CN107383190B

CN107383190B - Humanized anti-HIV gp41 specific antibody and application thereof

Info

Publication number: CN107383190B
Application number: CN201710657311.9A
Authority: CN
Inventors: 杨争; 孙泽华; 刘曦; 余卫业; 谭卫国
Original assignee: SHENZHEN CENTER FOR CHRONIC DISEASE
Current assignee: SHENZHEN CENTER FOR CHRONIC DISEASE
Priority date: 2017-08-03
Filing date: 2017-08-03
Publication date: 2020-09-25
Anticipated expiration: 2037-08-03
Also published as: CN107383190A

Abstract

The invention provides a human HIV gp41 specific antibody and application thereof. The invention utilizes single memory B cell sorting and antibody gene direct amplification method to obtain variable region segment of HIV virus gp41 specific antibody, constructs eukaryotic instant expression vector of complete IgG antibody by gene engineering method, and expresses and purifies antibody provided by IgG protein, belonging to fully human monoclonal antibody, the amino acid sequences of light chain variable region and heavy chain variable region are respectively shown in SEQ ID NO.1 and 3. The antibody specifically binds to a membrane-proximal region of HIV gp41 with high affinity, has an EC50 of 0.05 mu g/ml (0.333nM), can neutralize the infection of HIV virus on target cells, can mediate the killing of effector cells mainly comprising NK cells on HIV infected cells, can be used for developing a therapeutic drug for HIV infection, and can also be used for developing an HIV virus antigen detection reagent.

Description

Humanized anti-HIV gp41 specific antibody and application thereof

Technical Field

The invention relates to preparation and application of a human genetic engineering antibody for treatment, in particular to a specific antibody aiming at HIV gp41 and a preparation method and application thereof.

Background

Scientists have focused on HIV vaccine development since the discovery of Human Immunodeficiency Virus (HIV) in 1983. However, despite over 30 years of effort, no effective vaccine against HIV prevention has been marketed to date. One of the most important reasons is that the virus has an ultra-high mutation frequency, thereby putting it under pressure to evade the human immune system. Conventional therapy for HIV generally employs highly active antiretroviral therapy (HAART), also known as cocktail therapy, which involves the combined use of three or more of a reverse transcriptase inhibitor, an integrase inhibitor, a fusion inhibitor, a protease inhibitor, and the like. However, patients need to take medicines for a long time to effectively inhibit viruses, and the toxic and side effects of the medicines cause poor adherence of the patients. Some HIV-infected patients, although they carry the virus, have not progressed to acquired immunodeficiency syndrome (AIDS) even without any intervention, and scientists refer to such patients as "long term non-progressor (LTNP)" or elite suppressor (elitecontroller). Further research finds that the patients usually have high-titer functional antibodies in vivo, and can effectively prevent virus-infected cells and assist effector cells in killing and removing the infected cells. At present, a plurality of clinical trials are underway at home and abroad for HIV monoclonal antibody pharmaceutical preparations with broad-spectrum neutralization activity, and the pharmaceutical preparations show remarkable effects in the aspects of pre-infection prevention and post-infection treatment. The effect of an antibody is not only related to whether it can neutralize the virus, but also to whether the antibody can mediate killing of effector cells against virus-infected cells (ADCC), i.e. achieve clearance of the virus.

Fully human antibodies are currently obtained mainly by antibody library display screening techniques and single B cell sorting techniques. Compared with single cell sorting method, the monoclonal antibody obtained by screening antibody library has greatly reduced probability of belonging to original light and heavy chain pairing, and the original light and heavy chain pairing of the antibody can maximize the function of the antibody. The light and heavy chains of the antibody gene obtained by the single cell sorting method are from original pairing theoretically, so the probability of obtaining the high-efficiency functional antibody by the single cell sorting method is far higher than that of an antibody library display screening method.

Monoclonal antibodies are highly homogeneous immunoglobulins produced by a single B cell clone directed against only a particular epitope. Due to small side effects, the humanized monoclonal antibody is not required to be humanized when being applied to human disease treatment, so that the loss of affinity in the process is avoided. The human monoclonal antibody plays a great role in the prevention and control process of infectious diseases, can neutralize viruses and mediate killing of effector cells to virus infected cells, and the protection effect of the human monoclonal antibody is fully proved in animal infection models of HIV, MERS, dengue fever viruses, hantaviruses, measles viruses, RSV, rabies viruses and the like. In addition, the monoclonal antibody with high affinity and high specificity is also a key component of a rapid detection reagent of pathogen.

Since 2008, single B-cell sorting and antibody gene direct amplification technology has become one of the major approaches to human antibody screening (Tiller et al, Journal of Immunological Methods 329, (2008), 112-124). In 2010, Wu et al (Science 329, (2010)856-861) used a flow cytometric sorting technique to sort single memory B cells with antigen specificity in HIV-infected peripheral blood, further used a reverse transcription PCR technique to directly amplify antibody genes VH and V kappa/V lambda of single cells, and then inserted the above gene fragments into a full-length IgG recombinant vector to perform eukaryotic cell transfection and expression purification, thereby successfully obtaining a famous HIV broad-spectrum neutralizing monoclonal antibody VRC 01. Because the technology can obtain antibody genes and recombinant antibody proteins from single cells in the shortest time and can ensure the original pairing of heavy chains and light chains of the antibodies (the antibody functions are optimal), the single cell sorting and antibody gene direct amplification technology quickly becomes a heavy tool in the field of antibody development. So far, the technology is successfully applied to screening broad-spectrum neutralizing monoclonal antibodies of HIV, influenza, MERS, Ebola, dengue fever and other viruses, and a plurality of high-efficiency antibodies obtained by the technology enter clinical drug research in sequence. The genetic engineering antibody from a single B cell brings new hope and broad prospect for the fields of rapid antigen detection and antibody pharmacy.

Many neutralizing antibodies against HIV have been made abroad such as VRC01, PGT121 and 10E8, etc., and there are many treatments for HIV-infected patients entered clinical studies such as VRC01 (US clinical trial registration No.: NCT01993706) and 3BNC117 (US clinical trial registration No.: NCT 02018510). HIV antibody studies have mostly focused on neutralizing activity of antibodies and little on their mediated cell killing cytotoxicity (ADCC). The only clinically effective HIV vaccine trial to date, late stage study findings of RV 144: high titers of ADCC antibodies in the vaccinated subjects were significantly negatively correlated with infection rates, suggesting an important role for ADCC antibodies in the prevention of HIV infection. Several HIV monoclonal antibodies with ADCC function have been identified internationally, including CD 4-inducible conformation-specific antibody A32, CD 4-binding domain-specific antibody b12, and membrane proximal region (MPER) -specific antibody 2F 5.

Disclosure of Invention

The first objective of the invention is to provide the amino acid sequences of human anti-HIV gp41 specific antibodies and active fragments thereof.

The second object of the present invention is to provide a gene encoding the above antibody or an active fragment thereof.

The third purpose of the invention is to provide the application of the antibody and the active fragment thereof in preparing the medicines for treating or preventing the diseases infected by HIV.

The fourth purpose of the invention is to provide the application of the antibody and the active fragment thereof in preparing HIV diagnosis or detection reagents.

The invention adopts a multicolor fluorescence labeling flow cytometer sorting method to obtain HIV envelope protein antigen specificity memory B cells, utilizes a reverse transcription kit to obtain cDNA of a single B cell, utilizes an antibody specificity primer to amplify antibody gene variable region fragments, utilizes a genetic engineering technology to construct a eukaryotic IgG expression vector, utilizes a cellular engineering technology to obtain a purified target IgG antibody E10 through transient transfection and an antibody purification technology, namely the human HIV gp41 specificity antibody, wherein the antibody light chain variable region amino acid sequence contains an amino acid sequence shown in SEQ ID NO.1, or contains an amino acid sequence which is formed by replacing, deleting or adding one or more amino acids and has the same function and is obtained by the amino acid sequence shown in SEQ ID NO. 1; the heavy chain variable region amino acid sequence contains the amino acid sequence shown in SEQ ID NO.3, or the amino acid sequence with the same function formed by replacing, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 3.

Preferably, the light chain amino acid sequence of the human HIV gp41 specific antibody is shown in SEQ ID NO.5, or the amino acid sequence shown in SEQ ID NO.5 is an amino acid sequence with the same function formed by replacing, deleting or adding one or more amino acids; the heavy chain amino acid sequence is shown as SEQ ID NO.6, or the amino acid sequence shown as SEQ ID NO.6 is formed by replacing, deleting or adding one or more amino acids to form the amino acid sequence with the same function.

The specific antibody can specifically recognize an epitope, and the amino acid sequence of the epitope is shown in SEQ ID NO. 7.

The light chain and heavy chain genes of the HIV-specific antibody E10 of the present invention are derived from HIV-infected patient peripheral blood B cells. The variable region sequence characteristics of the antibody are formed by the combination of three CDR region sequences corresponding to the variable regions of the light chain and the heavy chain and the framework region sequences between the CDR regions, the variable region gene of the light chain of the antibody E10 belongs to the family IGKV3-20, and the heavy chain belongs to the family IGHV 1-69. The function of the antibody protein is determined by the specific nucleotide sequence and the complementary sequence thereof in the complementarity determining regions CDR1, CDR2 and CDR3 of the light chain and heavy chain variable regions of the antibody gene, and 6 corresponding CDR region amino acid sequences form the specific antigen binding region of the antibody, thereby determining the antigen binding characteristic and the anti-HIV functional characteristic of the antibody.

In addition, in consideration of the degeneracy of codons, for example, the gene sequence encoding the variable region of E10 can be modified in the coding region thereof without changing the amino acid sequence to obtain a gene encoding an antibody having the same function. One skilled in the art can artificially synthesize and modify genes according to the codon preference of the host for expressing the antibody so as to improve the expression efficiency of the antibody.

Further, the light chain variable region and the heavy chain variable region of the antibody E10 of the present invention may be recombined to form a Fab antibody of smaller molecular weight or a single chain antibody (SCFV) of smaller molecular weight. Fab antibodies and single chain antibodies also have the property of recognizing HIV surface antigens. The antibody with small molecular weight has strong penetrating power and is easy to enter local tissues or cells to play a role.

The gene encoding Fab antibody and the gene encoding SCFV antibody may be cloned into an expression vector, and the Fab antibody and single-chain antibody (SCFV) may be obtained by transforming a host and inducing expression

Further, the human HIV gp 41-specific antibody of the present invention is a single-chain antibody, Fab antibody, minibody, chimeric antibody, or whole antibody immunoglobulin IgG1, IgG2, IgG4, IgA, IgE, IgM, or IgD.

The invention provides a gene encoding the human HIV gp 41-specific antibody.

Preferably, the nucleotide sequences encoding the light chain variable region and the heavy chain variable region are the nucleotide sequences shown in SEQ ID No.2 and SEQ ID No.4, respectively.

The present invention provides an expression vector containing the above gene.

The invention provides a host bacterium, a host cell or an expression cassette containing the expression vector.

The invention provides a medicament or a detection reagent containing the human HIV gp41 specific antibody.

The invention provides application of the human HIV gp41 specific antibody in preparing a medicine for treating HIV infection.

The invention provides an application of the humanized HIV gp41 specific antibody in preparation of an HIV detection reagent.

Hybridoma cells producing antibodies specific for the human HIV gp41 are also within the scope of the invention.

The invention provides an antigen target combined with the human HIV gp41 specific antibody, and the amino acid sequence of the antigen target is shown as SEQ ID NO. 7.

Further, the invention provides application of the antigen target in preparing HIV detection reagents or diagnostic reagents.

The invention also provides a method for preparing the human HIV gp41 specific antibody, which comprises the steps of obtaining the light and heavy chain variable region gene of the HIV gp41 specific antibody by using a single memory B cell sorting and antibody gene direct amplification method, cloning the light and heavy chain variable region gene into a whole antibody expression vector, and carrying out whole antibody expression on the light and heavy chain variable region gene through an expression system to obtain the whole antibody protein of the light and heavy chain variable region gene, wherein the nucleotide sequence of the light and heavy chain variable region gene of the antibody is shown as SEQ ID NO.2 and SEQ ID NO. 4.

The present invention provides an immunotoxin comprising the human HIV gp41 specific antibody E10 linked to a cytotoxic agent.

The cytotoxic agent includes a chemical molecule, radioisotope, polypeptide or toxin having cell killing or cell death-inducing properties.

The present invention provides a bispecific or multispecific molecule comprising the human HIV gp 41-specific antibody E10 or the antigen-binding site of an antibody as described above.

The functional identification of the obtained IgG antibody E10 is carried out by methods such as SDS-PAGE, ELISA, in-vitro virus neutralization experiments, antibody-dependent cell-mediated cytotoxicity killing experiments and the like, and the result shows that the molecular weight of the human IgG antibody E10 expressed and purified accords with the expectation (see figure 2), and the antibody provided by the invention can be efficiently combined with the gp41 segment of HIV envelope protein, and the EC50 of the affinity of the antibody against bal gp41 is 0.05 mug/ml (0.333nM) (see figure 3). Specifically, in an ELISA experiment for detecting the affinity of the antibody E10 with HIV gp41 protein, when the concentration of the antibody is as low as 0.05 mu g/ml (0.333nM), the OD450 value of the antibody E10 can still reach half of the maximum value, namely 1.6.

The antibody provided by the invention can specifically recognize the Membrane Proximal End (MPER) of HIV gp 41. Through ELISA experiments, the IgGE10 has obvious affinity with gp41 Membrane Proximal End (MPER) short peptide F7 (amino acid sequence: QQELLELDKWANLWNWFDISNWLW), and the affinity with F7-Fc fusion protein is obviously higher than that of a CD4 specific neutralizing antibody b12 when the concentration of E10 is as low as 0.1 mu g/ml (see figure 4). The antibody provided by the invention can be used for developing HIV antigen detection reagents.

The antibody provided by the invention can effectively mediate killing of effector cells (such as NK cells) to HIV infected cells. The antibody provided by the invention can mediate killing (ADCC) of effector cells to an HIV envelope protein stable expression cell line TF228, and the killing percentage is kept above 19% when the concentration of the antibody is more than 0.1 mu g/ml (see figure 5) (reference: Srivastava, V).^#,Z.Yang^#Hung, J.xu, B.ZHEN, and M.Y.Zhang.identification of a dominant antibody-dependent cell-mediated cytoxicity epitopes on the magic pigment antibody of global H1N1 fluorescent virus. journal of virology2013.87: 5831-. The antibodies provided by the invention can be used for treating HIV infected patients.

The antibody provided by the invention has certain HIV virus neutralization capacity. The neutralization activity of E10 against 6 strains of HIV pseudoviruses was tested by a pseudovirus neutralization experiment with TZM-bl AS the target cell (ref: Zhang MY, Borges AR, Ptak RG, Wang Y, Dimitrov AS, Alam SM, Wieczorek L, Bouma P, Fouts T, Jiang S et al: Point and broad neural activity of a single chain antibody fragment against MAbs2010,2(3): 266-274). Wherein the inhibition rate of E10 on GXC44 and JRFL infection of target cells reaches 50% at the concentration of about 100 mug/ml (see figure 6). In general, the neutralizing activity and the broad spectrum of E10 are not as good as other broad-spectrum high-efficiency neutralizing antibodies such as b12 and VRC 01.

The humanized neutralizing HIV gene engineering antibody variable region gene obtained in the said way may be used in expression and production of Fab antibody, single chain antibody gene and full length IgG antibody in prokaryotic cell, eukaryotic cell and any recombinant protein expression system, or in the modification of the said variable region gene to obtain antibody product with high affinity, specific recognition of HIV, neutralizing HIV infection and killing HIV infected cell.

Drawings

FIG. 1 is a schematic diagram of a fully human monoclonal antibody technology platform based on single cell sorting and antibody gene amplification.

FIG. 2 is the electrophoresis chart of the non-denaturing and denaturing SDS-polyacrylamide gel electrophoresis of IgGE10 for detecting the molecular weight and purity of antibody.

FIG. 3 is a graph showing the effect of IgGE10 on affinity detection (ELISA method) of HIV gp 41. In which the gp41-Fc protein was coated at a concentration of 1. mu.g/ml on an ELISA plate and IgGE10 was diluted in a 3-fold gradient starting at 10. mu.g/ml. The concentration of antibody E10 when the OD450 value was half of the highest value was its EC50 value against gp 41. CD4 binding site specific neutralizing antibody IgGb12 served as a negative control. (Log10, ug/ml) indicates the antibody concentration (ug/ml) expressed as a base 10 Log.

FIG. 4 is a graph showing the effect of IgGE10 on affinity detection (ELISA method) of gp41 Membrane Proximal End (MPER) short peptide fusion protein F7-Fc. F7-Fc coated ELISA plates at 1. mu.g/ml, IgGE10 from 50. mu.g/ml initial 3 times gradient dilution. CD4 binding site specific neutralizing antibody IgGb12 served as a negative control. (Log10, ug/ml) indicates the antibody concentration (ug/ml) expressed as a base 10 Log.

FIG. 5 is a graph showing the effect of detecting ADCC activity of IgGE10 against TF228, a cell line stably expressing HIV envelope protein. Healthy human peripheral blood mononuclear cells as effector cells; the flow cytometer detects the killing status of the target cells. PKH67 and 7AAD double positive cells were circled and analyzed for differences in this ratio for different treatments. The treatment without antibody was negative control, and the TF228 cells treated at 85 ℃ for 5 minutes were positive control for cell killing. IgG 2F5 is an MPER specific neutralizing antibody and is known to have strong ADCC function, and IgG mz16 is an HIV neutralizing antibody and is used as a negative control of ADCC. (Log10, ug/ml) indicates the antibody concentration (ug/ml) expressed as a base 10 Log.

FIG. 6 is a graph showing the effect of detecting the neutralizing activity of IgGE10 against the pseudovirus 6. The neutralization activity is expressed in percent inhibition of infection on the ordinate and the logarithm of the antibody concentration (. mu.g/ml) is on the abscissa. (Log10, ug/ml) indicates the antibody concentration (ug/ml) expressed as a base 10 Log. Drawing notes: antibody names-HIV virus isolate (subtype) -IC50, e.g. E10-GXC44(C), i.e. the percentage neutralization activity/infection inhibition of antibody E10 against the subtype C virus strain GXC 44.

Detailed Description

The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

EXAMPLE 1 preparation of human anti-HIV gp 41-specific antibody

1. Labeling of the antigen. Expression of plasmid (pcDNA) containing HIV SF162 isolate full-length envelope protein (Access NO. EU123924) from 293F (Thermal, R79007) cells^TM3.1(+) Thermal, cat # V79020), purification of gp140 trimer in 293F supernatant by nickel column purification (SF 162). The trimeric protein was diluted to 0.5-2 mg/ml with PBS, and then biotin (EZ-Link) from Thermal corporation was used^TMSulfo-NHS-LC-Biotin, 21335) gp140 protein was labeled according to the kit protocol (ratio of number of molecules: protein: biotin 1: 20-100), incubating for 0.5-2 hours at room temperature in a dark place, centrifuging for 4-6 times at 8000g by using a 10kD centrifugal semi-permeable column (Merck Millipore, UFC501096), supplementing with sterile PBS, removing redundant biotin molecules, and using the labeled gp140 protein molecules for screening antigen-specific memory B cells.

2. Antigen-specific memory B cell sorting and reverse transcription

Peripheral blood mononuclear cells of a convalescent patient infected by HIV are separated, washed once by PBS, then resuspended to the power of 6-8 of 10 per ml by PBS (PBSA) containing 1% BSA, biotinylated gp140 protein is added to make the concentration reach 10 mu g/ml, after mixing well, the mixture is incubated at 4 ℃ for half an hour, then washed once by PBS, then the PBMC is resuspended by the same volume of PBSA, and then the ratio of the total volume of PBMC is 1: 50 to volume ratio of 50, mouse anti-human CD19(APC-H7), mouse anti-human IgG (APC), mouse anti-human IgM (Percp-cy5.5) and mouse anti-human CD27(FITC) were purchased from biolegend, while mixing them as follows: 200 volume ratio of invitrogen 7AAD (Percp-cy5.5) and Jackson immunolab streptavidin-PE (016. sup. 110. sup. 084) were added, and after mixing, incubation was carried out at 4 ℃ for half an hour. PBS was washed twice and PBSA was resuspended for cell sorting. Patient PBMCs were sorted for CD19+, IgM-, IgG +, CD27+, 7aad-, gp140/PE + positive cells (see FIG. 1) using BD FACS ariaIII, 1 cell per well, using 5ul repulpion buffer (SuperScript)^TMIII cell direct cDNA Synthesis System, invitrogen 18080-.

3. Amplification of variable regions of antibody genes.

The heavy chain variable region (VH) and light chain variable region (VK/VL) of the antibody gene were amplified using primers and amplification protocols described by Tiller, 2008, published in J Immunol Methods. VH, VK and VL are respectively subjected to two rounds of amplification, firstly, 3-5 ul of cDNA is taken as a template, and first round amplification is carried out by using antibody variable region gene leader sequence specific primers, specifically, primers 1-4 and primers 45 and 46 are used for amplifying heavy variable region (VH); primers 17, 18, 19 and primer 51 amplify the Kappa chain variable region (VK), and primers 31-37 and primer 57 amplify the Lambda chain variable region (VL). Then, performing second-round amplification by using a nested primer with an enzyme cutting site and using 5ul of a product of the first-round amplification as a template, specifically, amplifying heavy chain variable regions (VH) by using primers 5-16 and primers 48-50; primers 21-30 and 53-56 amplify the Kappa chain variable region (VK), and primers 38-43 and 58 amplify the Lambda chain variable region (VL). (reference: Tiller, T., Meffre, E., Yurasov, S., Tsuiji, M., Nussenzweig, M.C., & Wardemann, H. (2008.) the effective generation of monoclonal antibodies from single human b cells rt-pcr and expression cloning. j. immunological. methods 329,112-124.Journal of immunological methods 329(1-2), 112-124.).

The PCR system used Super HiFi PCR Mix (KT212)2 XPCR Mix from tiangen. The PCR program is 95 ℃ for 3 min; 30 seconds at 95 ℃, 30 seconds at 58-60 ℃,1 minute at 72 ℃ and 50 cycles; 10 minutes at 72 ℃; 10 minutes at 4 ℃. The amplified product was electrophoresed on 1.5% agarose gel (120V, 40min) to cut a variable region fragment of about 400 bp. In order to avoid false positives due to contamination, the cDNA in wells without sorted cells was used as a template for the first round of PCR and the reaction without added first round PCR product was used as a negative control for the second round of PCR. And (3) pairing the amplified heavy chain variable region and the light chain variable region, and performing the next enzyme digestion and vector construction only if the pairing of the heavy chain variable region and the light chain variable region is amplified simultaneously. The gene amplification of the antibody from a single cell and the construction of the antibody expression vector are shown in FIG. 1.

4. And (3) constructing a eukaryotic transient expression vector.

Transient expression vectors were constructed using the antibody gene transient expression vector system described in J Immunol Methods, published by tiller in 2008. The system comprises three vectors which are respectively used for expressing IgG1 heavy chain, Kappa chain and Lambda chain and are respectively named as IgH (Access Number DQ407610), IgK (Access Number rDQ407610) and IgL (Access Number FJ 517647). According to this system, the antibody capable of obtaining full-length IgG1 by transfection in eukaryotic cells can be obtained by double digestion, ligation, and transformation of the vector and the variable region fragment having the cleavage site. The heavy chain vector and the fragment are subjected to double enzyme digestion by AgeI and SalI, the kappa chain vector and the fragment are subjected to double enzyme digestion by AgeI and BsiwI, and the Lambda chain vector and the fragment are subjected to double enzyme digestion by AgeI and XhoI. After escherichia coli is transformed, selecting monoclonals, sequencing vector insertion sequences by using 5' absense primers, selecting 3-5 bacterial monoclonals for sequencing in each transformation, and successfully constructing the vector by using three sequencing results which are consistent, the insertion sequences and the vector can be translated into complete antibody fragments together, and the insertion sequences are different from the original vector enzyme digestion preorders.

5. And (3) preparing and purifying the monoclonal antibody.

2ml of E.coli containing successfully constructed vectors was inoculated into 200ml of 2YT medium (trypton16g/L, yeastextract 10g/L, NaCl 5g/L, Ampicillin 100. mu.g/ml) and cultured at 37 ℃ and 220rpm for 16 hours. The cells were collected by centrifugation at 6000g for 15 minutes, and a plasmid mass extraction kit (according to Thermal: (al))

HiPure plasmid maxiprep Kit, K210006), filtering and sterilizing for later use. By using

FreeStyle^TM293 Expression Medium (Thermal,12338018) culture based on CO at 37 ℃₂The shaking incubator cultures 293F (Thermal, R79007) cells, which are grown to a density of 1.0 × 10 according to the S-shaped growth curve⁶At/ml, transfection was performed. Each 30ml of 293F cells was transfected with 37.5ug of antibody plasmid, where the heavy and light chain plasmid mass ratio was 2: 3, culturing the transfected cells in a 37-degree shaking incubator containing 8% CO2 at 125rpm for 96-120 hours by shaking, and centrifuging to collect supernatant.

Monoclonal IgG antibodies were purified using Protein G Agarose Beads (13103-PNAE-RN) from Beijing Yinqiao Shenzhou, Inc. and following the protocol. The antibody was eluted with 0.2M glycine solution at pH 2.2d, immediately neutralized with 1/20 volumes of 1M tris-HCL (pH 9.2), and then centrifuged 4-6 times at 8000g using a 10kD semipermeable spin column (merck millipore, UFC501096), supplemented with sterile PBS and the excess glycine molecules were removed. Detecting OD280 value with Nanodrop spectrophotometer to determine antibody concentration, filtering with 0.22um filter for sterilization, subpackaging, and storing at-20 or-80 deg.C. The purified antibody was checked for purity and size by denaturing and non-denaturing polyacrylamide gel electrophoresis (see FIG. 2). The results show that the antibody expression is successful, and the size of the purified antibody is consistent with the expectation, namely two bands of 50kd +25kd in denaturing electrophoresis and 150kd in non-denaturing electrophoresis. The purity of the antibody reaches more than 90%.

Example 2 monoclonal antibody against gp41-Fc and its membrane proximal polypeptide F7-Fc fusion protein EC50 determination

The 293F cell (Thermal, R79007) is transfected by a pcDNA3.1(+) vector to express a fusion protein of gp41 envelope protein (access number: AY426110, 1533-2565bp) of HIV isolate bal and human IgG1 immunoglobulin Fc (constant region 2+ constant region 3) and is named as gp 41-Fc; the F7 (amino acid sequence: QQELLELDKWANLWNWFDISNWLW) and human IgG1 immunoglobulin Fc (constant region 2+ constant region 3) fusion protein was expressed using the same vector and cell line and was designated F7-Fc. Fc fusion proteins were purified using protein G resin. Purified gp41-Fc and F7-Fc proteins were diluted to 1. mu.g/ml with ELISA coating solution, respectively, and 100ul of each well was coated on ELISA plates (Corning, 9018) overnight at 4 ℃. PBST washing plate, 5% skim milk-PBS closed more than 2 hours. The monoclonal antibody prepared in example 1 was diluted with 5% skim milk-PBS at a 3-fold gradient starting from a starting concentration of 10 or 50. mu.g/ml and added to the blocked ELISA plate. And setting 7-8 gradients in three times, wherein the wells without the antibody are used as negative controls, and the wells with the IgG b12 diluted by the gradients are used as antibody controls. Goat anti-human IgGFab-HRP (1:10000, Jackson immunolab, 109-. And (3) taking the average value of the multiple wells of each gradient, drawing an OD 450-concentration curve, performing curve fitting according to a sigmoidal dose response model of GraphPad Prism, and calculating the corresponding antibody concentration when the OD450 value is half of the highest value (1.6), namely the EC50 value of the antibody for the specific antigen. The antibody binds HIV envelope protein gp41 with high efficiency, and the EC50 of antibody E10 for affinity to gp41 is 0.05. mu.g/ml (0.333nM) (see FIG. 3). E10 was able to efficiently recognize the membrane-proximal segment (MPER) short peptide F7 of gp41 (see FIG. 4).

EXAMPLE 3 determination of neutralizing Activity of monoclonal antibodies against HIV-1 pseudovirus

Pseudovirus packaging and antibody neutralization activity assay systems were performed AS described in the references Zhang MY, Borges AR, PtakRG, Wang Y, Dimitrov AS, Alam SM, Wieczorek L, Bouma P, Fouts T, Jiang S et al, patent and broad immunization activity of a single chain antibody fragment-free and cell-associated HIV-1.MAbs2010,2(3): 266-. Briefly, the PNL4-3 plasmid (containing the VSV viral backbone and containing the luciferase gene) and the HIV-1envelope protein plasmid were co-transfected with 293T cells to package HIV-1 pseudoviruses. After 72 hours of culture, virus supernatants were collected and virus titers were determined. Selecting a proper amount of virus to infect TZM-bl cells in a 96-well plate after incubation with an E10 antibody gradient diluent, cracking the cells after 72 hours, and detecting luciferase activity in the cracked cells by a luciferase luminescence detection system (Promega) to judge the inhibition effect of the antibody on pseudovirus infection. Wells without added virus were background controls and wells with added virus but no antibody were positive controls. Percent infection inhibition ═ (positive well reading-negative well reading-test well reading)/(positive well reading-negative well reading). Percent infection inhibition at 50% corresponds to antibody concentration as the IC50 value for antibody neutralization of the virus. The results showed that E10 had an infection inhibition rate of 73% against the C-subtype isolate GXC44 and 69% against the B-subtype isolate JRFL at a concentration of 150 ug/ml. The infection inhibition rate of E10 against other 4 strains of viruses does not reach 50% (see FIG. 5). In general, E10 has neutralizing activity against HIV-1. Reference is made to Yang, Z, J.Li, Q.Liu, T.Yuan, Y.Zhang, L.Q.Chen, Q.Lou, Z.Sun, H.ying, J.Xu, D.S.Dimitrov, and M.Y.Zhang.identification of Non-HIV infectious agent at any Bind to Germineb 12 producers and primers for evaluation of Cross-close diagnosis HIV-1antibodies.

TABLE 1 neutralization Activity of antibody E10 against different HIV isolates at different concentrations (percentage inhibition of infection)

Example 4 detection of antibody-dependent cell-mediated cytotoxic killing (ADCC) Activity of monoclonal antibodies

The TF228 cells were cultured in DMEM medium (the source and properties of the cells are described in Puri A, Hug P,

human erythrocytic glycophytes promoter HIV-1 enveloppe glycoprotien of CD4+ cells biochem Biophys ResCommun.1998Jan 6; 242(1):219-25.), the cell density is about 90%, the cells are resuspended by PBS washing after trypsinization, TF228 cells are labeled according to the PKH67 labeling kit (Sigma, PKH67GL-1KT) procedure, and the cell density is adjusted to 106/ml for use, the antibody E10 to be tested is diluted with DMEM medium gradient, mixed with 100ul labeled TF228 cells, placed in a cell culture chamber at 37 ℃, CO2 concentration 5% for 15 minutes, 100ul density 2 × 10 is added⁷A/ml freshly isolated healthy human Peripheral Blood Mononuclear Cells (PBMC), mixed and then placed at 37 ℃ in CO₂Culturing in 5% cell culture box for 4 hr; 1ul 7AAD (Thermal, 302232) was added to each reaction, mixed well and incubated for 15 minutes, and the cells were washed once with PBS and examined by flow cytometry. PKH67(Sigma, MINI67-1KT, green fluorescence) and 7AAD (Lifetech, 302232, red fluorescence) double positives, i.e. dead TF228 cell populations, were circled and analyzed for differences in this ratio for different treatments. The treatment without antibody was negative control, and the TF228 cells treated at 85 ℃ for 5 minutes were positive control for cell killing. The ADCC percentage is (the ratio of double positive cells of the sample to be tested to the ratio of double positive cells of the negative control)/(the ratio of double positive cells of the positive control to the ratio of double positive cells of the negative control). The results show that different concentrations of E10 can mediate remarkable toxic killing on target cells, the percentage of killing function is more than 17%, and especially under the condition of low concentration (0.08ug/ml), the killing effect is remarkableHigher than the putative cell killing antibody 2F5 (see fig. 6).

TABLE 2 percentage of cell killing mediated by three antibodies at different concentrations

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

SEQUENCE LISTING

<110> Shenzhen City center for preventing and treating chronic diseases

<120> human anti-HIV gp41 specific antibody and application thereof

<130>KHP171114310.9

<160>7

<170>PatentIn version 3.5

<210>1

<211>110

<212>PRT

<213> E10 light chain variable region

<400>1

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

15 10 15

Glu Arg Ala Thr Val Ser Cys Arg Ala Ser Gln Thr Val Ala Ser Asn

20 25 30

Ser Leu Ala Trp Tyr Gln His Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Ser Ala Ala Ser Ser Arg Ala Thr Gly Ile Ala Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser Arg Val Glu

65 70 75 80

Pro Glu Asp Ile Ala Val Tyr Phe Cys Gln Arg Tyr Gly Thr Ser Pro

85 90 95

Pro Tyr Ser Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

<210>2

<211>336

<212>DNA

<213> E10 light chain variable region

<400>2

gtacattcag aaattgtgtt gacacagtct ccaggcaccc tgtctttgtc tccaggggaa 60

agagccaccg tctcctgcag ggccagtcag actgtcgcca gcaactcctt agcctggtac 120

cagcacaaac ctggccaggc tcccaggctc ctcatctccg ctgcatccag cagggccact 180

ggcatcgcag acaggttcag tggcagtgga tctgggacag acttcagtct caccatcagc 240

agagtggagc ctgaagatat tgcagtatat ttctgtcaac gatatgggac gtcacctcca 300

tatagttttg gccaggggac caaggtggaa atcaaa 336

<210>3

<211>124

<212>PRT

<213> E10 heavy chain variable region

<400>3

Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Arg Lys Pro Gly Ser

1 5 10 15

Ser Val Arg Val Ser Cys Gln Ala Ser Gly Gly Thr Phe Asn Asn Tyr

20 25 30

Ala Leu His Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Asp Trp Met

35 40 45

Gly Arg Ile Leu Pro Ile Pro Asp Ile Thr Lys Tyr Ala Gln Lys Phe

50 55 60

Glu Gly Arg Ile Thr Ile Thr Ala Asp Arg Ser Thr Ser Ser Ala Phe

65 70 75 80

Leu Glu Val Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Trp Ser Arg Arg Asp Gly His Asn Ser His Phe Asp

100 105 110

Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>4

<211>383

<212>DNA

<213> E10 heavy chain variable region

<400>4

gtacattctc aggtgcagct ggtggagtct ggggctgagg tgaggaagcc tgggtcctcg 60

gtgagggtct cctgccaggc ctctggaggc accttcaaca attatgctct ccactgggtg 120

cgacaggccc ctggagaggg gcttgattgg atgggaagaa tcctgcctat ccctgatata 180

accaaatacg cccagaagtt cgagggcaga atcacgatta ccgcggacag atccacgagt 240

tcagccttct tggaagtgag cagcctgaga tctgaagaca cggccgtcta ttattgtgcg 300

agaagtcttt ggtcccgtcg agatggccac aactctcact ttgactcctg gggccaggga 360

accctggtca ccgtctcctc agc 383

<210>5

<211>216

<212>PRT

<213> full Length of E10 light chain

<400>5

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Val Ser Cys Arg Ala Ser Gln Thr Val Ala Ser Asn

20 25 30

Ser Leu Ala Trp Tyr Gln His Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Ser Ala Ala Ser Ser Arg Ala Thr Gly Ile Ala Asp ArgPhe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser Arg Val Glu

65 70 75 80

Pro Glu Asp Ile Ala Val Tyr Phe Cys Gln Arg Tyr Gly Thr Ser Pro

85 90 95

Pro Tyr Ser Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210>6

<211>454

<212>PRT

<213> E10 heavy chain full Length

<400>6

Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Arg Lys Pro Gly Ser

1 5 10 15

Ser Val Arg Val Ser Cys Gln Ala Ser Gly Gly Thr Phe Asn Asn Tyr

20 25 30

Ala Leu His Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Asp Trp Met

35 40 45

Gly Arg Ile Leu Pro Ile Pro Asp Ile Thr Lys Tyr Ala Gln Lys Phe

50 55 60

Glu Gly Arg Ile Thr Ile Thr Ala Asp Arg Ser Thr Ser Ser Ala Phe

65 70 75 80

Leu Glu Val Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Trp Ser Arg Arg Asp Gly His Asn Ser His Phe Asp

100 105 110

Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser SerLys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu GluGln Tyr Asn

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

325 330 335

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

355 360 365

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

370 375 380

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

405 410 415

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

420 425 430

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

435 440 445

Ser Leu Ser Pro Gly Lys

450

<210>7

<211>24

<212>PRT

<213>HIV

<400>7

Gln Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Asn Leu Trp Asn Trp

1 5 10 15

Phe Asp Ile Ser Asn Trp Leu Trp

20

Claims

1. A human HIV gp41 specific antibody is characterized in that the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 1; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 3.

2. The specific antibody of claim 1, wherein the light chain amino acid sequence is set forth in SEQ ID No. 5; the amino acid sequence of the heavy chain is shown as SEQ ID NO. 6.

3. The specific antibody according to claim 1, which is a single chain antibody, Fab antibody, minibody, chimeric antibody or whole antibody immunoglobulin IgG1, IgG2, IgG4, IgA, IgE, IgM or IgD.

4. A gene encoding the specific antibody according to any one of claims 1 to 3.

5. An expression vector, host cell or expression cassette comprising the gene of claim 4.

6. A pharmaceutical or a detection reagent comprising the specific antibody according to any one of claims 1 to 3.

7. Use of the specific antibody according to any one of claims 1 to 3 for the preparation of a medicament for the treatment of HIV infection or for the preparation of a reagent for the detection of HIV.