眼肌结合抗体的测定及其致免疫损伤机制的研究

用牛和猪眼肌抗原建立了检测眼肌结合抗体（ＥＭｂ Ａｂ ） 的ＥＬＩＳＡ法。证实甲亢患者血清中存在ＥＭｂ Ａｂ。通过ＥＭｂ Ａｂ 特异免疫复合物、补体复合物解离活性抗体依赖的细胞毒或补体介导的细胞毒（ＡＤＣＣ或ＣＭＡＣ） 测定, 以及ＥＭｂ Ａｂ 与眼肌细胞抗原结合的实验观察, 表明此种ＥＭｂ Ａｂ 可能在与已受某种因素损伤后的眼肌结合时, 通过ＡＤＣＣ反应加剧眼肌损伤。     

CD4 mimetics sensitize HIV-1-infected cells to ADCC

HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 has evolved a sophisticated mechanism to avoid exposure of ADCC-mediating Env epitopes by down-regulating CD4 and by limiting the overall amount of Env at the cell surface. Here we report that small-molecule CD4-mimetic compounds induce the CD4-bound conformation of Env, and thereby sensitize cells infected with primary HIV-1 isolates to ADCC mediated by antibodies present in sera, cervicovaginal lavages, and breast milk from HIV-1-infected individuals. Importantly, we identified one CD4 mimetic with the capacity to sensitize endogenously infected ex vivo-amplified primary CD4 T cells to ADCC killing mediated by autologous sera and effector cells. Thus, CD4 mimetics hold the promise of therapeutic utility in preventing and controlling HIV-1 infection.Worldwide, it is estimated that more than 35 million people are living with HIV. In 2013 alone, around 2.1 million people became newly infected with HIV, and 1.5 million people died from AIDS (1). Measures to prevent HIV-1 transmission are desperately needed. Prevention of HIV-1 transmission and progression likely requires approaches that can specifically target and eliminate HIV-1-infected cells. Interestingly, there is increasing evidence supporting a role of antibody (Ab)-dependent cell-mediated cytotoxicity (ADCC) in controlling HIV-1 transmission and disease progression (2–8). Analysis of the correlates of protection in the RV144 vaccine trial suggested that increased ADCC activity was linked with decreased HIV-1 acquisition (9), and Abs with potent ADCC activity were isolated from some RV144 vaccinees (10). Recent studies reported that the viral accessory proteins Nef and Vpu protect HIV-1-infected cells from anti-HIV-1 envelope (Env)-mediated ADCC responses (11–14). Importantly, we and others reported that Env in the CD4-bound conformation was preferentially targeted by ADCC-mediating Abs and sera from HIV-1-infected individuals (11, 12, 15, 16), which represent a significant proportion of anti-Env Abs elicited during natural HIV infection (11, 17). However, the vast majority of circulating HIV-1 strains worldwide express functional Nef and Vpu proteins, which limit the exposure of CD4-induced (CD4i) Env epitopes at the surface of infected cells, likely preventing ADCC responses.Theoretically, agents promoting the CD4-bound Env conformation should expose CD4i epitopes that are readily recognized by ADCC-mediating Abs and sera from infected individuals (11, 12, 15, 16, 18), resulting in the sensitization of HIV-1-infected cells to ADCC. Importantly, modulating Env conformation at the surface of HIV-1-infected cells has become feasible as a result of the availability of small CD4-mimetic compounds (CD4mc). The prototypes of such compounds, NBD-556 and NBD-557, were discovered in a screen for inhibitors of gp120-CD4 interaction (19). These small-molecule ∼337-Da compounds and recent derivatives (DMJ-I-228, JP-III-48) bind in the Phe-43 cavity (20–22), a highly conserved ∼150-ų pocket in the gp120 glycoprotein located at the interface of the inner domain, outer domain, bridging sheet, and CD4 receptor (23). CD4mc block gp120-CD4 interaction and induce thermodynamic changes in gp120 similar to those observed during CD4 or soluble CD4 (sCD4) binding (24). Accordingly, these small molecules, as well as sCD4, can promote the transition of Env to the CD4-bound conformation, thus sensitizing HIV-1 particles to neutralization by otherwise nonneutralizing CD4i Abs (17, 25). Additional strategies using scaffolded miniproteins targeting critical gp120 elements required for CD4 interaction allowed the identification of CD4 mimetics with nanomolar affinity for gp120 (26). One of these variants, M48U1, displayed remarkably potent neutralization of three HIV-1 isolates (27). Its crystal structure in complex with HIV-1 gp120 was recently solved, showing that M48U1 engages the Phe-43 cavity in a manner similar to that of CD4 (28); thus, M48U1 might induce gp120 to adopt the CD4-bound conformation and expose CD4i epitopes. Previous studies exploring the antiviral properties of CD4mc were performed on viral particles (17, 25, 27). However, whether these compounds are able to engage the large amounts of Env present at the surface of infected cells and modulate Env conformation in a way that allows exposure of ADCC-mediating epitopes is currently not known. In this study, we show that CD4mc strongly sensitize HIV-1-infected primary CD4 T cells to ADCC mediated by sera, cervicovaginal fluids, and breast milk from HIV-1-infected individuals, as well as help eliminate infected, ex vivo-expanded primary CD4 T cells from HIV-1-infected individuals. Therefore, CD4mc possess three valuable complementary antiviral properties: direct inactivation of viral particles, sensitization of viral particles to neutralization by otherwise nonneutralizing Abs, and sensitization of HIV-1-infected cells to ADCC-mediated killing.     

Nimotuzumab: beyond the EGFR signaling cascade inhibition

One of the most known oncogenes is the epidermal growth factor receptor (EGFR) family. It activates multiple signaling cascades that promote carcinogenesis and immune evasion. Therefore, these molecules have been extensively targeted in cancer immunotherapy. Beyond EGFR signaling cascade inhibition, some of these agents are able to induce T-cell activation, transforming a passive therapy into a vaccine-like effect. Nimotuzumab is an IgG1 humanized monoclonal antibody directed against the extracellular domain of the EGFR blocking the binding to its ligands. It possesses unique pharmacodynamic properties, which allow treating patients for long-term periods and with very low toxicity. Based on its clinical effect, nimotuzumab has been approved in Cuba and abroad for the treatment of different epithelial tumors. Recently, new potential mechanisms of action of nimotuzumab involving the activation of the innate and adaptive immune response have been reported. This review summarizes the main properties of nimotuzumab in comparison with other EGFR-specific monoclonal antibodies, highlighting its capacity to activate an effective immune response. In addition, differential clinical effects of this antibody and ongoing clinical trials to deeply characterize the biomarkers of clinical benefit are shown.     

A Fc engineering approach to define functional humoral correlates of immunity against Ebola virus

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Failure of ADCC to predict HIV-associated disease progression or outcome in a haemophiliac cohort.

Antibody-dependent cell-mediated cytotoxicity (ADCC) has been described in a number of virus infections and occurs in HIV infection. In spite of numerous studies on the ability of HIV-positive sera to mediate ADCC, it remains unclear whether ADCC has any functional significance. Here we examine serial serum samples from a cohort of haemophilia patients who became infected from a common source in 1984, and show that there is no significant difference in ADCC values between those who remained asymptomatic and those who progressed to disease. This study does not compare effector cell function and clinical status and does not exclude activity against different target cell lineages in vivo.     

Antitumor activities against breast cancers by an afucosylated anti-HER2 monoclonal antibody H2Mab-77-mG2a-f

Breast cancer patients with high levels of human epidermal growth factor receptor 2 (HER2) expression have worse clinical outcomes. Anti-HER2 monoclonal antibody (mAb) is the most important therapeutic modality for HER2-positive breast cancer. We previously immunized mice with the ectodomain of HER2 to create the anti-HER2 mAb, H₂Mab-77 (mouse IgG₁, kappa). This was then altered to produce H₂Mab-77-mG_2a-f, an afucosylated mouse IgG_2a. In the present work, we examined the reactivity of H₂Mab-77-mG_2a-f and antitumor effects against breast cancers in vitro and in vivo. BT-474, an endogenously HER2-expressing breast cancer cell line, was identified by H₂Mab-77-mG_2a-f with a strong binding affinity (a dissociation constant [K_D]: 5.0 × 10⁻⁹ M). H₂Mab-77-mG_2a-f could stain HER2 of breast cancer tissues in immunohistochemistry and detect HER2 protein in Western blot analysis. Furthermore, H₂Mab-77-mG_2a-f demonstrated strong antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) for BT-474 cells. MDA-MB-468, a HER2-negative breast cancer cell line, was unaffected by H₂Mab-77-mG_2a-f. Additionally, in the BT-474-bearing tumor xenograft model, H₂Mab-77-mG_2a-f substantially suppressed tumor development when compared with the control mouse IgG_2a mAb. In contrast, the HER2-negative MDA-MB-468-bearing tumor xenograft model showed no response to H₂Mab-77-mG_2a-f. These findings point to the possibility of H₂Mab-77-mG_2a-f as a treatment regimen by showing that it has antitumor effects on HER2-positive breast tumors.     

Host-dependent variables: The missing link to personalized medicine

Individualized medicine has the potential to tailor anticancer therapy with the best response and highest safety margin to provide better patient care. However, modern targeted therapies are still being tested through clinical trials comparing preselected patient cohorts and assessed upon behaviour of group averages. Clinically manifesting malignant disease requires identification of host- and tumour-dependent variables such as biological characteristics of the tumour and its microenvironment including immune response features, and overall capacity of the host to receive, tolerate and efficiently utilize treatment. Contemporary medical oncology including clinical trial design need to refocus from assessing group averages to individuality taking into consideration time dependent host-associated characteristics and reinventing outliers to be appreciated as naturally occurring variables collectively determining the ultimate outcome of malignant disease.     

A novel antibody-like TCRγδ-Ig fusion protein exhibits antitumor activity against human ovarian carcinoma

TCRγ9δ2(OT3) is a tumor-specific TCR with an unique complementarity-determining region 3 (CDR3) sequence, referred to as OT3, in its δ2 chain. This region was identified in tumor-infiltrating lymphocytes (TILs) from human ovarian epithelial carcinoma. We demonstrated that TCRγ9δ2(OT3)-Fc, a fusion protein composed of the complete extracellular domains of the γ9 and δ2 chains linked to the Fc domains of human IgG1, exhibited successful binding to multiple human carcinoma cell lines. In vitro, TCRγ9δ2(OT3)-Fc mediated cell killing via antibody-dependent cellular cytotoxicity (ADCC) in a dose-dependent manner. In vivo, TCRγ9δ2(OT3)-Fc significantly inhibited tumor growth and enhanced survival in human ovarian carcinoma xenograft models. Our findings suggest that the TCRγ9δ2(OT3)-Fc fusion protein possesses both the antigen-recognition properties of TCR γδ and the Fc-mediated effector functions of the antibody.     

Antibody to cloned HSV glycoproteins B and D plus adult human leukocytes protect neonatal mice from lethal HSV infection

Antisera produced by HSV infection or following vaccination of guinea pigs with the cloned herpes simplex virus (HSV) glycoproteins gB and gD were compared for in vitro antibody-dependent cellular cytotoxicity (ADCC) activity and for in vivo protection. Antibody from guinea pigs was able to participate in ADCC with human mononuclear cells in vitro, anti-gBgD serum being equivalent to HSV convalescent sera. In vivo, each of the guinea pig sera was able to protect neonatal mice from a fatal HSV-1 infection when given with human mononuclear cells but not when given alone. The anti-gBgD serum was the most effective in vivo, protecting 15 of 17 (88%) neonatal mice when given at a 10⁻⁴ dilution with human mononuclear cells and was the only guinea pig serum protective at a 10⁻⁶ dilution (5 of 7 neonatal mice).     

Different types of FCgamma-receptors are involved in anti-Lewis Y antibody induced effector functions in vitro

Stimulation of monocytes by interaction of monoclonal antibodies (mAbs) with Fc gamma receptors (FcgammaRs) results in the activation of various monocyte effector functions. In the present investigation we show that the anti-Lewis Y (LeY) anti-tumour mAb ABL 364 and its mouse/human IgG1 chimaera induce both antibody-dependent cellular cytotoxicity (ADCC) and the release of tumour necrosis factor alpha (TNF-alpha) during mixed culture of monocytes with LeY+ SKBR5 breast cancer cells in vitro. Although anti-LeY mAb-mediated TNF-alpha release paralleled ADCC activity, cytokine release required a higher concentration of sensitizing mAb than the induction of cytolysis. The determination of the FcgammaR classes involved in the induction of the distinct effector functions showed that anti-LeY mAb-induced cytolysis was triggered by interaction between anti-LeY mAbs and FcgammaRI. In contrast, mAb-induced TNF-alpha release mainly depended on the activation of monocyte FcgammaRII. Neutralization of TNF-alpha showed no influence on monocyte ADCC activity towards SKBR5 target cells. Our data indicate an independent regulation of anti-LeY mAb induced effector functions of ADCC and TNF-alpha release which seemed to be triggered by activation of different types of FcgammaR.