WO2024125271A1 - Recombinant protein against hiv-1 and use thereof - Google Patents

Abstract

The present invention relates to the field of molecular biology, specifically, to a fusion protein, and more specifically, to a recombinant protein against HIV-1 and use thereof. The present invention provides a recombinant protein comprising: 1) a D1D2 domain of CD₄; 2) ND and CRD domains of DC-SIGN; and 3) an IgG1Fc fragment. The recombinant protein provided by the present invention possesses a good inhibitory effect against HIV-1 with broad-spectrum activity, as well as inhibitory effects on a range of early HIV-1 strains.

Description

Recombinant protein against HIV-1 and its application

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based on the Chinese patent application with application number 202211603567.9 and application date December 13, 2022, and claims the priority of the Chinese patent application. The entire contents of the Chinese patent application are hereby incorporated into this application by introduction.

Technical Field

The invention belongs to the field of molecular biology, and in particular, relates to a fusion protein, and more particularly, to an anti-HIV-1 recombinant protein and an application thereof.

Background technique

Human acquired immunodeficiency syndrome (AIDS), abbreviated as AIDS, is caused by infection with human immunodeficiency virus (HIV).

So far, all HIV-1 vaccine research has ended in failure. An important early achievement in drug treatment for HIV-1 is cocktail therapy (Highly active antiretroviral therapy, HAART). Continuous cocktail therapy can maintain the viral load in HIV-1 infected people at a low level for a long time, but it cannot eliminate the virus. Patients need to take drugs for a long time. HIV-1 infected people and AIDS patients will have serious and complex side effects after using such drugs, and long-term medication will lead to the production of HIV-1 resistant strains. Although HIV-1 broad-spectrum neutralizing antibodies (bNAbs) can also inhibit the infection of multiple HIV-1 strains in hosts, they have been used as drug candidates for preclinical or clinical trials in recent years, but their cost is very expensive, and even if they are successful, they are difficult to use on a large scale. Currently, anti-HIV-1 drugs, such as reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors, etc., are mostly chemical drugs. Biological drugs have the advantages of low toxicity and continuous or controllable expression in patients, but biological drugs currently used in clinical practice are extremely scarce, and there are still problems such as poor broad-spectrum and inhibitory efficacy. There are problems such as poor results, serious side effects and the existence of resistant strains.

There is a need in the art for a product that has broad spectrum and good effect in inhibiting HIV-1 and is safe to use.

Summary of the invention

In view of this, in a first aspect, the present invention provides a recombinant protein, comprising:

1) D1D2 (domain 1 domain 2) domain of CD4;

2) ND (neck domain) and CRD (carbohydrate recognition domain) of DC-SIGN; and

3) IgG1 Fc segment.

Furthermore, the recombinant protein is sequentially 1), 2), 3), or 1), 3), 2), or 3), 1), 2), or 3), 2), 1), or 2), 1), 3), or 2), 3), 1).

Furthermore, the recombinant protein is preferably 1), 2), 3), or 1), 3), 2) from N-terminus to C-terminus.

Furthermore, the recombinant protein is more preferably 1), 3), 2) from N-terminus to C-terminus.

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

In some specific embodiments, the linker can be (G ₄ S) _n , AADP, (EAAAK) _n , KESGSVSSEQLAQFRSLD, EGKSSGSGSESKST, etc.

Furthermore, the sequence of the D1D2 domain of CD4 is:

Furthermore, the sequences of ND and CRD of DC-SIGN are:

Furthermore, the sequence of the IgG1 Fc segment is:

The use of the recombinant protein of the present invention has a significant inhibitory effect on HIV-1 and is safe to use.

In some specific embodiments, the recombinant protein is a protein shown in SEQ ID NO.1 to 4.

In some preferred embodiments, the recombinant protein is a protein shown in SEQ ID NO.2~4.

The use of the above-mentioned recombinant protein has a better inhibitory effect on HIV-1 and has a good broad spectrum.

In some more preferred embodiments, the recombinant protein is a protein as shown in SEQ ID NO.2~3.

The use of these recombinant proteins has the best inhibitory effect on HIV-1 and has a better broad spectrum, and also has a good inhibitory effect on many early strains of HIV-1.

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.

In some specific embodiments, the nucleic acid molecule is as shown in SEQ ID NO. 5 to 8 Nucleic acid.

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.

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

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).

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.

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.).

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

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 culture of the cultured host cell.

In certain preferred embodiments, the method comprises:

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

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

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

4) Recovering the recombinant protein from the cultured host cell.

In another aspect, the present invention provides use of the recombinant protein of the present invention in preparing a drug for resisting HIV-1 infection.

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.

The recombinant protein of the present invention or 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.

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: the recombinant protein of the present invention is mixed with the necessary dose in an appropriate solvent, 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 and sterilization. In addition, sterile injection solutions can be prepared as sterile lyophilized powders (e.g., 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.

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.

The recombinant protein or pharmaceutical composition of the present invention can be administered by any suitable method known in the art, including but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic, inguinal, intravesical, topical (e.g., powder, ointment or drops) administration. The preferred route of administration is parenteral administration (e.g., intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled person will appreciate that the route and/or mode of administration will vary depending on the intended purpose. In a preferred embodiment, the recombinant protein or pharmaceutical composition of the present invention is administered by intravenous infusion or injection.

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

The pharmaceutical composition or recombinant protein provided by the present invention can be used alone or in combination, or can be used in combination with another pharmaceutically active agent (e.g., a viral inhibitor). In certain preferred embodiments, the recombinant protein of the present invention is used in combination with other HIV-1 inhibitors to prevent and/or treat HIV-1 infection.

The pharmaceutical composition 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" refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease (e.g., progression to AIDS). A "therapeutically effective amount" refers to 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 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 simultaneously, etc.

Therefore, in another aspect, the present invention provides a kit comprising the recombinant protein of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows (A) HIV-1 p24 concentration in the culture medium of human foreskin inner plate tissue in different treatment groups; (B) the inhibition rate of ②, ③ and ④ proteins against BaL in human foreskin inner plate tissue. The data shown are mean ± standard deviation.

Detailed ways

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to Carriers and/or excipients that are physically and/or physiologically compatible with the subject and the active ingredient are well known in the art and include, but are not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH regulators include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin.

As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human.

As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., HIV-1 infection) refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease (e.g., progression to AIDS); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on 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 simultaneously, etc.

The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are used to illustrate the present invention, rather than to limit the present invention.

Example 1: Construction of the recombinant protein plasmid of the present invention

Different combinations of gene sequences (SEQ ID NO.5-8) were inserted into plasmid pcDNA3.1(+) to construct plasmids expressing target proteins. All recombinant plasmids were double-digested with endonucleases BamHI and XhoI, and then identified by 1% agarose gel electrophoresis at 120V for 30 minutes.

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

Express

1) Materials:

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

Cells: HEK 293F cell line

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

2) Operation steps:

(1) Transfection was performed when the density of HEK 293F cells was 1-1.2×10 ⁶ cells/mL, and 1-1.2 μg of plasmid was transfected per million cells. The ratio of plasmid to PEI (1 mg/mL) was 1:3;

(2) Taking a 250 mL shake flask as an example (the total volume of cells planted 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;

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

(4) Use a pipette to gently pipette up and down the mixture three times, while shaking the flask, add the PEI-DNA mixture to the cell culture flask with the culture medium replaced to ensure that the PEI-DNA complex is evenly distributed to prevent local excessive concentration. After culturing for 2 to 3 hours, add 25 mL of fresh complete culture medium and continue culturing at 37°C and 8% _CO2 ;

(5) 4 days after transfection, collect samples, centrifuge at 1200 rpm for 5 min, collect cell supernatant, and detect the expression of target protein by Western blot.

purification

1) Materials

Protein A Resin (GenScript, L00210) purified protein;

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

Elution Buffer: 0.1 M glycine, pH 3.0;

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

2) Operation steps:

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

(2) After the resin settles naturally, add 5 mL of Binding/Wash Buffer for balance at a flow rate of 1 mL/min;

(3) Mix the sample and Binding/Wash Buffer in 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.

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

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

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

Example 3: Evaluation of the neutralizing activity of the recombinant protein of the present invention

1) Materials

DEAE (Diethylaminoethyl, Sigma, D9885);

NP40 (Sigma-Aldrich, I8896;

Luciferase Assay System(Promega, E1501)

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

2) Determine the concentration of DEAE to be used:

(1) 100 μL of TZM-bl cells were plated into a 96-well flat-bottom cell culture plate at a density of 1 × 10 ⁶ cells/mL and cultured in a 37°C CO ₂ cell culture incubator;

(2) The next day, the DEAE solution was diluted by a factor of 3, with 11 dilutions. The 12th well was filled with complete medium without DEAE as a blank, and four replicates were performed;

(3) Prepare a sufficient volume of virus solution with a known high toxicity;

(4) Aspirate the old culture medium and add 100 μL/well of culture medium containing DEAE and 100 μL of virus solution;

(5) After 48 hours, read the absorbance value using the same method as HIV-1 TCID50;

(6) Analyze the data. The concentration of DEAE corresponding to the well with the largest reading is the working concentration.

3) Evaluation of neutralization effect using TZM-bl cell line

(1) After the cultured TZM-bl cells were made into a single cell suspension, they were added into a 96-well plate at a concentration of 1×10 ⁴ cells/well, with a final volume of 100 μL, and cultured in a 37°C CO ₂ cell culture incubator;

(2) The next day, dilute HIV-1 to 200 TCID ₅₀ /mL;

(3) The purified target protein was diluted to 9 μg/mL as the initial dilution gradient, and then diluted backwards for 10 gradients with a dilution factor of 3, with the 12th column being complete culture medium;

(4) Mix 60 μL of protein or antibody dilution solution with 60 μL of HIV-1 dilution solution and incubate them together in a 37°C CO ₂ cell culture incubator for 1 h. Perform 3 replicates for each experimental well.

(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;

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

(7) Centrifuge at 1000 rpm for 5 min at room temperature and use a pipette to take 50 μL of the supernatant into each well of a 96-well white plate;

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

(9) Data were analyzed. The inhibition rate at each concentration was calculated with the well without protein added as 0% inhibition rate. The _IC50 value was calculated using GraphPad Prism 8.0.

The neutralization test results for HIV-1 strains NL4-3 and BaL are shown in Table 1 (①～④ correspond to protein sequences SEQ ID NO.1～4):

Table 1

Example 4: Neutralizing activity of the recombinant protein of the present invention against different virus strains

Using the method of Example 3, the neutralizing activity of multiple different strains was evaluated, and the specific results are shown in Table 2.

Table 2

Note: a. IC ₅₀ , 50% inhibition concentration, unit nM, molar concentration is calculated based on the molecular weight of protein monomer (② and ③: 170 kDa; CLD (fusion protein with only CD ₄ and DC-SIGN): 60 kDa).

b.No, no inhibitory effect.

c.ND, not tested.

Example 5: Evaluation of HIV-1 neutralization effect using human foreskin tissue blocks

(1) Washing human foreskin tissue several times with serum-free RMPI-1640 medium;

(2) Carefully remove the fat tissue and cut it into tissue blocks of approximately 3 mm × 3 mm;

(3) Culture in a 48-well plate, one tissue per well, add 500 μL RMPI-1640 complete medium to each well, add 50 μg/mL gentamicin, and culture in a 37°C CO ₂ cell culture incubator.

(4) Dilute the purified protein to a final concentration of 100 μg/mL using RPMI-1640 complete medium, and dilute HIV-1 to 2000 TCID ₅₀ /mL using RPMI-1640 complete medium;

(5) Mix 250 μL of protein dilution solution or RPMI-1640 complete medium with 250 μL of HIV-1 dilution solution and incubate them together in a 37°C cell culture incubator for 1 h. Perform 3 replicates for each experimental well.

(6) Add 500 μL of the incubated mixed solution to the tissue block and culture in a cell culture incubator at 37°C for 1 h;

(7) Wash five times with 500 μL/well PBS buffer;

(8) Remove PBS, add 500 μL of RPMI-1640 complete medium containing gentamicin (final concentration 50 μg/mL), and culture in a cell culture incubator at 37°C;

(9) 7 days after infection, take 100 μL of culture medium, add TritonX-100 (final concentration 1%), incubate in a cell culture incubator at 37°C for 1 h, and detect the p24 content in the supernatant by ELISA. After each sample, add 100 μL of fresh RPMI-1640 complete medium containing gentamicin (final concentration 50 μg/mL) to the culture plate.

The specific neutralization results are shown in Figure 1, (A) is the concentration of HIV-1 p24 in the culture medium of the inner plate tissue of the human foreskin in different treatment groups. HIV-1 p24 is the main structural protein of HIV-1 virus particles. The detection of p24 can represent the virus content. The higher the p24 concentration, the more HIV-1. Taking the culture medium without antiviral protein as the control, it can be seen that the p24 content of ②, ③ and ④ is significantly lower than that of the control group.

(B) is the inhibition rate of ②, ③ and ④ proteins against BaL in human foreskin inner plate tissue. The inhibition rate of ②, ③ and ④ proteins was calculated with the control group of culture medium without protein added as 0% inhibition rate.

Claims (10)

A recombinant protein comprising: 1) D1D2 domain of _CD4 ; 2) ND and CRD domains of DC-SIGN; and 3) IgG1 Fc segment. The recombinant protein according to claim 1, wherein the recombinant protein is 1), 3), and 2) from the N-terminus to the C-terminus. The recombinant protein according to claim 1, wherein the recombinant protein further comprises a linker. The recombinant protein according to claim 1, wherein the recombinant protein is a sequence as shown in SEQ ID NO.1~4. An isolated nucleic acid molecule comprises a nucleotide sequence encoding the recombinant protein according to any one of claims 1 to 4. A vector comprising the isolated nucleic acid molecule according to claim 5. A host cell comprising the isolated nucleic acid molecule of claim 5 or the vector of claim 6. A method for preparing a recombinant protein, comprising culturing the host cell according to claim 7 under conditions allowing expression of the recombinant protein, and recovering the recombinant protein from the culture of the cultured host cell. Use of the recombinant protein according to any one of claims 1 to 4 in the preparation of a drug for resisting HIV-1 infection. A pharmaceutical composition comprising The recombinant protein according to any one of claims 1 to 4; and Pharmaceutically acceptable carriers and/or excipients.