WO2014206336A1

WO2014206336A1 - A hiv-1 fusion inhibitor with long half-life

Info

Publication number: WO2014206336A1
Application number: PCT/CN2014/080953
Authority: WO
Inventors: Shibo Jiang; Lu LU; Wei Xu; Rui Wang; Jin Huang
Original assignee: Fudan University; Beijing Prosperous Biopharm Co., Ltd.
Priority date: 2013-06-28
Filing date: 2014-06-27
Publication date: 2014-12-31
Also published as: CN103755810A; CN103333255A; CN103755810B

Abstract

The present invention provides a long-effect membrane-fusion anti-HIV1 macromolecule comprising two or three parts. The first and the second parts are anti-HIV1 membrane-fusion peptides and an antibody mimetic capable of binding serum albumin, respectively. The third part, a linker molecule, can be added between the first part and the second part to improve the macromolecule" s anti-HIV1 activity. The macromolecule has long blocking effect on the virus-mediated membrane fusion.

Description

A HIV-1 fusion inhibitor with long half-life

Priority Information:

This application claims priority to Chinese application 201310264732.7 filed on June 28, 2013 and Chinese application 201310655778.1 filed on December 6, 2013.

Technical Field:

This application relates to inhibitors of human immunodeficiency virus (HIV), especially long-effect membrane-fusion polypeptides and methods of designing such polypeptides.

Background

HIV (Human Immunodeficiency Virus) is the virus that causes AIDS (Acquired Immune Deficiency Syndrome). Lack of effective drugs and vaccines against HIV infection is one of the most difficult problems in the world. To date, drugs treating HIV infection can be divided into four major classes: reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors and HIV fusion inhibitors. These drugs interfere or block different processes of virus production to ultimately inhibit HIV virus infection. HIV fusion inhibitor is the newest one among the four classes.

Most HIV fusion inhibitors are peptides or proteins. They rapidly degrade after being administered into a subject. Some traditional half-life extending technology can resolve the degradation problem to a certain extent.

However, traditional half-life extension technologies (e.g., PEG modification, serum albumin/Fc fusion) have to introduce an unrelated foreign group 10 times larger than the HIV inhibitor themselves, resulting in the inhibitors losing activities, yet still only achieving limited improvement in half-life. Summary

The present application relates to a macromolecule, comprising a first part and a second part, wherein the first part is a peptide or protein with biological function, wherein the sequence of the first part is, or is at least 50% homologous to, one of these sequences: (a) SEQ ID NO: 1, (b) SEQ ID NO: 5, and (c) SEQ ID NO: 6; wherein the second part is a serum albumin targeting peptide or protein, where the sequence of the second part is, or is at least 50% homologous to the sequence of SEQ ID NO: 3.

In one embodiment, the macromolecule further comprises a linker molecule as a third part between the first part and the second part, wherein the molecular weight of the linker molecule is between 300 and 5,500 kilo Dalton.

In another embodiment, the first part, the second part and the third part are fused together via the form of a fusion protein, or via conjugation.

In yet another embodiment, the linker molecule is a non-peptide.

In yet another embodiment, the linker molecule is polyethylene glycol, polypropylene glycol, (ethylene / propylene) copolymer glycol, polyoxyethylene, polyurethane, polyphosphazene, polysaccharides, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyl ethyl ether, polypropylene amides, polypropylene, a cyano group, a lipid polymer, chitin, hyaluronic acid, and heparin, or a combination thereof.

In yet another embodiment, the linker molecule is a peptide, wherein the linker molecule is composed of natural amino acids or non-natural amino acids.

In yet another embodiment, the linker molecule is composed of natural amino acids.

In yet another embodiment, the natural amino acids are those capable of forming protein.

In yet another embodiment, the natural amino acids are those coded by genetic codes.

In yet another embodiment, the sequence of the linker molecule is, or at least 50% homologous to, the sequence of SEQ ID NO: 2. In yet another embodiment, the sequence of the linker molecule is at least 60% homologous to the sequence of SEQ ID NO: 2.

In yet another embodiment, the sequence of the linker molecule is at least 70% homologous to the sequence of SEQ ID NO: 2.

In yet another embodiment, the sequence of the linker molecule is at least 80% homologous to the sequence of SEQ ID NO: 2.

In yet another embodiment, the sequence of the linker molecule is at least 90% homologous to the sequence of SEQ ID NO: 2.

In yet another embodiment, the sequence of the linker molecule is at least 95% homologous to the sequence of SEQ ID NO: 2.

In yet another embodiment, the sequence of the linker molecule is at least 99% homologous to the sequence of SEQ ID NO: 2.

The present application also relates to a macromolecule, wherein the first part, the peptide or protein with biological function, is at least 60% homologous to one of these sequences: (a) SEQ ID NO: 1, (b) SEQ ID NO: 5, and (c) SEQ ID NO: 6; wherein the second part, the serum albumin targeting peptide or protein, is at least 60% homologous to the sequence of SEQ ID NO: 3.

In one embodiment, the first part, the peptide or protein with biological function, is at least 70% homologous to one of one of these sequences: (a) SEQ ID NO: 1, (b) SEQ ID NO: 5, and (c) SEQ ID NO: 6; wherein the second part, the serum albumin targeting peptide or protein, is at least 70% homologous to the sequence of SEQ ID NO: 3.

In another embodiment, the first part, the peptide or protein with biological function, is at least 80% homologous to one of one of these sequences: (a) SEQ ID NO: 1, (b) SEQ ID NO: 5, and (c) SEQ ID NO: 6; wherein the second part, the serum albumin targeting peptide or protein, is at least 80% homologous to the sequence of SEQ ID NO: 3.

In yet another embodiment, the first part, the peptide or protein with biological function, is at least 90% homologous to one of one of these sequences: (a) SEQ ID NO: 1, (b) SEQ ID NO: 5, and (c) SEQ ID NO: 6; wherein the second part, the serum albumin targeting peptide or protein, is at least 90% homologous to the sequence of SEQ ID NO: 3.

In yet another embodiment, the first part, the peptide or protein with biological function, is at least 95% homologous to one of one of these sequences: (a) SEQ ID NO: 1, (b) SEQ ID NO: 5, and (c) SEQ ID NO: 6; wherein the second part, the serum albumin targeting peptide or protein, is at least 95% homologous to the sequence of SEQ ID NO: 3.

In yet another embodiment, the first part, the peptide or protein with biological function, is at least 99% homologous to one of one of these sequences: (a) SEQ ID NO: 1, (b) SEQ ID NO: 5, and (c) SEQ ID NO: 6; wherein the second part, the serum albumin targeting peptide or protein, is at least 99% homologous to the sequence of SEQ ID NO: 3.

The present application also relates to an isolated nucleic acid, wherein the isolated nucleic acid encodes for the peptide or protein in the macromolecule described above.

The present application also relates to an expression vector, comprising the nucleic acid that encodes for the peptide or protein in the macromolecule described above.

The present application also relates to an expression vector, wherein the expression vector is capable of expressing the peptide or protein in the macromolecule described above.

The present application also relates to a pharmaceutical composition or vaccine, comprising the macromolecule described above, or the nucleic acid described above, or the expression vector described above.

Brief Description of Drawings

FIG. l shows SDS-PAGE electrophoresis result of ABT and AB proteins.

FIG.2 shows the activity of ABT and AB in affecting the N36 C34 six-helix bundle formation, demonstrating that ABT (not AB) effectively inhibits the formation of six-helix bundle.

FIG.2A shows utilizing FN-PAGE to measure ABT and AB's ability to form six-helix with N36.

FIG.2B shows the inhibitory effect of ABT, AB and CP38 on six-helix formation.

FIG.3 via p24-based ELISA result shows the inhibitory effect of ABT, AB and

CP38 on HIV-1 IIIB infection by using MT-2 cells, demonstrating that ABT and

CP38 (but not AB) efficiently inhibits HIV -1 IIIB infection.

FIG.4 via p24-based ELISA result shows the inhibitory effect of ABT, AB and

CP38 on HIV-1 Bal infection, demonstrating that the IC50 values of ABT, CP38 and T20 were 11.85 nM, 3.72nM and 8.85 nM, respectively.

FIG.5 shows the PK curves of ABT, CP38 and T20 in SD rats, demonstrating that the half-life of ABT (32.62 hours) is much longer than those of CP38 and T20

(10.47 hours and 3.80 hours, respectively).

FIG.6 shows the serum concentrations of ABT, CP38 and T20 in rats by ELISA method.

Detailed Descriptions

This application relates to a long-acting HIV fusion inhibitor macromolecule. The macromolecule includes two parts, one part for inhibiting HIB infection and the other parts for extending its half-life.

The part of the macromolecule for inhibiting HIV infection can be one or more peptides or proteins. Such inhibitor peptides or proteins can disrupt or block one of steps (e.g., membrane fusion step) during the entry of HIV into the host cells. In addition to membrane fusion step, the inhibitor peptides or proteins or other agents can also interfere with other steps during HIV entry into host cells. For example, clinical stage drugs can inhibit reverse transcriptase, protease and integrase, to achieve the anti-HIV effect. These drugs target different steps of the HIV entry and replication process. HIV fusion inhibitor is a new class HIV drug that achieve its anti-HIV effect via inhibiting HIV membrane fusion process. Human immunodeficiency vims type I (HIV-1) invades its target cells by a glycoprotein-mediated membrane fusion process. During this process, the HIV envelope glycoprotein surface subunit gpl20 is responsible for the recognition of and binding to receptors, while the transmembrane subunit gp41 mediates fusion between the virus envelope and host cell's membrane. The two heptad repeat region on gp41 (HR1 and HR2) play important roles in this process. When HIV-1 infects its target cells, gp41 undergoes a series of conformational changes and finally forms a hairpin trimer (also called a six-helix bundle) structure. In the hairpin trimer structure three HR1 regions of the gp41 form a central trimeric coiled coil, while three HR2 regions pack as antiparallel helices that fit into the hydrophobic grooves of the HR1 central trimeric coiled coil. It is believed that there is a hairpin precursor intermediate state before the final hairpin formation (and while the HR1 and HR2 regions are exposed). At this intermediate state the HR1 and HR2 are capable of binding exogenous HR1, HR2 or similar peptides. Binding to exogenous HR1 and HR2 causes the failure of intermediate state gp41 indigenous HR1 and HR2 to bind to each other, thereby results in the failure of gp41 to transform to the final hairpin trimer structure, which ultimately inhibits the membrane fusion.

T20 (brand name: Fuzeon) is the first FDA-approved membrane fusion inhibitor. It is currently used as a last resort to treat HIV-infected patients who do not respond well to other antiretroviral drugs. T20 contains 36 amino acids, including seven repeat binding domain (HBD) and a tryptophan-rich region (TRD). As the first-generation HIV membrane fusion inhibitor, T20 has high inhibitory activities against different sub strains of HIV-1. However, T20 is limited by its short half-life and occurrence of resistant strains.

A third-generation HIV membrane fusion inhibitor, CP38, contains 38 amino acids. It is based on the T651 peptide, which increases the a-helix and helix-6 stability and has improved pharmacokinetic properties. It is so far the most effective membrane fusion inhibitor, especially against T20-resistant strains. However, for peptides, polypeptides or polynucleotides agents such as CP38, one of the obvious limitations is the short serum half-life. In order to maintain a therapeutically effective concentration, high-frequency and high-dose administrations become necessary. Therefore, developing long half-life HIV inhibitors is a major challenge the field faces today.

In addition to inhibit HIV infection, this application also relates to a serum albumin targeting peptide. In recent years, half-life extension by binding to albumin through small peptides becomes an important area of research. This technology utilizes small artificial modified protein molecules (typically about 100 residues) that have the ability of targeting serum albumin. Fusing such peptides with peptide drugs in need of longer half-life does not cause significant reduction in the peptide drugs' activity. When entering into the circulation system most of the fusion proteins bind with serum albumin with a small amount of the fusion proteins remaining free. As such the fusion proteins avoid degradation (and subsequent excretion) by reversibly binding to serum albumin (half-life 19-20 days). As the free-state fusion proteins being degraded and leave the circulation system, serum-albumin-bound fusion proteins dissociate with the serum albumin and become free-state, such that the concentration of the free fusion protein in the blood is maintained, thereby making the fusion protein a long-acting drug. Some examples of the artificial targeting prototype proteins are Staphylococcus aureus protein A domain (US5831012, EP0739353), human fibronectin (US6818418, EP 1266025). However, techniques in these examples do not utilize the latest antibody mimetic technology to take advantage of the structural information in the template proteins. That is, these techniques do not actively and systematically assess the ability of a template protein to produce antibody mimetic. The serum-albumin-targeting peptides or proteins disclosed in the present application utilizes the latest antibody mimetic technology, fully take advantage the structural information of the template protein itself as well its family of proteins, to arrive at a most effective serum-albumin targeting protein or peptide.

The macromolecule disclosed in the present application can also include a linker molecule. The main purpose of the linker is to spatially separate the two parts discussed above (the part that inhibits HIV and the part that targets serum albumin) to achieve optimal biological effect. Because the role of the linker is only to separate the two parts of the fusion protein, chemical composition of the linker is not as important. On the other hand, the size of the linker impacts the separating effect (and ultimately the biological functions of the macromolecule). Therefore, the linker can be a non-peptide or s peptide. Non-peptide linkers can be natural or non-natural. For instance, non-peptide linkers can be (but not limited to) polyethylene glycol, polypropylene glycol, (ethylene/propylene) copolymer glycol, polyoxyethylene, polyurethane, polyphosphazene, polysaccharides, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyl ethyl ether, polyacrylamide, polypropylene, polycyanoacrylate, lipid polymer, chitin, hyaluronic acid and heparin. Peptide linkers can be any amino acids, natural and non-natural. The amino acids can be D or L amino acids. The amino acids include those that are involved in forming proteins and those that are not. Moreover, the amino acids can be those that are coded by genetic codes and those that are not.

Example 1

1. Cloning and Expression of ABT

1.1 Materials:

PCR reagents: PrimeSTARDNAPolymerase(Takara) lOXbuffer (Mg+)(Takara) dNTP(Takara);

Sterile water: High-pressure deionized water ;

PCR primers: synthesized by Shanghai Jie Lee Corporation ;

Plasmids: PHFT plasmid provided by Beijing Prosperous Biopharm Company.

PGEX-6P-1MD1.1-L35-CP38 plasmids were constructed by the inventors.

Restriction Enzymes: BamHI, Xho and Bglll;

T4 Ligase: purchased from Takara Company;

30% Bis-Acr polyacrylamide gel: purchased from Bio-Rad company

Ni purification column: Purchased from Qiagen

Competent E. coli FIB 101 and BL21 (DE) 3 : purchased from Beijing Tiangen Company;

Other chemical reagents were of analytical grade. Experimental Steps:

1.2.1 Cloning and construction of ABT

To get long half-life fusion peptide ABT (SEQIDNO: 4), enzyme stitching PCR method is used. Two peptides (SEQ ID NO: 1 and SEQ ID NO: 3) are ligated together via the common Bglll restriction sites at ends of the two peptides. In order not to affect the activity of these two peptides, an effective linker sequence (SEQ ID NO: 2) was added between the two peptides.

Nucleic acid primers for AB gene:

AB-BamHI: 5'-CGCGGATCCGTTTCTTCTGTGATG-3 (SEQIDNO: 12) AB-Bgl 11: 5'-GGAAGATCTGGTACGGTAGTTAATC-3 '(SEQ ID NO: 13).

Nucleic acid primers for L35-CP38 gene (SEQ ID NO: 1 and SEQ ID NO: 2) are: 5'-GGAAGATCTGGAGGAGGAGGAAG-3 '(SEQ ID NO: 14)

L35-CP38-XhoI: 5'-CGGCTCGAGCTATAATTCCCTTA AG-3 ' (SEQ ID NO: 15)

AB fragment was cloned by using AB-PHFT plasmid as a template as well as AB-BamHI and AB-Bglll as primers. L35-CP38 fragment was cloned using PGEX-6P-1MD1.1-L35-CP38 plasmid as a template as well as

L35-CP38-Bglll and L35-CP38 -Xhol as primers.

AB and L35-CP38 fragments are recovered and purified with gel purification kit. Concentration of the recovered/purified nucleic acids is measured by Nanodrop. The recovered/purified nucleic acid fragments were digested by Bgl II . Total reaction system has a volume of 20 μΐ. The reaction system contained 2 μΐ of 10X restriction buffer, 1 μΐ of Bgl II and 17 μΐ of nucleic acid fragments. After overnight incubation at 37 ^°C, nucleic acid fragments resulting from enzyme digestion is recovered with PCR product recovering kit. The nucleic acid fragments are ligated with T4 DNA Ligase overnight at 4 ^°C . The ligated nucleic acid fragments are amplified via PCR, with 2 μΐ ligated product as template, AB-BamHI and L35-CP38-XhoI as primers. The PCR product of nucleic acid sequence (SEQ ID NO: 4) was purified with DNA agarose gel recovery kit. The PCR product and the plasmid AB-PHFT are then digested with BamHI and Xhol. The digestion is run overnight at 37 ^°C . Digested PCR product and plasmid AB-PHFT are recovered with gel purification kit. They are then ligated with T4 ligase overnight at 4 ^°C . The next day 5 μΐ of ligation products was added to competent E.Coli HB 101 for transformation. The competent E.Coli was incubated for 16 to 20 hours at 37 ^°C . Positive colonies are picked and further identified by PCR method. The sequences of the positive clones were confirmed by direct DNA sequencing. The final product is named ABT-PHFT.

1.2.2 Expression of ABT protein and human fibronectin (AB)

The sequence-confirmed plasmids ABT-PHFT and sequence-confirmed AB- PHFT were transformed into the expression strain E. coli BL21 (DE3). After incubation of 16 to 20 hours, 4-5 colonies were selected and added to LB medium with Kanamycin and incubated overnight. The following day, 16 ml of the overnight bacteria culture was added to 500 ml LB medium for incubation at 30^°C for about 4 hours at 220ipm/min speed. When the OD value reached 0.6-0.8, the protein expression was induced with IPTG at 0.2 mm/ml, and kept at 16 ^°C for about 12 hours for continued induction. The bacteria cells were collected by centrifugation at 4500rpm/min. The collected bacteria cells were re-suspended with 20 ml of PBS and centrifuged again. The supernatant is discarded. The pellet bacteria cells were stored at -80 ^°C .

The proteins were purified with Ni Sepharose column as follows:

(1) The frozen pellets were thawed in room temperature and re-suspended in 30 ml of binding buffer (20 mM sodium phosphate, pH7.4,; 500 mM sodium chloride; 15 mM imidazole) and vortexed for 10 minutes. 150 ml 10% Triton-PBS was then added to the re-suspended cells. The cells were mixed well and put on ice.

(2) ultra-sonication of the cells. Sonication conditions are as follows: ultrasonic power 300W, working time 3 s, interval 5s, totaling ultrasound time 30min. 12000rpm/min centrifuged for 20 minutes. .

(3) The supernatant after centrifugation was filtered by 0.45μπι membrane. Pre-balanced Ni purification column is then provided. 1 ml of Ni column material is mixed with the supernatant. The mixture is then vortexed for another 45 minutes on ice.

(4) The mixture was added to the column while Ni agarose gradually settle down. The supernatant was passed through the column at least twice so that the protein sufficiently bound with Ni column.

(5) The column was washed with washing buffer (50mM NaH₂P04, pH8.0, 300mMNaCI, and 60 mM imidazole). The volume of the washing buffer was approximately 40 ml.

(6) The column was eluted with striping buffer (50mM NaH₂P04, pH8.0, 300mMNaCI, 300mM imidazole). Every fraction of the elution was collected. The elution was dialyzed overnight at 4 ° C with PBS, and subsequently frozen in -80 ^°C freezer.

(7) After the purify procedure, the column was refreshed with about 5ml 6M guanidine to rid any proteins off the column. After equilibrium with binding buffer, 20% ethanol solution was added to the column which was then stored at 4^°C .

Figure 1 shows the AB and ABT electrophoresis bands. Example 2

2. FN-PAGE (Fluorescence native polyacrylamide gel electrophoresis) to detect ABT and AB inhibition of six bundle formation

2.1 Materials Non-denaturing polyacrylamide gel (PAGE) gel electrophoresis kit: purchased from Beijing Tianenze company; N36, C34, FAM-C34 peptide synthesizer by the

Biosystems 433A protein synthesis

2.2 Experimental procedure

(1) Preparation of 18% separating gel, 5% stacking gel.

(2) Preparation of N36, F-C34, ABT, AB, N36+ABT, N36+AB and other

polypeptides, each polypeptide with a final concentration of 40um, 37^°C for 30 minutes in the dark at room temperature under conditions of a voltage 125 V.

Electrophoresis for 2 hours.

(3) Imaged with Fluorchem8800 (UV).

(4) PAGE gel stained with Coomassie blue.

As shown in FIG.2, AB peptide did not compete with C34 or N36 for 6-HB formation, while ABT competed with C34 or N36 for 6-HB formation.

Example 3

Measurement of Anti-HIV-1 Activity against Laboratory-adapted HIV-1 Strains and primary HIV-1 isolates

3.1 Materials

Cells: MT-2 cells, M7 cell culture: 1640, 1640+10% FBS

Plates: 96-well flat plate (corning), 96 well round bottom plates (corning) Lysates: 5% TritonX-100

Virus: Laboratory strains HIV-1 IIIB, HIV-1 Bal viruses and various HIV-1 primary strains.

3.2 Experimental Steps:

(1) ABT, AB, CP38 and T20 polypeptide proteins were series diluted in 96 well plates, while setting positive controls (wells without polypeptide proteins) and negative controls (including cell control and virus control). (2) Sufficiently mix the vims which was thawed from -80 ^°C refrigerator. Added 100 times TCID50 (50% tissue culture infective dose) virus into the wells.

(3) The MT-2 or M7 cells were adjusted to lxlO⁵ cells / ml, 100 μΐ cell was loaded per well. Incubation was performed at 37^°C with 5% C0₂, overnight.

(4) Next day, 150 μΐ medium was withdrawn and 150 μΐ fresh 1640+10%FBS medium was added. Incubation continued for four days. 50 μΐ cell supernatant was withdrawn and loaded to 96-well plates five days post-infection. 5% Triton X-100 was added to lyse the cells at 4^°C overnight.

(5) p24 antigen content was tested using enzyme-linked immunosorbent assay (ELISA). Chromogenic reaction was done with TMS. OD450 was read with Ultra386 (Tecan).

As shown in Table 1, compared with CP38 and T20, ABT had a similar or better IC50 (i.e., inhibitory activity) against the cellular entry by HIV. Moreover, as shown in Fig.3, inhibitory activities of ABT, CP38 and T20 is much higher than that of AB (i.e., the antibody mimic template protein AB in the fusion protein ABT). ABT, CP38 and T20 inhibited HIV-1 IIIB with mean IC50 values at 47.55nM, 38.9nM and 34.06nM, respectively. Similarly, ABT, CP38 and T20 had much higher inhibitory activity against HIV-1 Bal than that of protein AB (FIG.4).

Table 1. Inhibitory activity of AB, ABT, CP38 and T20 on infection by primary HIV-1 isolates.

IC₅₀(nM)

AB ABT CP38 T20

92UG029(A, X4) >1000 4.3±0.0 4.6±0.4 17.5±3.3

94US_33931N(B, R5) >1000 51.6±0.3 11.5±1.6 37.8±9.3

93IN101(C, R5) >1000 19.5±3.3 8.1±0.1 43.3±11.1

92UG024(D, X4) >1000 26.1±7.3 13.1±0.6 44.1±1.6

92TH009(A/E, R5) >1000 25.7±7.8 20.5±0.4 50.7±9.7

NP1525(A/E, X4 and R5) >1000 6.5±9.1 7.6±5.5 51.5±1.1

93/BR/020(F, X4 and R5) >1000 31.2±0.1 17.2±1.8 35.1±9.3

BCF02(O, R5) >1000 52.5±4.6 52.6±4.8 34.3±4.3 Example 4

4. Detection of inhibition of 6-HB formation by ELISA

4.1 Materials

Coating buffer: 0.1M Tris-cl pH8.8

Antibodies: Rabbit anti-NY364, monoclonal antibody NC-1, rabbit anti-mouse HRP (DAKO)

Peptide: N36, C34, ABT, AB, CP38

(1) Coating was done with ΙΟΟμΙ rabbit anti-gp41 antibody (NY364) at 2 ug/ml in pH 8.8 Tris-Hcl buffer at 4^°C overnight.

(2) Blocking: The plate was blocked with 200 μΐ blocking buffer (2% glutin in pH 7.2 PBS) at 37°C for 1 h and washed 3 times.

(3) 30 μΐ series dilution peptides and control peptides were added to the wells. 2 μΜ 30 μΐ N36 was then added to each well. Incubation was done at 37 ^°C for 30 minutes. 60 μΐ 1 μΜ C34 was then added and incubation continued at 37^°C for 30 minutes.

(4) Take 100 μΐ mixture to the well and incubated at 37^°C for 1 hour. Washed 3 times.

(5) Added lug/ml monoclonal antibody NC-1 100 μΐ per well and incubated at 37^°C for 1 hour. Washed 3 times.

(6) Added 1 :3000 diluted rabbit anti-mouse IgG-HRP 100 μΐ per well and incubated at 37^°C for 1 hour. Washed 6 times.

(7) Added 3,3 ', 5,5' - tetramethylbenzidine (TMB) solution 100 μΐ per well.

Chromogenic reaction was done for 3-10 minutes.

(8) Stop the reaction with 1M H₂S04 (50μ1 per well).

As shown in Fig. 2B, fusion protein ABT participated in the interaction between HRl and HR2 in forming the six helices. In other words, ABT and CP38 had a similar activity in interrupting C34 to form 6-HB with N36, while AB does not have such activity. Example 5

Pharmacokinetics studies with rat

5.1 Materials: SD-rats, T20, CP38, ABT and other peptide drugs

MT-2 cells, HIV-IIIB virus,

P24 detection reagents.

Experiment steps:

SD rats received intravenous injections of each peptide.

(1) Before intravenous injection, serum was taken from rats as a negative control.

(2) T20, Tl 144 and FLT at the same molar concentration (1.0, 1.0, and 4.9 mg/kg), respectively.

(2) T20, Tl 144 and FLT were administered via intravenous injection at the same molar concentration (234μΜ), i.e., T20 (lmg/ml/kg), CP38 (lmg/ml/kg), ABT (4.9mg/ml/kg), respectively.

(3) 200 μΐ Serum samples were collected via orbital blood collection method at 0, 0.5, 1.5, 3, 6, 9, 12, 24, 48, 72, 96 and 120 hours after intravenous injection.

(4) Keep in room temperature for 2 hours, serum was separated and frozen at -80 ^°C freezer.

(5) Serum complement and enzymes were inactivated at 56 ^°C for 1 hour. Serum was diluted 1 :80, 1 : 160, 1 :320, 1 :640, 1 : 1280, 1 :2560, 1 :5120, and 1 : 10240. Virus inhibition experiments were performed as in Example 3. The peptides' drug effects were determined via measuring P24 level in the serum.

As described above, binding with serum albumin can substantially improve the half-life of peptides or proteins. The half-life of ABT, CP38 and T20 were compared. As shown in Fig.5, the half-life of ABT is much longer than CP38 and T20. The serum half-lives of ABT, CP38 and T20 (ti_/2) in SD rats was 32.62 hours, 10.47 hours and 3.80 hours, respectively. Because human albumin half-life (about 19 to 21 days) is much longer than rat albumin half-life (about 1.2 day), in human serum ABT is likely to have an even longer half-life than CP38 and T20.

Example 6 Serum concentration detection by ELISA method

6.1 Materials: T20, CP38, ABT and other peptide drugs

Rabbit anti-T20 antibody, rabbit anti-CP38 antibody, rabbit anti ABT antibody, mouse anti-T20 antibody, mouse anti-CP38 antibody, mouse anti ABT antibody, TMB solution and polystyrene plates (Corning, highbinding)

(1) T20 ELISA Test Method

Coating with NHS chromatography purified ^g/ml rabbit anti-T20 antibody, 50μ1 per well, 4°C overnight. Washing 3 times with PBST and then blocking with 2% milk-PBS for T20, 37°C, 2 hours. Washing 3 times with PBST, adding 1 :20 dilution serum samples for 45 minutes at 37°C. The ELISA plates are washed three times. Addingl : 1200 dilution mouse anti-T20 antibody 50μ1 per well and incubating for 45 minutes and washing 3 times with PBST, each time lasting 5 minutes. Lastly, 1 :3000 dilution Rabbit anti mouse-HRP antibody (i.e., HRP labeled secondary antibody) is added and incubated for another 45 minutes. Washing 5 times with PBST, each time lasting 5 minutes. TMB was used as substrate solution. After 5 minutes, terminated the reaction by adding 2M H₂S04 50 μΐ. Absorbance at 450 nm (A450) was measured using an ELISA reader.

(2) CP38 ELISA Test Method

Coating with NHS chromatography purified ^g/ml rabbit anti- CP38 antibody, 50μ1 per well, 4°C overnight. Washing 3 times with PBST and then blocking with 2% milk-PBS for CP38, 37°C, 2 hours. Washing 3 times with PBST, adding 1 :20 dilution serum samples for 45 minutes at 37°C. The ELISA plates are washed three times. Addingl : 1000 dilution mouse anti- CP38 antibody 50μ1 per well and incubating for 45 minutes at 37°C and washing 3 times with PBST. Lastly, 1 :3000 dilution Rabbit anti mouse-HRP antibody (i.e., HRP labeled secondary antibody) is added and incubated for another 45 minutes at 37°C. Washing 5 times with PBST, each time lasting 5 minutes. TMB was used as substrate solution. After 5 minutes, terminated the reaction by adding 2M H₂S04 50 μΐ. Absorbance at 450 nm (A450) was measured using an ELISA reader. (3) ABT ELISA Test Method

Coating with purified 5μ /ηι1 rabbit anti- ABT antibody, 50μ1 per well, 4°C overnight. Washing 3 times with PBST, each time lasting 5 minutes. Blocking with 0.5 % geltin-PBS for ABT, 37°C, 2 hours. Washing 3 times with PBST, adding 1 :50 dilution serum samples for 45 minutes at 37°C. Addingl : 1500 dilution mouse anti- ABT antibody 50μ1 per well and incubating for 45 minutes at 37°C and washing 3 times with PBST. Lastly, 1 :3000 dilution Rabbit anti mouse-HRP antibody (i.e., HRP labeled secondary antibody) is added and incubated for another 45 minutes at 37°C. Washing 5 times with PBST. TMB was used as substrate solution. After 5 minutes, terminated the reaction by adding 2M H₂S04 50 μΐ. Absorbance at 450 nm (A450) was measured using an ELISA reader.

As shown in FIG.6, the half-lives of ABT (fusion protein), CP38 and T20 in rats are 27.097 hours, 7.479 hours and 1.372 hours, respectively. Compared with CP38 and T20, the half-life of ABT is substantially improved. In other words, the non-CP38 domain in ABT antibody fusion protein plays an important role in stabilizing the fusion protein.

Claims

A HIV-1 fusion inhibitor with long half-life What is claimed is:

1. A macromolecule, comprising a first part and a second part, wherein the first part is a peptide or protein with biological function, wherein the sequence of the first part is, or is at least 50% homologous to, one of these sequences: (a) SEQ ID NO: 1, (b) SEQ ID NO: 5, and (c) SEQ ID NO: 6; wherein the second part is a serum albumin targeting peptide or protein, where the sequence of the second part is, or is at least 50% homologous to the sequence of SEQ ID NO: 3.

2. The macromolecule of claim 1, wherein the macromolecule further comprises a linker molecule as a third part between the first part and the second part, wherein the molecular weight of the linker molecule is between 300 and 5,500 kilo Dalton.

3. The macromolecule of claim 2, wherein the first part, the second part and the third part are fused together via the form of a fusion protein, or via conjugation.

4. The macromolecule of claim 2, wherein the linker molecule is a non-peptide.

5. The macromolecule of claim 4, wherein the linker molecule is polyethylene glycol, polypropylene glycol, (ethylene / propylene) copolymer glycol, polyoxyethylene, polyurethane, polyphosphazene, polysaccharides, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyl ethyl ether, polypropylene amides, polypropylene, a cyano group, a lipid polymer, chitin, hyaluronic acid, and heparin, or a combination thereof.

6. The macromolecule of claim 2, wherein the linker molecule is a peptide,

wherein the linker molecule is composed of natural amino acids or non-natural amino acids.

7. The macromolecule of claim 6, wherein the linker molecule is composed of natural amino acids.

8. The macromolecule of claim 7, wherein the natural amino acids are those

capable of forming protein.

9. The macromolecule of claim 8, wherein the natural amino acids are those coded by genetic codes.

10. The macromolecule of claim 9, wherein the sequence of the linker molecule is, or at least 50% homologous to, the sequence of SEQ ID NO: 2.

11. The macromolecule of claim 10, wherein the sequence of the linker molecule is at least 60% homologous to the sequence of SEQ ID NO: 2.

12. The macromolecule of claim 11, wherein the sequence of the linker molecule is at least 70% homologous to the sequence of SEQ ID NO: 2.

13. The macromolecule of claim 12, wherein the sequence of the linker molecule is at least 80% homologous to the sequence of SEQ ID NO: 2.

14. The macromolecule of claim 13, wherein the sequence of the linker molecule is at least 90% homologous to the sequence of SEQ ID NO: 2.

15. The macromolecule of claim 14, wherein the sequence of the linker molecule is at least 95% homologous to the sequence of SEQ ID NO: 2.

16. The macromolecule of claim 15, wherein the sequence of the linker molecule is at least 99% homologous to the sequence of SEQ ID NO: 2.

17. A macromolecule as in any one of claims 1-16, wherein the first part, the

peptide or protein with biological function, is at least 60% homologous to one of these sequences: (a) SEQ ID NO: 1, (b) SEQ ID NO: 5, and (c) SEQ ID NO: 6; wherein the second part, the serum albumin targeting peptide or protein, is at least 60% homologous to the sequence of SEQ ID NO: 3.

18. The macromolecule of claim 17, wherein the first part, the peptide or protein with biological function, is at least 70% homologous to one of one of these sequences: (a) SEQ ID NO: 1, (b) SEQ ID NO: 5, and (c) SEQ ID NO: 6; wherein the second part, the serum albumin targeting peptide or protein, is at least 70% homologous to the sequence of SEQ ID NO: 3.

19. The macromolecule of claim 18, wherein the first part, the peptide or protein with biological function, is at least 80% homologous to one of one of these sequences: (a) SEQ ID NO: 1, (b) SEQ ID NO: 5, and (c) SEQ ID NO: 6; wherein the second part, the serum albumin targeting peptide or protein, is at least 80% homologous to the sequence of SEQ ID NO: 3.

20. The macromolecule of claim 19, wherein the first part, the peptide or protein with biological function, is at least 90% homologous to one of one of these sequences: (a) SEQ ID NO: 1, (b) SEQ ID NO: 5, and (c) SEQ ID NO: 6; wherein the second part, the serum albumin targeting peptide or protein, is at least 90% homologous to the sequence of SEQ ID NO: 3.

21. The macromolecule of claim 20, wherein the first part, the peptide or protein with biological function, is at least 95% homologous to one of one of these sequences: (a) SEQ ID NO: 1, (b) SEQ ID NO: 5, and (c) SEQ ID NO: 6; wherein the second part, the serum albumin targeting peptide or protein, is at least 95% homologous to the sequence of SEQ ID NO: 3.

22. The macromolecule of claim 21, wherein the first part, the peptide or protein with biological function, is at least 99% homologous to one of one of these sequences: (a) SEQ ID NO: 1, (b) SEQ ID NO: 5, and (c) SEQ ID NO: 6; wherein the second part, the serum albumin targeting peptide or protein, is at least 99% homologous to the sequence of SEQ ID NO: 3.

23. An isolated nucleic acid, wherein the isolated nucleic acid encodes for the

peptide or protein in the macromolecule as in any one of claims 1-22.

24. An expression vector, comprising the nucleic acid of claim 23.

25. An expression vector, wherein the expression vector is capable of expressing the peptide or protein in the macromolecule as in any one of claims 1-22.

26. A pharmaceutical composition or vaccine, comprising the macromolecule as in any one of claims 1-22, or the nucleic acid of claim 23, or the expression vector as in any one of claims 24-25.