE buffer after loading. Elute the target protein with 0.05 mol / L borate in TEG buffer (elution 1), 0.075 mol / L borate in TEG buffer (elution 2), and 0.1 mol / L borate in TEG buffer (elution 3). Regenerate the column with HS packing buffer. CIP the column with 0.5 mol / L sodium hydroxide solution and store it in 20% ethanol. Combine the elution 2 and elution 3 fractions to form the anion exchange chromatography fraction B.

[0108] 3) Poros 50HQ AEC

[0109] A material

[0110] Buffer: 0.5mol / L sodium hydroxide solution, TE buffer, HS packing buffer, LS packing buffer, 20% ethanol solution, Low TEG buffer, 0.05mol / L NaCl in low TEG buffer, 0.15mol / L NaCl in low TEG buffer

[0111] Filler: Poros 50HQ

[0112] B. Process Description

[0113] Sample dilution: Dilute the collected anion exchange chromatography solution of B 4-fold with TE buffer.

[0114] Chromatography process: Equilibrate the chromatography column with TE buffer and then load the sample. Re-equilibrate with Low TEG buffer after loading. Elute the target protein with 0.05 mol / L NaCl in low TEG buffer (elution 1) and 0.15 mol / L NaCl in low TEG buffer (elution 2). Regenerate the chromatography column with HS packing buffer. CIP the chromatography column with 0.5 mol / L sodium hydroxide solution and preserve the chromatography column with 20% ethanol solution. Check the purity of the collected solutions of elution 1 and elution 2 (purity ≥ 98%, combine the collected solutions and proceed to the next step).

[0115] 4) Conductivity adjustment and dilution

[0116] A material

[0117] Buffer: 0.5mol / L NaCl in low TEG buffer, 0.15mol / L NaCl in low TEG buffer

[0118] B. Process Description

[0119] The conductivity of the anion exchange chromatography collected solution was adjusted with 0.5 mol / L NaCl in low TEG buffer, and the protein concentration was diluted to ≥ 0.8 mg / ml with 0.15 mol / L NaCl in low TEG buffer (stock buffer).

[0120] 5) Stock solution filtration

[0121] A material

[0122] Consumables: 0.22μm sterilizing filter

[0123] The stock solution (filter) sample was filtered through a 0.22 μm filter to obtain the VG712 stock solution.

[0124] Example 3: Experiment on killing T cells by the recombinant protein of the present invention

[0125] 1. Materials:

[0126] Culture medium: RPMI 1640 (supplemented with 10% fetal bovine serum (Gibico, F9665-500ML), 100 U / mL penicillin and 100 U / mL streptomycin (Sigma))

[0127] Cells: Jurkat cell line

[0128] VG712: prepared as in Example 2.

[0129] 2. Operation steps:

[0130] a) Jurkat cells were harvested, centrifuged at 1000 rpm for 5 min, and the cells were counted.

[0131] b) Press 1×10 6 cells were added to 1×10 4 The virus and cells were mixed evenly at a ratio of TCID50 to HIV-1, incubated in a 37°C CO2 cell culture incubator for 1 h, then the cells were washed three times with PBS and resuspended in fresh complete culture medium.

[0132] c) Press 1×10 5 Cells were inoculated into 24-well plates at 1 mL per well and cultured in a 37°C CO2 cell culture incubator for 48 h.

[0133] d) Add VG712 (final concentration 0.2 nM) or VG712 buffer to the 24-well plate and continue culturing.

[0134] e) Cell culture fluid was collected 8 h, 12 h, 24 h, and 72 h after addition of VG712, and centrifuged at 1000 rpm for 5 min. The supernatant was collected and a portion of the supernatant was used to determine the TCID50 value of the virus in the culture medium.

[0135] The recombinant protein VG712 of the present invention has a strong killing effect on T cells. CCK8 cell activity assays show that cells begin to apoptosis 8 hours after VG712 treatment. The data are shown in Table 1. The ratios of the 4-hour and 8-hour positive control groups are distorted due to the shorter cell culture time. After 14 hours, after cell proliferation, it can be observed that the ratios of the positive controls are relatively stable. The results of VG712 killing (0.1nM) / T cells show an overall downward trend, which is consistent with the results observed under the microscope. Microscopic observation shows that cells gradually undergo apoptosis.

[0136] Table 1

[0137] After T cells were treated with VG712 for 24 h, 48 h, and 72 h, the proliferation of cells treated with VG712 was significantly arrested, and the cells showed obvious apoptosis ( FIG2 ). Compared to HIV-1-infected Jurkat cells treated with VG712 buffer, the infectious virus titer in the culture supernatant of cells treated with VG712 for 12 hours was slightly lower (TCID50: 54 vs. 39.3). The infectious virus titer in the culture supernatant of cells treated with VG712 for 24 hours was not different from that of the 12-hour treatment, while the infectious virus titer in the culture supernatant of cells treated with VG712 buffer for 24 hours increased significantly (TCID50 = 809). No infectious virus was detected in the culture supernatant of cells treated with VG712 buffer for 72 hours, while the infectious virus titer in the culture supernatant of cells treated with VG712 buffer for 72 hours continued to increase significantly (TCID50 = 20200) (Figure 3). These results indicate that VG712 kills T cells while also inhibiting HIV-1 proliferation.

[0138] Example 4. Preparation and purification of virus invasion inhibitors

[0139] 1. VG627 protein expression

[0140] 1) Materials:

[0141] Culture medium: FreeStyle 293 Expression Medium (Gibico, 12338018, supplemented with 100 U / mL penicillin and 100 U / mL streptomycin (Sigma)

[0142] Cells: HEK 293F cell line

[0143] Transfection reagent: PEI (Polyetherimide: Polysciences, 23966-1)

[0144] 2) Protein expression steps:

[0145] (1) The density of HEK 293F cells is 1-1.2×10 6 Transfection was performed at 100 cells / mL, with 1-1.2 μg of plasmid transfected per million cells. The ratio of plasmid to PEI (1 mg / mL) was 1:3.

[0146] (2) Taking a 250 mL shake flask as an example (the total volume of cells seeded before transfection is 25 mL, and the final total culture volume is 50 mL), add 50 μg of plasmid to 2.5 mL of sterile saline, gently mix the solution, and then add 50-200 μL of PEI transfection reagent;

[0147] (3) Vortex for 5 seconds and let stand for 10 minutes to allow the PEI-DNA complex to form;

[0148] (4) Gently pipette the mixture up and down three times, shaking the flask while adding the PEI-DNA mixture to the cell culture flask with the replaced medium to ensure uniform distribution of the PEI-DNA complex and prevent local over-concentration. After culturing for 2-3 hours, add 25 mL of fresh complete medium and continue culturing at 37°C with 8% CO2.

[0149] (5) 4 days after transfection, the samples were collected and centrifuged at 1200 rpm for 5 min. The cell supernatant was collected and the expression of the target protein was detected by Western blot.

[0150] 2. VG627 protein purification

[0151] 1) Materials

[0152] Protein A Resin (GenScript, L00210) was used to purify the protein;

[0153] Binding / Wash Buffer: 0.15M NaCl, 20mM Na2HPO4, pH 7.0;

[0154] Elution Buffer: 0.1M glycine, pH 3.0;

[0155] Neutralization Buffer: 1M Tris-HCl, pH 8.5; peristaltic pump

[0156] 2) Operation steps:

[0157] (1) Pre-add 1 mL of Binding / Wash Buffer to the column, completely resuspend the Protein A Resin, and then pipette 2 mL of the mixture into the column.

[0158] (2) Allow the resin to settle naturally, then add 5 mL of Binding / Wash Buffer for equilibrium at a flow rate of 1 mL / min.

[0159] (3) Mix the sample with Binding / Wash Buffer at a volume ratio of at least 1:1 to form a sample diluent, and add it to the column at a flow rate of 1 mL / min;

[0160] (4) Wash the equilibrated column with 30 mL of Binding / Wash Buffer and drain the buffer at 2 mL / min;

[0161] (5) Elute with 15-20 mL of Elution Buffer at a flow rate of approximately 1 mL / min, collect the target solution, and immediately neutralize to pH 7.4 with 1 / 10 the total volume of the collected solution using Neutralization Buffer;

[0162] (6) Reduced SDS-PAGE electrophoresis was performed, and then Coomassie Brilliant Blue staining was used to detect the purification of the target protein.

[0163] Example 5: Evaluation of the neutralizing activity of VG627 against free HIV-1

[0164] 1. Materials

[0165] DEAE (Diethylaminoethyl, Sigma, D9885);

[0166] NP40 (Sigma-Aldrich, I8896);

[0167] Luciferase Assay System (Promega, E1501)

[0168] VG627: Prepare it yourself

[0169] Cells: TZM-bl cell line

[0170] Culture medium: Dulbecco's modified Eagle medium-High, Sigma, D6429-500ML, supplemented with 100U / mL penicillin and 100U / mL streptomycin (Sigma), 10% fetal bovine serum (Gibico, F9665-500ML)

[0171] 2. Operation steps

[0172] (1) The day before the neutralization experiment, cultured TZM-bl cells were made into a single cell suspension at a concentration of 1×10 4 cells / well were added to a 96-well plate with a final volume of 100 μL and cultured in a 37°C CO2 cell culture incubator;

[0173] (2) The purified VG627 was diluted backward in 5 gradients with a dilution factor of 3 (corresponding to VG627 concentrations of 6480, 2160, 720, 240, and 80 ng / mL, respectively, and a control without VG627 (concentration of 0) was set;

[0174] (3) The culture supernatant of cells treated with VG712 for different time periods in Example 3 was used as the starting dilution gradient, and then diluted backward for 5 gradients with a dilution factor of 3;

[0175] (4) Mix 60 μL of the diluted VG627 solution from step (2) with 60 μL of the collected supernatant. Mix 60 μL of the collected supernatant solution and the diluted supernatant solution from step (3) with 60 μL of VG627 (240 ng / mL). Incubate the mixture in a 37°C CO2 cell culture incubator for 1 h. Perform three replicates for each experimental well.

[0176] (5) Discard the old cell culture medium, add 100 μL of fresh DMEM medium containing DEAE, and then add 100 μL of the incubated mixed solution and culture in a cell culture incubator at 37°C;

[0177] (6) After 48 h, the culture medium was discarded, the cells were washed once with 200 μL / well PBS buffer, and 100 μL / well 0.5% NP40 lysis buffer was added and lysed at room temperature for 5–10 min.

[0178] (7) Centrifuge at 1000 rpm for 5 min at room temperature, and pipette 50 μL of supernatant into each well of a 96-well white plate;

[0179] (8) Add 50 μL / well of luciferase substrate and read the absorbance using a microplate reader;

[0180] (9) Analyze the data and calculate the inhibition rate at each concentration, taking the case without VG627 as 0% inhibition rate.

[0181] Recombinant protein VG627 has a strong neutralizing effect on free HIV-1. Neutralization assay results for HIV-1 strain BaL are shown in Figure 4. VG627 was used to neutralize HIV-1 in cell culture supernatants treated with VG712 and VG712 buffer for 12 and 24 hours, respectively. As shown in Figure 5, different concentrations of VG627 effectively neutralized the virus in these supernatants. The combination of VG627 and VG712 significantly reduced the titer of infectious virus in the cell culture supernatant after 24 hours of treatment (TCID50 = 0), as shown in Figure 8.

[0182] Example 6: Evaluation of the neutralizing activity of neutralizing antibody N6 against free HIV-1

[0183] The experimental process is as described in Example 5. Jurkat cells are infected with the HIV-1 experimental strain BaL. The cells are treated with VG712 (final concentration of 0.2nM) or VG712 buffer 48h after infection, and then the cell culture supernatant is collected 12h and 24h after treatment. The supernatant samples are then incubated with different concentrations of N6 at 37 degrees for 1h, and the incubated mixture is added to a 96-well plate pre-plated with TZM-bl cells. After further culture for 48h, the luciferase activity is detected and the virus inhibition rate is calculated. As shown in Figures 6A-D, N6 can neutralize the viruses in these supernatants very well. The combination of neutralizing antibodies N6 and VG712 significantly reduces the titer of infectious viruses in the cell culture supernatant treated for 24h (TCID50=0), as shown in Figure 8.

[0184] Example 7: Evaluation of the neutralizing activity of ART drugs against free HIV-1

[0185] The experimental process is as described in Example 5. Jurkat cells were infected with the HIV-1 experimental strain BaL. 48 hours after infection, the cells were treated with VG712 (final concentration of 0.2nM) or VG712 buffer, and the cell culture supernatant was collected 12 hours and 24 hours after treatment. The supernatant samples were then incubated with different concentrations of cocktail therapy reagents (lamivudine (100ng / mL), tenofovir (2μg / mL), ribavirin (15ng / mL)) at 37 degrees for 1 hour, and the incubated mixture was added to a 96-well plate pre-plated with TZM-bl cells. After further culture for 48 hours, luciferase activity was detected and the virus inhibition rate was calculated. As shown in Figures 7A-D, the reagents used in the cocktail therapy can well neutralize the viruses in these supernatants. The combination of ART drugs and VG712 significantly reduced the titer of infectious virus in the cell culture supernatant treated for 24 hours (TCID50=0), as shown in Figure 8.

Claims

1. A pharmaceutical composition comprising a recombinant protein and an HIV virus inhibitor, wherein: The recombinant protein comprises: 1) scFV of anti-CD3 antibody; and 2) Diphtheria toxin.

2. The pharmaceutical composition according to claim 1, wherein The scFV of the anti-CD3 antibody has the light chain CDR sequences shown in SEQ ID NOs. 1 to 3 and the heavy chain CDR sequences shown in SEQ ID NOs. 4 to 6.

3. The pharmaceutical composition according to claim 1, wherein The diphtheria toxin has an amino acid sequence as shown in SEQ ID NO.

10.

4. The pharmaceutical composition according to claim 1, wherein The sequence of the recombinant protein is shown in SEQ ID NO.

11.

5. The pharmaceutical composition according to claim 1, wherein The HIV virus inhibitor is an agent used in cocktail therapy, or a virus invasion inhibitor.

6. The pharmaceutical composition according to claim 1, wherein The HIV virus inhibitor is an HIV virus integrase inhibitor, an HIV virus protease inhibitor, an HIV virus fusion inhibitor, an HIV virus reverse transcriptase inhibitor, a neutralizing antibody and / or an antibody fragment, or a molecule that competitively binds to a cell surface receptor with HIV.

7. The pharmaceutical composition according to claim 1, wherein The antibody has a light chain CDR sequence as shown in SEQ ID NOs. 12 to 14; and a heavy chain CDR sequence as shown in SEQ ID NOs. 15 to 17; The antibody has a light chain CDR sequence as shown in SEQ ID NOs. 18 to 20; and a heavy chain CDR sequence as shown in SEQ ID NOs. 21 to 23; or The antibody has light chain CDR sequences shown as SEQ ID NOs. 24-26; and heavy chain CDR sequences shown as SEQ ID NOs. 27-29.

8. The pharmaceutical composition according to claim 1, wherein The molecule that competes with HIV for binding to the cell surface receptor has a sequence as shown in SEQ ID NO.

42.

9. The pharmaceutical composition according to claim 1, wherein The pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

10. A carrier, characterized in that The method comprises a nucleotide sequence encoding the pharmaceutical composition according to any one of claims 1 to 9.

11. Use of the pharmaceutical composition according to any one of claims 1 to 9 for preparing an agent for treating HIV infection.

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