Defucosylated Bispecific Multivalent Antibodies Mediating ADCC For HIV-1 Eradication And ADCC Effect In The Plasma From HIV-1 Infected Patients

Background and objectiveHIV-1 can be integrated into the genome of some long-lived CD4+resting T lymphocyte and form a stable viral reservoir.The viremia will rebound soon once the HAART was disrupted,which become a major barrier for eradicating HIV-1 infection.The concept of "functional cure" was proposed based on the difficulty of "sterilizing cure" for HIV-1.A 'functional' cure for HIV-1 is defined as a long-term host-mediated control of viral replication and remission of the symptoms of HIV-1 infection in the absence of antiretroviral therapy,even if replication competent viruses remain in the body.The most widely discussed approach for eliminating the latent reservoir is the so-called "shock and kill" strategy.Specifically,this strategy seeks to activate HIV-1 from latent reservoirs using latency-reversing agents(LRAs)and then to target HIV-1-infected cells for eradication via the host immune response and/or cytotoxic drugs.Broadly neutralizing antibodies(bNAbs)can not only effectively neutralize free viral particles but also mediate antibody-dependent cell-mediated cytotoxicity(ADCC)through their Fc fragments to kill HIV-1 infected cells.Based on the study of bNAbs and Env target proteins,bispecific or even trispecific broad-spectrum neutralizing antibodies were prepared by genetic engineering methods which exhibited superior antiviral properties.Our partner,the National Cancer Institute developed a defucosylated bispecific multivalent CD4-antibody fusion protein,LSEVh-LS-F,which composed of two engineered domains,mD1.22 and m36.4.mD1.22,an engineered mutant of the D1 extracellular domain of CD4,selectively binds to the gp120 CD4-binding site,while m36.4,an antibody domain,targets the highly conserved CD4-induced(CD4i)gp 120 coreceptor-binding site.Although our US-China cooperative project partner,the National Cancer Institute,has reported the neutralization and ADCC effects of LSEVL-LS-F.Nevertheless,HIV-1 is a highly mutated virus and there is still a lack of systematic evaluation of the neutralization ability of LSEVL-LS-F.The neutralizing effect of LSEVL-LS-F on HIV-1 clinical isolates in China is also unknown.Further research is also needed to determine whether the ADCC effect of LSEVL-LS-F can be used to clear the activated latent infection cells.Therefore,this study systematically identified the neutralization width and breadth of LSEVL-LS-F and focused on the analysis of the neutralization ability of LSEVL-LS-F against clinical isolates in China.We also assessed the effect of LSEVh-LS-F on NK-mediated killing of HIV-1 Env-expressing cell lines and HIV-1-infected cells.We finally explored the potential of LSEVL-LS-F to kill reactivated latently infected cells,especially resting CD4+ T lymphocytes isolated from patients receiving HAART cells,in combination with LRAs.Compared with the non-defucosylated molecule LSEVh-LS at the same time.This study provides a scientific basis for the combination of antibodies and LRAs to reduce the HIV-1 virus reservoir and the functional cure of HIV-1/AIDS.In addition,this study also systematically analyzed the correlation between the ability of plasma-mediated ADCC in HIV-1 infected individuals and its ability to bind to target cells and its neutralization intensity.Methods1.Isolation and preparation of HIV-1 clinical virusPeripheral blood mononuclear cells were isolated by density gradient centrifugation and the HIV-1 infected PBMCs and healthy human PBMCs were co-cultured to isolate and replicate HIV-1 virus.GHOST cell lines were used to detect the use of co-receptors in clinical isolates.Viral RNA was extracted from the virus isolated from the PBMCs for the near full-length genome(NFLG)amplification and sequencing.The subtype of the isolated virus was analyzed by constructing a phylogenetic tree.293T cells were transfected with pNL4-3 and pWT/Bal plasmids to obtain NL4-3 and Bal laboratory standard strains.The titers of isolated or replicated viruses were determined on TZM-bl and PBMCs cells.2.TZM-bl-or PBMCs-based neutralization assayIn a neutralization assay based on TZM-bl or PBMCs,HIV-1 virus was pre-incubated with a series of 3-fold diluted LSEVh-LS-F then TZM-bl or PBMCs cells were added.The 50%inhibitory concentration(IC50)and 90%inhibitory concentration(IC90)of the antibody were calculated by detecting the relative light units(RLU)of TZM-bl cells or supernatant p24 content.HIV-1 infected cells were pre-incubated with a series of 3-fold diluted LSEVh-LS-F and TZM-bl cells were added.The inhibition of the virus through cell-cell contact infection by LSEVh-LS-F were analyzed by detecting the RLU value of TZM-bl cells.3.Detection of NK cell activation and antibody ADCC effectThe level of NK cells activation was analyzed by measuring the expression of CD 107a on the surface of NK cells and the secretion level of cytokines and chemokines in supernatant.The affinity of LSEVh-LS-F and LSEVh-LS to Env-expressing cell lines and HIV-1 infected cells were measured by flow cytometry.LSEVh-LS-F-mediated ADCC in the presence of NK cells against Env-expressing cell lines and HIV-1-infected cells,was measured using a lactate dehydrogenase(LDH)cytotoxicity assay or flow cytometry and compared with fucosylated polyvalent bispecific antibody(LSEVh-LS)-mediated ADCC effect.HIV-1 infected target cells was co-incubated with LSEVh-LS-F and effector cells,the virus inhibition rate was calculated by measuring the content of p24 in the supernatant to analyze antibody-dependent cell-mediated viral inhibition(ADCVI)effect of the LSEVh-LS-F.4.Reactivation and elimination of HIV-1 latently infected cellsLatently infected ACH2 and U1 cells were used to determine the reactivation activity of three different HDACIs,including SAHA,Apicidin,or Chidamide,as LRAs.The killing effect of LSEVh-LS-F on activated ACH2 cells was analyzed by detecting the release of LDH.Resting CD4+ T latently infected cell model was prepared by using laboratory standard strains and Chinese clinical isolates.The killing effect of LSEVh-LS-F to reactivated resting CD4+ T latently infected cell model was analysed by measuring the level of supernatant p24 or the supernatant RLU value after infection with TZM-bl.The resting CD4+ T latent infected cells were isolated from HAART-treated HIV-1 infected patients and reactivated with PHA or LRAs.The killing effect of LSEVh-LS-F to reactivated resting CD4+ T latently infected cells isolated from HAART-treated HIV-1 infected patients was analysed by measuring the level of supernatant p24.5.ADCC effect in HIV-1 infected patients plasmaThe ADCC activity of 62 non-antiretroviral-treated HIV-1 infected plasmas on 8E5 or NL4-3 infected CEM.NKR.CCR5 target cells were detected by flow cytometry-based cytotoxicity assay.The affinity of HIV-1 infected plasma to target cells also detected by flow cytometry.The neutralization intensity of HIV-1 infected plasmas was determined by neutralization of 14 different subtypes of HIV-1 pseudovirus.The correlation between the ADCC effect of HIV-1 infected plasmas and their affinity to target cells and neutralization intensity was analyzed.Results1.LSEVh-LS-F exhibited exceptionally potent and broad neutralizing activities against Chinese HIV-1 clinical isolatesThe neutralization test results based on TZM-bl cells showed that LSEVh-LS-F potently neutralized 49 newly isolated Chinase clinical strains(100%),and the geometric mean of neutralization of IC50 was 4.8 times lower than that of VRC01(4.7 nM).In the PBMCs cell-based neutralization assay which much closer to the human condition,21 strains(100%)of newly isolated clinical strains were neutralized by LSEVh-LS-F,and the geometric mean of neutralization IC50 was 4.2 times lower than that of VRC01(0.9 nM).LSEVh-LS-F neutralized all the 14 strains of pseudoviruses,which from international neutralizing antibody detection platform.The geometric mean of IC50 was 11 times lower than that of VRC01,which was 0.28 nM;and IC90 was neatly 4 times lower than that of VRC01,which was 3.9 nM.LSEVh-LS-F have similar neutralization intensity with N6 but have better neutralization breadth.The neutralizing effect of LSEVh-LS-F on NL4-3 and Bal(HIV-1 laboratory adapted strains)was superior to that of VRC01,with IC50 of 0.005 nM and 0.05 nM,respectively.In addition,LSEVh-LS-F could also efficiently inhibit cell-to-cell transmission.Defucosylation has no effect on the neutralization activity of LSEVh-LS-F.2.Specific and efficient killing of Env-expressing target cells mediated by LSEVh-LS-FLSEVh-LS-F can effectively activate NK cell degranulation and activate NK cells to release antiviral factors such as IFN-? and p-chemokines.LSEVh-LS-F and LSEVh-LS can specifically bind to Env-expressing cell lines(8E5 and CHOZA-gp160sc cells)and HIV-1 infected cells(CD4+ T and CEM.NKR.CCR5 cells),and there is no difference in the binding ability between them.LSEVh-LS-F can specifically kill the cells expressing Env protein(Env-expressing cell lines and HIV-1 infected cells)through ADCC effect,and its killing effect is significantly better than that of LSEVh-LS and VRC01.LSEVh-LS-F can also effectively mediate the ADCC effect against 8E5 cells by using HIV-1 infected PBMCs as effector cells.The inhibitory effect of LSEVh-LS-F on the replication of HIV-1 in CD4+ T and CEM.NKR.CCR5 cells persisted in the presence of effector cells,while the inhibitory activity of LSEVh-LS decreased with time.3.Reactivation and killing of HIV-1 latently infected cellsAfter treatment of ACH2 and U1 cells with three LRAs:SAHA,Apicidin,and Chidamide,the p24 content in the supernatant was positively related to the concentration of three LRAs.The expression level of on ACH2 cells was also concentration-dependent within a certain range.LSEVh-LS-F mediates effective ADCC on reactivated ACH2 cells and is superior than that of LSEVh-LS.LSEVh-LS-F mediated ADCC effect against reactivated ACH2 cells is correlated with the level of Env protein expressed on ACH2.In the LSEVh-LS-F treatment group,the p24 level on NL4-3 and GX2016EU03 latently infected CD4+ T cell models was reduced 40-fold and 5-fold,respectively,compared with the level of the non-antibody control group.Detection of RLU values in cell supernatants after infection with TZM-bl also yielded similar results,with a decrease of 85-fold and 27-fold,respectively.HIV-1 latently infected resting CD4+ T cells were isolated from patients receiving HAART and reactivated by PHA or or a combination of specific LRAs,including SAHA and Apicidin.The content of p24 in LSEVh-LS-F treatment group was significantly lower than that in the non-antibody control group or M336,VRC01,and LSEVh-LS treatment group.The results suggested that can kill reactivated resting cells in the clinical patients.4.ADCC effect in the plasma from HIV-1 infected patientsMost HIV-1-infected plasmas showed some neutralization activity against different subtypes of pseudotyped virus and NL4-3,but the difference in neutralization intensity was greater.The HIV-1 infected plasma has distinctly different characteristics for the ADCC effects when using 8E5 or NL4-3 infected CEM.NKR.CCR5 as target cells.HIV-1 infected plasma mediate ADCC effect against 8E5 cells was positively correlated with its binding to target cells and its neutralization intensity.However,its effect against NL4-3 infected CEM.NKR.CCR5 cells was negatively correlated with its binding to target cells and had no correlation with its neutralization intensity.The conformation of Env protein on the membrane surface of 8E5 cells was changed and entered the "open" conformation state after co-incubation with CD4+ T cells.The characteristics of HIV-1 infected plasma mediate ADCC effect against Env "opened" 8E5 cells was changed.ConclusionsThis novel bispecific multivalent molecule LSEVh-LS-F exhibited broad and potent neutralizing activity in vitro.It also has good neutralizing effect on HIV-1 clinical isolates in China.LSEVh-LS-F can activate NK cells and effectively mediate the ADCC effects on HIV-1 infected cells.Especially,LSEVh-LS-F can also mediate strong ADCC effects when the untreated and treated HIV-1 infected PBMCs were used as effector cells.LSEVh-LS-F was more effective in NK-mediated killing of latency reactivation agents-reactivated ACH2 cells,reactivated latently infected resting CD4+ T cell line,and more importantly resting CD4+ T lymphocytes isolated from patients receiving HAART.This study provides a scientific basis for the combination of antibodies and LRAs to reduce the HIV-1 virus reservoir and the functional cure of HIV-1/AIDS.The conformation of the Env target protein has an important influence on plasma-mediated ADCC in HIV-1 infected patients.The HIV-1 gp120 CD4-bound conformation is preferentially targeted by ADCC-mediating antibodies in sera from HIV-1-Infected patients.HIV-1 infected plasma with weaker neutralization mediates stronger ADCC effects.These results laid the foundation for further understanding of the ADCC characteristics of HIV-1 infected plasma and the development of novel anti-HIV-1 antibodies.