Inhibition of complement activity by humanized anti-C5 antibody and single-chain Fv

Activation of the complement system contributes significantly to the pathogenesis of numerous acute and chronic diseases. Recently, a monoclonal antibody (5G1.1) that recognizes the human complement protein C5, has been shown to effectively block C5 cleavage, thereby preventing the generation of the pro-inflammatory complement components C5a and C5b-9. Humanized 5G1.1 antibody, Fab and scFv molecules have been produced by grafting the complementarity determining regions of 5G1.1 on to human framework regions. Competitive ELISA analysis indicated that no framework changes were required in the humanized variable regions for retention of high affinity binding to C5, even at framework positions predicted by computer modeling to influence CDR canonical structure. The humanized Fab and scFv molecules blocked complement-mediated lysis of chicken erythrocytes and porcine aortic endothelial cells in a dose-dependent fashion, with complete complement inhibition occurring at a three-fold molar excess, relative to the human C5 concentration. In contrast to a previously characterized anti-C5 scFv molecule, the humanized h5G1.1 scFv also effectively blocked C5a generation. Finally, an intact humanized h5G1.1 antibody blocked human complement lytic activity at concentrations identical to the original murine monoclonal antibody. These results demonstrate that humanized h5G1.1 and its recombinant derivatives retain both the affinity and blocking functions of the murine 5G1.1 antibody, and suggest that these molecules may serve as potent inhibitors of complement-mediated pathology in human inflammatory diseases.     

Improvement in affinity and HIV-1 neutralization by somatic mutation in the heavy chain first complementarity-determining region of antibodies triggered by HIV-1 infection

We assessed the impact of somatic hypermutation in the framework region 1 (FR1) and complementarity-determining region 1 (CDR1) of three clonally-related heavy chains from the human monovalent antigen-binding fragments Fab S19, S8 and S20 on gp120 binding and HIV-1 neutralization capacity. Nucleotide changes were introduced in the heavy chains to revert single and multiple amino acid residues, and two Fab libraries were constructed with the same light chain to express equivalent amounts of parental and reverted phage Fab. We studied the contribution of each amino acid replacement to antigen binding by calculating the frequency of phage Fab retrieval after competitive library selection on gp120. Whereas mutations in FR1 had no effect on antigen binding, somatic replacements in the CDR1 of the heavy chain (HCDR1) appeared to produce significant changes. In S19 HCDR1, somatic mutation of residue 32 reduced gp120 binding. In Fab S20, the Arg(30) and Asp(31) somatically replaced residues in HCDR1 improved antigen binding. Both of these residues are necessary to increase Fab binding to gp120; reversion of either residue alone results in a decrease in binding. The impact of these two replacements was confirmed by the greater neutralization capacity of S20 compared to the other Fab. Molecular modeling of S20 HCDR1 suggests that Arg(30) and Asp(31) are the main interaction sites for gp120, increasing antibody affinity and promoting the enhanced neutralization ability of S20. These findings are consistent with a gp120-driven process, supporting a role for affinity maturation and intraclonal evolution of HIV-1 neutralizing antibodies.     

Ex vivo neutralization of HIV-1 quasi-species by a broadly reactive humanized monoclonal antibody KD-247

By immunizing mice sequentially with six different V3 peptides we obtained a murine monoclonal antibody (MAb) C25, and its humanized counterpart KD-247. The MAb recognizes the sequence IGPGRA at the tip of the V3 loop and displays broad neutralizing activity against a variety of HIV-1 isolates. KD-247 was tested in an ex vivo neutralization assay to determine its capability to contain the spread of a quasi species population of clade B HIV-1 derived from two patients. The epitope of KD-247 was generally matching with the V3 sequences of various clones isolated from plasma and peripheral blood mononuclear cells (PBMC) of two patients. Complete or strong inhibition of viral replication was observed when the patients' PBMC were cultured with a high concentration of KD-247. Some neutralization escape variants, which had mutations in the V3 or outside of the V3 loop, emerged only at a low concentration of the MAb. These results suggest that KD-247 could be a good candidate for immunotherapy against HIV-1 in vivo.     

Humanized Antibodies: Enhancing Therapeutic Utility Through Antibody Engineering

The promise of antibody therapeutics has been greatly expanded by the development of monoclonal antibody technology and more recently antibody humanization. By transferring the mouse antibody binding site into a human antibody gene, we can engineer a “human antibody” which retains the specificity and biological effects of the original mouse antibody but has the potential to be nonimmunogenic in humans. Additionally, antibody effector functions can be improved through manipulation of the antibody constant region genes.

We have produced a humanized version of OKT3 with human IgG4 and kappa constant regions. This antibody retains all of the in vitro characteristics of murine OKT3 including induction of cytokine release and T cell activation markers. Humanized OKT3 has an affinity of 1.4 × 10⁹ M^?1 relative to a 1.2 × 10⁹ M^?1 affinity of murine OKT3. Substitution of a glutamic acid for leucine at residue 235 in the antibody constant region abrogates FcR I binding and causes a marked reduction of T cell activation. The humanized FcR mutant of OKT3 has potential to be an improved therapeutic for transplantation and may have applications in autoimmune disease treatment.     

The importance of antibody isotype in HIV-1 virus capture assay and in TZM-bl neutralization

The binding of murine IgM mAbs to five different clades of HIV-1 was examined using a modified ELISA-based virus capture assay. Two murine multispecific IgM mAbs that exhibit both lipid and gp41 epitope specificities, and one murine IgM mAb that exhibits lipid-binding specificity, were utilized. The binding of the IgG and the IgM isotypes of human mAb 2F5 to clades A through AE were also evaluated. The binding of 2F5 to HIV-1 was dependent upon the antibody isotype. Monoclonal IgM antibodies bound significantly lower amounts of HIV-1 than the corresponding IgG isotype. Although murine IgM mAbs bound HIV-1 to varying degrees in the virus capture assay, they failed to neutralize HIV-1 in a TZM-bl pseudovirus assay. In contrast, 2F5-IgM mAb bound certain HIV-1 isolates, and also neutralized them, although not as efficiently as the 2F5-IgG isotype. Studies on the relationship between virus binding and neutralization in a TZM-bl pseudovirus assay indicated that in most cases, mAbs that exhibited neutralization also bound the virus.     

A single backmutation in the human kIV framework of a previously unsuccessfully humanized antibody restores the binding activity and increases the secretion in cos cells

Humanization of rodent mAbs by CDR-grafting (also called "reshaping") is now a standard procedure for reducing immunogenicity and recruiting human effector functions. However, the design of the humanized mAb can sometimes prove circuitous. Attempts were made to humanize L-25, a mouse antibody against the human alpha-4 integrin subunit using the usual protocols. Despite reaching eight backmutations in the light chain, it was not possible to recover the binding activity to the level of the chimeric. In an effort to restore the binding activity, an analysis of the human kappa IV acceptor frameworks was undertaken. This analysis highlighted the Asp at position 9 in framework 1, which although a common amino acid in human kappa IV frameworks, was an unusual residue in mouse kappa frameworks. Backmutating this position to the mouse amino acid completely restored the binding of the humanized antibody and as a by-product also increased the secretion levels in cos cells. Mutating position 9 to the consensus residue for human kappa I also restored the binding and secretion levels although not to the same extent. The resulting humanized antibody had a light chain with only a single backmutation to the mouse sequence.     

Neutralization kinetics of sensitive and resistant subtype B primary human immunodeficiency virus type 1 isolates

The aim of the study was to determine if sensitive and resistant human immunodeficiency virus type 1 (HIV-1) subtype B primary isolates have different neutralization kinetics. Neutralization assays were undertaken where either the time allowed for virus to react with antibodies or the subsequent period of this mixture's exposure to target cells were varied. The relative neutralization sensitivity/resistance is a reproducible property of the isolates. In a minority of combinations, the titre falls exponentially for as long as the free virions are exposed to antibody. In the remainder, neutralization kinetics shows deviations which may be attributed to events occurring after the virus-antibody mixture is added to the target cells: significant neutralization with minimal exposure of the free virions to antibody; a plot where reduction in virus titre is parallel to the duration of the incubation phase of the assay. Neutralization rate constants are similar for primary HIV-1 SF33, HIV-1 SF162, and HIV-1 89.6, reaching 5 x 10(5)-1 x 10(6)/M sec for the monoclonal antibody IgG1 b12. However, although increased antibody levels produced greater reductions in virus titre the rate of neutralization was not proportional to the antibody concentration. Neutralization of either the free virion or cell-associated virus does not correlate with the resistance/sensitivity of primary subtype B isolates. The target cells play an active role, so that in designing neutralization assays with primary isolates of HIV-1, events following the virus-antibody complex binding to the cell surface have to be taken into consideration.     

Production and characterization of high-affinity human monoclonal antibodies to human immunodeficiency virus type 1 envelope glycoproteins in a mouse model expressing human immunoglobulins

Human (Hu) monoclonal antibodies (MAbs) against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) are useful tools in the structural and functional analysis of Env, are under development both as potential prophylaxis and as therapy for established HIV-1 infection, and have crucial roles in guiding the design of preventative vaccines. Despite representing more than 50% of infections globally, no MAbs have been generated in any species against C clade HIV-1 Env. To generate HuMAbs to a novel Chinese C clade Env vaccine candidate (primary isolate strain HIV-1(97CN54)), we used BAB5 mice that express a human immunoglobulin (Ig) M antibody repertoire in place of endogenous murine immunoglobulins. When immunized with HIV-1(97CN54) Env, these mice developed antigen-specific IgM antibodies. Hybridoma fusions using splenocytes from these mice enabled the isolation of two Env-specific IgM HuMAbs: N3C5 and N03B11. N3C5 bound to HIV-1 Env from clades A and C, whereas N03B11 bound two geographically distant clade C isolates but not Env from other clades. These HuMAbs bind conformational epitopes within the immunodominant region of the gp41 ectodomain. N3C5 weakly neutralized the autologous isolate in the absence of complement and weakly enhanced infection in the presence of complement. N03B11 has no effect on infectivity in either the presence or the absence of complement. These novel HuMAbs are useful reagents for the study of HIV-1 Env relevant to the global pandemic, and mice producing human immunoglobulin present a tool for the production of such antibodies.     

Anti-HIV-1 humoral immune response in Brazilian patients

Data on humoral immune response against Brazilian human immunodeficiency virus type 1 (HIV-1) isolates have been collected and reviewed in reference to data published in the international literature. The results obtained up to now indicate that the Brazilian humoral immune response can be divided into at least three antibody binding serotypes: anti-Bbr, anti -B/F and anti-C/F. No neutralization serotypes have been distinguished up to now, just different degrees of resistance to neutralizing antibody (NAb), similar to data obtained by neutralization analyses of European/North American HIV-1isolates. No correlation between V3 binding antibodies, genotype and HIV-1 NAb could be observed.     

Humanization of an anti-ICAM-1 antibody with over 50-fold affinity and functional improvement

The monoclonal antibody 1A6 binds to human intercellular adhesion molecule 1 (ICAM-1, CD54) and inhibits infection by 90% of human rhinovirus (HRV) serotypes. To make a therapeutic molecule for preventing and treating HRV infection, we humanized a single chain antibody (scFv), 1A6, by a structure-guided complementarity-determining region (CDR) grafting procedure. Our final humanized 1A6 scFv does not retain any mouse back mutations in the framework. Without changing the CDR sequences, the humanized 1A6 scFv demonstrates over 50-fold improvement in both affinity for ICAM-1 and protection efficacy against HRV infection in vitro.     

Zymosan enhances the immune response to DNA vaccine for human immunodeficiency virus type-1 through the activation of complement system

In the present study, the adjuvant effect of zymosan on human immunodeficiency virus type-1 (HIV-1)-specific DNA vaccine and the mechanism of this enhancement were studied in a murine model. We coinoculated zymosan with our candidate HIV-1 specific DNA vaccine (pCMV160IIIB) into skeletal muscles of BALB/c mice. Higher levels of both humoral immune response and HIV-specific delayed-type hypersensitivity (DTH) response were observed when zymosan was coinoculated with pCMV160IIIB compared with that obtained using pCMV160IIIB alone. HIV-specific cytotoxic T lymphocyte (CTL) activity was also enhanced. This enhancing activity was suppressed when coinoculated to the fifth complement (C5)-deficient DDD and AKR mice. The enhanced activity was also suppressed when anti-C3 antibody was inoculated to mice intramuscularly. There was significant induction of immunoglobulin G2a (IgG2a) and interferon-gamma (IFN-gamma) in pCMV160IIIB vaccine with zymosan. These results suggest that zymosan-mediated DNA vaccination enhances helper T cell (Th) 1-mediated immunity. The effect is suggested to be based on the consequences of its recruitment and activation of macrophages, dendritic cells or antigen-presenting cells (APC) through complement activation, especially through the alternative pathway. Taken together, these results suggest that zymosan can be an effective immunological adjuvant in DNA vaccination against HIV-1.     

构建预设CDR3基因噬菌体抗体库筛选抗人整合素ανβ3 mAb的人源化Fab

目的:构建预设CDR3基因的噬菌体抗体库,通过抗原表位导向选择方法筛选抗人整合素ανβ3单克隆抗体(mAb)人源化Fab。方法:将鼠mAbLCDR3重组到人轻链可变区文库中并与人/鼠嵌合重链Fd基因配对构建杂合噬菌体抗体库,用固相人整合素ανβ3抗原筛选人源化轻链基因。再用所获人轻链基因与移植有鼠mAbHCDR3的人重链Fd基因配对构建人源噬菌体抗体库,筛选人源化Fab。结果:分别构建了库容为2.1×106和2×107的杂合噬菌体抗体库和人源噬菌体抗体库,筛选到3株人源Fab克隆。经间接ELISA及竞争抑制ELISA证实,能特异结合整合素ανβ3抗原,其中人源D5株Fab克隆的基因序列表明,人轻链可变区基因属VKIII亚群,人重链可变区基因属VH1亚群。结论:利用噬菌体抗体库技术,成功地进行了鼠抗人整合素ανβ3mAbE10人源化的改造,为进一步临床应用奠定了基础。     

The neutralization sensitivity of viruses representing human immunodeficiency virus type 1 variants of diverse subtypes from early in infection is dependent on producer cell, as well as characteristics of the specific antibody and envelope variant

Neutralization properties of human immunodeficiency virus (HIV-1) are often defined using pseudoviruses grown in transformed cells, which are not biologically relevant HIV-1 producer cells. Little information exists on how these viruses compare to viruses produced in primary lymphocytes, particularly for globally relevant HIV-1 strains. Therefore, replication-competent chimeras encoding envelope variants from the dominant HIV-1 subtypes (A, C, and D) obtained early after infection were generated and the neutralization properties explored. Pseudoviruses generated in 293T cells were the most sensitive to antibody neutralization. Replicating viruses generated in primary lymphocytes were most resistant to neutralization by plasma antibodies and most monoclonal antibodies (b12, 4E10, 2F5, VRC01). These differences were not associated with differences in envelope content. Surprisingly, the virus source did not impact neutralization sensitivity of most viruses to PG9. These findings suggest that producer cell type has a major effect on neutralization sensitivity, but in an antibody dependent manner.     

Vernier zone residue 4 of mouse subgroup II kappa light chains is a critical determinant for antigen recognition.

BACKGROUND: During the conversion of murine monoclonal antibodies directed against the human chorionic gonadotropin (hCG) into bacterially expressed single chain fragments (scFv), we found a major reduction of binding activity upon introduction of a primer encoded mutation. OBJECTIVES: In this study we tried to determine which mutation was responsible and on what manner this mutation affected antigen binding (structural effect versus direct involvement of the residue in binding). RESULTS: No binding could be detected, when the wild type residue methionine at position 4 within the Framework region 1 of the Vkappa light chain was substituted by serine in two antibodies with a subgroup II kappa light chain. However, a similar replacement within an anti-hCG antibody with a subgroup IV kappa light chain and thereby having leucine as wild type residue, did not affect the binding characteristics. The mutant scFv's derived from both AB-s sensitive for substitution by serine never reacted with antigen in ELISA. Analysis with surface plasmon resonance revealed a residual binding only on a sensorchip with a high density coating of antigen; however, an increased dissociation, relative to that of the wild type scFv and the absence of reactivity in ELISA suggest a drastically altered affinity. CONCLUSION: A structural explanation for the changed binding characteristics can be the influence of the position 4 residue, as being a constituent of the Vernier zone, on the position of the CDR1 loop of Vkappa, which might harbour residues that directly bind to antigen, or indirectly positions other variable loops of the binding pocket. An increased sensitivity for trypsin digestion supported the hypothesis of a local conformational change in the serine mutant of the subgroup II kappa containing antibody.     

Interaction between virion-bound host intercellular adhesion molecule-1 and the high-affinity state of lymphocyte function-associated antigen-1 on target cells renders R5 and X4 isolates of human immunodeficiency virus type 1 more refractory to neutralization

The oligomeric nature of the viral envelope proteins has been partly held responsible for the observed differences in neutralization sensitivity between primary and laboratory-adapted strains of human immunodeficiency virus type 1 (HIV-1). However, recent evidence suggests that host factors can also modify the sensitivity of HIV-1 particles to neutralization. Having previously demonstrated that the acquisition of host-encoded intercellular adhesion molecule (ICAM)-1 proteins by newly formed viruses has a functional significance for the life cycle of HIV-1, we investigated whether the acquisition of host-derived ICAM-1 by HIV-1 could affect the virus sensitivity to neutralization. In this study, we have first shown that the physical presence of host cell membrane ICAM-1 on HIV-1 was not modifying virus sensitivity to neutralization by either two different anti-gp120 monoclonal antibodies (0.5beta and 4.8D) or soluble CD4. However, the ability of the F105 anti-gp120 monoclonal antibody (specific for the CD4-binding site) to neutralize ICAM-1-bearing virions was diminished when target cells were pretreated with an lymphocyte function-associated antigen-1 (LFA-1)-activating antibody. Interestingly, ICAM-1/POS progeny viruses were found to be slightly more resistant to neutralization by individual human sera in target cells expressing a low-affinity form of LFA-1 than viruses devoid of host-encoded ICAM-1 proteins. This resistance was markedly enhanced when target cells expressed an activated LFA-1 form on their surface. These results suggest that the interaction between virally embedded host ICAM-1 and target cell surface LFA-1 should be considered a factor modulating neutralization sensitivity of HIV-1 by human sera from HIV-1-infected individuals.     

Enhanced antiglobulin-mediated neutralization of herpes simplex virus-IgG complexes by complement and heterologous anti-immunoglobulin

Summary The ability of IgG anti-Fc and anti-Fab to neutralize infectious herpes simplex virus-IgG (HSV-IgG) complexes was determined. When limiting amounts of antiglobulin were used, antibody directed against the Fab portion of human IgG was significantly more effective than anti-Fc antibodies in neutralizing the HSV-IgG complexes. The detection of viral bound antibody was enhanced by the incorporation of heterologous antiglobulin or complement in the antiglobulin neutralization test. Specifically, HSV-IgG which had been incubated with rabbit antihuman globulin was further neutralized by goat antirabbit IgG or guinea pig serum complement. This augmented neutralization test could prove useful in detecting small amounts of antibody bound to virus in infectious isolates from patients or experimental animals with viral diseases.With 4 Figures     

Infection of human peripheral blood mononuclear cells by erythrocyte-bound HIV-1: effects of antibodies and complement

Two human monoclonal antibodies, 4E10 and b12, were examined for antibody-dependent neutralization, or antibody-dependent complement (C)-mediated neutralization, of infection of peripheral blood mononuclear cells (PBMC) by either free HIV-1 or trans infection by HIV bound to erythrocytes. Neutralization of free HIV-1 by b12 was stronger than by 4E10, but b12 neutralized erythrocyte-bound HIV-1 less efficiently than cell-free virus. 4E10 did not neutralize erythrocyte-bound HIV-1 and at a low concentration it caused enhancement of infection. Antibody (4E10)-dependent C activation inhibited trans infection by erythrocyte-bound HIV-1, but caused enhanced infection with cell-free HIV-1 in the presence of erythrocytes. No effects of C were observed with b12. C-dependent enhancement in the presence of erythrocytes is proposed as due to binding of C3b-4E10-cell-free-HIV or C3d-4E10-cell-free-HIV to C receptor type 1 (CR1) on erythrocytes, or C receptor type 2 (CR2) on B cells in the PBMC, followed by trans infection of susceptible cells.     

Humanization of the anti-CD18 antibody 6.7: an unexpected effect of a framework residue in binding to antigen

Humanization of monoclonal antibodies by complementary determinant region (CDR)-grafting has become a standard procedure to improve the clinical usage of animal antibodies. However, antibody humanization may result in loss of activity that has been attributed to structural constraints in the framework structure. In this paper, we report the complete humanization of the 6.7 anti-human CD18 monoclonal antibody in a scFv form. We used a germline-based approach to design a humanized VL gene fragment and expressed it together with a previously described humanized VH. The designed humanized VL has only 14 mutations compared to the closest human germline sequence. The resulting humanized scFv maintained the binding capacity and specificity to human CD18 expressed on the cell surface of peripheral blood mononuclear cells (PBMC), and showed the same pattern of staining T-lymphocytes sub-populations, in comparison to the original monoclonal antibody. We observed an unexpected effect of a conserved mouse-human framework position (L37) that hinders the binding of the humanized scFv to antigen. This paper reveals a new framework residue that interferes with paratope and antigen binding and also reinforces the germline approach as a successful strategy to humanize antibodies.     

HIV-1 neutralization by chimeric CD4-CG10 polypeptides fused to human IgG1

The envelope glycoprotein of HIV-1 is the principal target for entry inhibitors. The use of soluble CD4 has been found to be impractical as most clinical isolates are resistant to neutralization at feasible concentrations. CG10 is one of a small group of monoclonal antibodies specific to CD4-induced epitopes, which are structurally associated with the chemokine receptor-binding site and are capable of blocking the interaction of gp120 with its obligatory co-receptor. We have reasoned that fusing the single chain Fv of CG10 with CD4 can lead to increased HIV-1 neutralization activity and that this effect could be further enhanced by engrafting this chimeric construct onto an IgG Fc. Here we report the cloning of the genes encoding the variable regions of CG10 heavy and light chains and demonstrate that when attached to human IgG1 Fc, the single chain Fv of CG10 retains the binding properties of the original mouse antibody. Fusing CG10 single chain Fv with the gp120-binding portion of CD4 on a human IgG1 Fc backbone results in stronger binding of gp120 of different tropisms and in enhanced neutralization of laboratory-adapted strains and most, but not all, clade B and clade C isolates tested. Our findings underscore the potential use of CD4-based fusion proteins in the design of HIV immuno-therapeutics.