Use of pharmaceutical composition for preparing Anti-HIV infection drug

Abstract

The present invention relates to the field of molecular biology, specifically provides a use of a pharmaceutical composition, and more specifically provides a use of an anti-HIV infection pharmaceutical composition. Provided is a use of a pharmaceutical composition for preparing an anti-HIV infection drug. The pharmaceutical composition comprises a recombinant protein as shown in the following: 1) a scFv of an anti-CD3 antibody; and 2) diphtheria toxin. The recombinant protein can be used to specifically bind to CD3 on the surfaces of CD3+ cells as an HIV virus reservoir. After binding, the diphtheria toxin functional group in the recombinant protein enters the CD3+ cells under the transposition action of CD3, leading to cell apoptosis, thereby destroying the HIV virus reservoir, avoiding subsequent repeated infection and amplification, and also achieving the effect of inhibiting HIV.

Description

Application of a pharmaceutical composition for preparing a drug for resisting HIV infection Technical Field

[0001] The present invention belongs to the field of molecular biology, and in particular, relates to the application of a pharmaceutical composition, and more particularly, relates to the application of a pharmaceutical composition for resisting HIV infection. Background Art

[0002] Acquired Immunodeficiency Syndrome (AIDS), abbreviated as AIDS, is caused by infection with the human immunodeficiency virus (HIV).

[0003] 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.

[0004] 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 entry or 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 neutralize free HIV in body fluids, inhibiting viral invasion of cells and protecting against HIV at its source. However, they are ineffective against cells already infected with HIV. These infected cells can still produce new HIV viruses, leading to subsequent re-infection and proliferation.

[0005] 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.

[0006] Summary of the Invention

[0007] In view of this, in a first aspect, the present invention provides a pharmaceutical composition for use in preparing a drug for anti-HIV infection, wherein the pharmaceutical composition comprises a recombinant protein as shown below:

[0008] 1) scFV of anti-CD3 antibody; and

[0009] 2) Diphtheria toxin.

[0010] Furthermore, the recombinant protein heavy chain is 1), 2), or 2), 1) from N-terminus to C-terminus.

[0011] Furthermore, the recombinant protein heavy chain from N-terminus to C-terminus is 2) and 1).

[0012] Furthermore, the recombinant protein also includes linkers of different types and lengths.

[0013] In some specific embodiments, the linker can be PW, (G4S) n ,AADP,(EAAAK) n , KESGSSVSSEQLAQFRSLD, EGKSSGSGSESKST, etc.

[0014] Furthermore, the scFV of the anti-CD3 antibody has the light chain CDR sequences shown in SEQ ID NOs. 1 to 3.

[0015] Furthermore, the scFV of the anti-CD3 antibody has the heavy chain CDR sequences shown in SEQ ID NOs. 4 to 6.

[0016] Furthermore, the scFV of the anti-CD3 antibody has a light chain variable region sequence as shown in SEQ ID NO.7.

[0017] Furthermore, the scFV of the anti-CD3 antibody has a heavy chain variable region sequence as shown in SEQ ID NO.8.

[0018] Furthermore, the scFV of the anti-CD3 antibody has an amino acid sequence as shown in SEQ ID NO.9.

[0019] The recombinant protein 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.

[0020] In a specific embodiment, the diphtheria toxin has the amino acid sequence shown in SEQ ID NO.10.

[0021] In some specific embodiments, the structure of the recombinant protein is shown in FIG1 , and its sequence is shown in SEQ ID NO.11.

[0022] 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.

[0023] Furthermore, the composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0024] Furthermore, the HIV is HIV-1 or HIV-2.

[0025] Furthermore, the pharmaceutical composition also includes another pharmaceutically active agent for combined use.

[0026] Furthermore, the dosage of the pharmaceutical composition used is no more than 50 μg / kg / person / day.

[0027] Furthermore, the dosage of the pharmaceutical composition used is no more than 22.5 μg / kg / person / day.

[0028] In another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a recombinant protein of the present invention. In certain preferred embodiments, the isolated nucleic acid molecule encodes a recombinant protein of the present invention.

[0029] 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.

[0030] In certain preferred embodiments, the vector comprises a nucleotide sequence encoding the recombinant protein of the present invention.

[0031] 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 recombinant protein of the present invention in a subject (eg, a mammal, such as a human).

[0032] 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.

[0033] 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, e.g., mouse cells, human cells, etc.).

[0034] 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.

[0035] In another aspect, the present invention provides a method for preparing the recombinant protein of the present invention, comprising culturing the host cell of the present invention under conditions allowing expression of the recombinant protein, and recovering the recombinant protein from the cultured host cell.

[0036] In certain preferred embodiments, the method comprises:

[0037] 1) constructing an expression vector comprising a nucleotide sequence encoding the recombinant protein of the present invention;

[0038] 2) transforming the expression vector described in step 1) into a host cell;

[0039] 3) culturing the host cell described in step 2) under conditions that allow expression of the recombinant protein of the present invention; and

[0040] 4) Recovering the recombinant protein from the cultured host cell.

[0041] In another aspect, the present invention provides a pharmaceutical composition comprising the recombinant protein of the present invention and a pharmaceutically acceptable carrier and / or excipient.

[0042] The recombinant protein or 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.

[0043] 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.

[0044] In addition, the recombinant protein of the present invention can be present in a pharmaceutical composition in a unit dosage form, so that it can be used. 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 (for example, 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.

[0045] 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.

[0046] In the present invention, the subject may be a mammal, such as a human.

[0047] 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.

[0048] Therefore, in another aspect, the present invention provides a kit for treating HIV, comprising the pharmaceutical composition of the present invention and instructions, wherein the instructions include instructions for using the pharmaceutical composition of the present invention for treating HIV.

[0049] Furthermore, the instructions include the method of using the pharmaceutical composition of the present invention for treating HIV, as well as the dosage of the active ingredient used.

[0050] Furthermore, the instructions include that the dosage of the active ingredient used is not more than 50 μg / kg / person / day.

[0051] 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

[0052] FIG1 is a schematic diagram of the structure of the recombinant protein of the present invention;

[0053] FIG2 shows the cell status of Jurkat cells after treatment with the recombinant protein of the present invention for 24, 48, and 72 hours;

[0054] 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. DETAILED DESCRIPTION

[0055] 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.

[0056] Example 1: Recombinant protein used in the present invention

[0057] The sequences used in the present invention are shown in Table 1 below.

[0058] Table 1

[0059] 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.

[0060] Example 2: Expression and purification of the recombinant protein of the present invention

[0061] 1. Fermentation culture

[0062] 1). Materials

[0063] 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)

[0064] Strain: VG712 working library strain

[0065] 2). Operation steps

[0066] 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.

[0067] 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.

[0068] 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.

[0069] D. When the DO peak appears, add 75% glycerol solution.

[0070] E. After a period of cultivation, start feeding methanol and 10% yeast extract and continue feeding until the end of the cultivation.

[0071] F. Use hollow fiber columns to collect the culture supernatant and transfer it to downstream for subsequent protein purification.

[0072] 2. Purification process

[0073] 1)Butyl 650M HIC

[0074] A material

[0075] Buffer: 0.5 mol / L sodium hydroxide solution, TE buffer, 4*BHIC binding buffer, BHIC binding buffer

[0076] TEG buffer, HS packing buffer, LS packing buffer, 20% ethanol solution

[0077] Filler: TOYOPEARL Buty1-650M

[0078] B. Process Description

[0079] Sample dilution: Adjust the conductivity of the clarified harvest solution to 34-42 mS / cm with 4*BHIC banding buffer;

[0080] 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.

[0081] 2)Borate Poros 50HQ AEC

[0082] A material

[0083] 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

[0084] Filler: Poros 50HQ

[0085] B. Process Description

[0086] Sample dilution: Dilute the hydrophobic chromatography collection solution 8 times with TE buffer.

[0087] 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 elutions from elutions 2 and 3 to form the anion exchange chromatography fraction B.

[0088] 3) Poros 50HQ AEC

[0089] A material

[0090] 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

[0091] Filler: Poros 50HQ

[0092] B. Process Description

[0093] Sample dilution: Dilute the collected anion exchange chromatography solution of B 4-fold with TE buffer.

[0094] Chromatography process: Equilibrate the chromatography column with TE buffer and then load the sample. After loading, re-equilibrate with Low TEG buffer. Elute the target protein with 0.05mol / L NaCl in low TEG buffer (elution 1) and 0.15mol / L NaCl in low TEG buffer (elution 2). Regenerate the chromatography column with HS packing buffer. CIP the chromatography column with 0.5mol / 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).

[0095] 4) Conductivity adjustment and dilution

[0096] A material

[0097] Buffer: 0.5mol / L NaCl in low TEG buffer, 0.15mol / L NaCl in low TEG buffer

[0098] B. Process Description

[0099] 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).

[0100] 5) Stock solution filtration

[0101] A material

[0102] Consumables: 0.22μm sterilizing filter

[0103] The stock solution (filter) sample was filtered through a 0.22 μm filter to obtain the VG712 stock solution.

[0104] Example 3: Experiment on killing T cells by the recombinant protein of the present invention

[0105] 1. Materials:

[0106] Culture medium: RPMI 1640 (supplemented with 10% fetal bovine serum (Gibico, F9665-500ML), 100 U / mL penicillin and 100 U / mL streptomycin (Sigma))

[0107] Cells: Jurkat cell line

[0108] VG712: prepared as in Example 2.

[0109] 2. Operation steps:

[0110] a) Jurkat cells were harvested, centrifuged at 1000 rpm for 5 min, and the cells were counted.

[0111] b) Press 1×10 6 cells were added to 1×10 4 The virus and cells were mixed evenly at a ratio of TCID50 HIV, 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.

[0112] 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.

[0113] d) Add VG712 (final concentration 0.2 nM) or VG712 buffer to the 24-well plate and continue culturing.

[0114] 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.

[0115] 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.

[0116] Table 1

[0117] After treatment of T cells with VG712 for 24, 48, and 72 hours, proliferation of VG712-treated cells was significantly arrested, and the cells exhibited a clear state of apoptosis (Figure 2). Compared to HIV-infected Jurkat cells treated with VG712 buffer, the titer of infectious virus in the culture supernatant of cells treated with VG712 for 12 hours was slightly lower (TCID50: 54 vs. 39.3). The titer of infectious virus in the culture supernatant of cells treated with VG712 buffer for 24 hours remained unchanged, while the titer of infectious virus 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, but the titer of infectious virus in the culture supernatant of cells treated with VG712 buffer for 72 hours continued to increase significantly (TCID50 = 20200) (Figure 3). The above results show that VG712 kills T cells while also inhibiting the proliferation of HIV.

Claims

1. A pharmaceutical composition for use in preparing a drug for resisting HIV infection, the pharmaceutical composition comprising a recombinant protein as shown below: 1) scFV of anti-CD3 antibody; and 2) Diphtheria toxin.

2. The use according to claim 1, wherein: The recombinant protein heavy chain from N-terminus to C-terminus is 2) and 1).

3. The use 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.

4. The use according to claim 1, wherein: The scFV of the anti-CD3 antibody has a light chain variable region sequence as shown in SEQ ID NO.7 and a heavy chain variable region sequence as shown in SEQ ID NO.

8.

5. The use according to claim 1, wherein: The scFV of the anti-CD3 antibody has the amino acid sequence shown in SEQ ID NO.

9.

6. The use according to claim 1, wherein: The diphtheria toxin has an amino acid sequence as shown in SEQ ID NO.

10.

7. The use according to claim 1, wherein: The sequence of the recombinant protein is shown in SEQ ID NO.

11.

8. The use according to claim 1, wherein: The dosage of the pharmaceutical composition used is not more than 50 μg / kg / person / day.

9. A kit for treating HIV, comprising a pharmaceutical composition and instructions; wherein: The pharmaceutical composition comprises the following recombinant proteins: 1) scFV of anti-CD3 antibody; and 2) diphtheria toxin; wherein, The instructions include instructions for use of the pharmaceutical composition for treating HIV.

10. The kit according to claim 9, wherein The instructions include the method of using the pharmaceutical composition for treating HIV, and the dosage of the active ingredient to be used.

11. The kit according to claim 10, wherein The dosage of the active ingredient used is not more than 50 μg / kg / person / day.

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