IgG1 anti‐epidermal growth factor receptor antibodies induce CD8‐dependent antitumor activity

Anti‐EGFR monoclonal antibodies (mAb) like Cetuximab are commonly used for treatment of EGFR⁺ solid tumors mainly by exerting their therapeutic effect through inhibition of signal transduction. Additionally, IgG1 is a potent mediator of antibody‐dependent cytotoxicity (ADCC). In case of the IgG1, Cetuximab induction of ADCC in vivo is controversially discussed. In our study, we investigated the efficiency of Cetuximab‐mediated ADCC in a humanized mouse tumor model in vivo and analyzed the contribution of immunologic processes toward antitumor activity. Therefore, we used immunodeficient NOD/Scid mice transgenic for human MHC class I molecule HLA‐A2 and adoptively transferred human HLA‐A2⁺ PBMC after engraftment of human epidermoid cell carcinoma A431. Here, we show that high doses of anti‐EGFR mAb induced strong tumor regression independent of the immune system. However, tumor regression by low doses of anti‐EGFR mAb treatment was ADCC dependent and mediated by tumor infiltrating CD8⁺ T effector cells. This novel mechanism of ADCC conducted by CD8⁺ T effector cells was restricted to IgG1 anti‐EGFR mAb, dependent of binding to CD16 on T cells and could be inhibited after EGFR blockade on tumor cells. Furthermore, CD8⁺ T effector cell‐mediated ADCC was enhanced in the presence of IL‐15 and strongly improved after glycosylation of anti‐EGFR mAb indicating the potential of glycoengineered therapeutic mAb as efficient biologicals in cancer therapy.     

An Fc‐optimized NKG2D‐immunoglobulin G fusion protein for induction of natural killer cell reactivity against leukemia

Recruitment of Fc‐receptor‐bearing effector cells, such as natural killer (NK) cells, is a feature critical for the therapeutic success of antitumor antibodies and can be improved by the modifications of an antibody's Fc part. The various ligands of the activating immunoreceptor NKG2D, NKG2DL) are selectively expressed on malignant cells including leukemia. We here took advantage of the tumor‐associated expression of NKG2DL for targeting leukemic cells by NKG2D–immunoglobulin G (IgG)1 fusion proteins containing modified Fc parts. Compared to NKG2D–Fc containing a wild‐type Fc part (NKG2D–Fc–WT), our mutants (S239D/I332E and E233P/L234V/L235A/ΔG236/A327G/A330S) displayed highly enhanced (NKG2D–Fc–ADCC) and abrogated (NKG2D–Fc–KO) affinity to the NK cell Fc receptor, respectively. Functional analyses with allogenic as well as autologous NK cells and primary malignant cells of leukemia patients revealed that NKG2D–Fc–KO significantly reduced NK reactivity by blocking immunostimulatory NKG2D–NKG2DL interaction. NKG2D–Fc–WT already enhanced antileukemia reactivity by inducing antibody‐dependent cellular cytotoxicity (ADCC) with NKG2D–Fc–ADCC mediating significantly stronger effects. Parallel application of NKG2D–Fc–ADCC with Rituximab caused additive effects in lymphoid leukemia. In line with the tumor‐associated expression of NKG2DL, no NK cell ADCC against resting healthy blood cells was induced. Thus, NKG2D–Fc–ADCC potently enhances NK antileukemia reactivity despite the inevitable reduction of activating signals upon binding to NKG2DL and may constitute an attractive means for immunotherapy of leukemia.     

Recombinant humanized anti-CD40 monoclonal antibody triggers autologous antibody-dependent cell-mediated cytotoxicity against multiple myeloma cells

Multiple myeloma (MM) is currently incurable, and novel therapies are needed. In this study, we examined a novel recombinant humanized monoclonal antibody against CD40 (rhuCD40 mAb) and demonstrate for the first time that rhuCD40 mAb induces antibody-dependent cell-mediated cytotoxicity (ADCC) against CD40-positive MM cells. Importantly, we show that rhuCD40 mAb induces autologous ADCC against primary patient MM cells, without triggering ADCC against normal B cells. This study, therefore, both demonstrates that rhuCD40 mAb triggers autologous ADCC against patient MM cells and provides the framework for the clinical evaluation of rhuCD40 mAb immunotherapy to improve patient outcome in MM.     

HIV-1 Envelope Glycoproteins Proteolytic Cleavage Protects Infected Cells from ADCC Mediated by Plasma from Infected Individuals

The HIV-1 envelope glycoprotein (Env) is synthesized in the endoplasmic reticulum as a trimeric gp160 precursor, which requires proteolytic cleavage by a cellular furin protease to mediate virus-cell fusion. Env is conformationally flexible but controls its transition from the unbound “closed” conformation (State 1) to downstream CD4-bound conformations (States 2/3), which are required for fusion. In particular, HIV-1 has evolved several mechanisms that reduce the premature “opening” of Env which exposes highly conserved epitopes recognized by non-neutralizing antibodies (nnAbs) capable of mediating antibody-dependent cellular cytotoxicity (ADCC). Env cleavage decreases its conformational transitions favoring the adoption of the “closed” conformation. Here we altered the gp160 furin cleavage site to impair Env cleavage and to examine its impact on ADCC responses mediated by plasma from HIV-1-infected individuals. We found that infected primary CD4+ T cells expressing uncleaved, but not wildtype, Env are efficiently recognized by nnAbs and become highly susceptible to ADCC responses mediated by plasma from HIV-1-infected individuals. Thus, HIV-1 limits the exposure of uncleaved Env at the surface of HIV-1-infected cells at least in part to escape ADCC responses.     

In vivo induction of functional FcγRI (CD64) on neutrophils and modulation of blood cytokine mRNA levels in cancer patients treated with G-CSF (rMetHuG-CSF)

Neutrophils from 13 children who received G-CSF for the collection of peripheral blood progenitors while they were in haematological steady state were studied at various times after G-CSF injection for FcγR expression (FcγRI or CD 64, FcγRII or CD32, and FcγRIII or CD16) and for their ability to exert antibody-dependent cell cytotoxicity (ADCC) through FcγRI. Changes in IFNγ, IL8, IL10, MCP1 and TNFα mRNA levels in peripheral blood cells were also studied 4 h and 24 h after the first G-CSF injection. FcγRI expression increased strongly after 24 h and then remained at the same level throughout treatment. In contrast, FcγRIII expression sharply decreased at day 1 and diminished even further thereafter. No change in FcγRII was observed. ADCC exerted by neutrophils through FcγRI started to increase after 24 h with the peak level at day 5. Cytokine mRNA analyses indicated a reproducible and strong increase of IL8 mRNA (11/13 children) after 24 h, whereas the changes in the mRNA levels of the other cytokines tested were more heterogenous (IFNγ: three; IL10: six; MCP1: five; TNFα: four, of the 13 children). Therefore this study opens the way to an optimized therapeutic schedule for the combined use of G-CSF and monoclonal antibodies in adjuvant immuno-intervention.     

小鼠感染后不同时间血清对日本血吸虫童虫细胞介导的细胞毒作用

用抗体依赖、细胞介导的细胞毒试验(ADCC)观察了小鼠感染后不同时间血清在体外杀伤系统中对日本血吸虫童虫的作用规律。未加补体时,嗜酸粒细胞或巨噬细胞介导的童虫杀伤作用于感染后4wk出现明显作用,分别于5—7wk和6—8wk达高峰,然后下降,至11wk时降至4wk时的水平;而未加补体时无明显的中性粒细胞介导的杀伤作用。在补体参与下,这三种细胞均能介导对童虫的杀伤作用,且作用增强。证实ADCC是日本血吸虫获得性抵抗力的一个重要组分,嗜酸粒细胞、巨噬细胞介导的对童虫的细胞毒作用是不依赖补体的,而中性粒细胞介导的作用是依赖补体的。结果对选择免疫预防适宜时间和评价候选疫苗效果有参考意义。     

HIV-1 Gag和Env蛋白特异性ADCC反应与AIDS病程进展的关系

目的了解艾滋病病毒I型（HIV-1）Gag和Env蛋白特异性抗体依赖性细胞介导的细胞毒作用（ADCC）反应,与艾滋病（AIDS）疾病进展的相关性。方法采集58份未经抗病毒治疗的、感染时间1年以内的男男性行为人群（MSM）标本,利用HIV-1特异性肽库刺激和流式细胞染色技术进行ADCC检测。结果病毒载量与HIV-1Gag特异性CD＋2IFN-γ＋细胞占CD-3细胞的比例呈显著的正相关（r=0.2919,P=0.0339）;而01_AE亚型感染者的病毒载量与HIV-1Env特异性CD＋2CD107a＋细胞占CD-3细胞的比例呈显著负相关（r=-0.3454,P=0.0454）。结论在HIV-1感染早期,感染者体内可以产生Gag蛋白特异性的ADCC反应,其中Env蛋白特异性的ADCC反应具有控制疾病进展的作用。     

实验性恙虫病立克次体感染或免疫鼠的ADCC活性和cAMP水平变化

本文用~(61)Cr释放试验和~3H-cAMP蛋白竞争结合分析法研究了实验性恙虫病立克次体感染或免疫鼠的ADCC活性和cAMP水平变化。结果发现强毒株(B株)和减毒株(49株)接种后均能导致鼠脾细胞的ADCC水平增高及cAMP水平降低,但灭活立克次体接种后对鼠脾细胞的ADCC活性及cAMP水平则无明显影响。     

Balance of cellular and humoral immunity determines the level of protection by HIV vaccines in rhesus macaque models of HIV infection

A guiding principle for HIV vaccine design has been that cellular and humoral immunity work together to provide the strongest degree of efficacy. However, three efficacy trials of Ad5-vectored HIV vaccines showed no protection. Transmission was increased in two of the trials, suggesting that this vaccine strategy elicited CD4+ T-cell responses that provide more targets for infection, attenuating protection or increasing transmission. The degree to which this problem extends to other HIV vaccine candidates is not known. Here, we show that a gp120-CD4 chimeric subunit protein vaccine (full-length single chain) elicits heterologous protection against simian-human immunodeficiency virus (SHIV) or simian immunodeficiency virus (SIV) acquisition in three independent rhesus macaque repeated low-dose rectal challenge studies with SHIV162P3 or SIVmac251. Protection against acquisition was observed with multiple formulations and challenges. In each study, protection correlated with antibody-dependent cellular cytotoxicity specific for CD4-induced epitopes, provided that the concurrent antivaccine T-cell responses were minimal. Protection was lost in instances when T-cell responses were high or when the requisite antibody titers had declined. Our studies suggest that balance between a protective antibody response and antigen-specific T-cell activation is the critical element to vaccine-mediated protection against HIV. Achieving and sustaining such a balance, while enhancing antibody durability, is the major challenge for HIV vaccine development, regardless of the immunogen or vaccine formulation.There are formidable difficulties for developing a vaccine against a retrovirus such as HIV because of the integration of its genes into the DNA of the host target cells upon infection. For HIV, this problem is compounded by HIV-induced immune suppression and the development of variants that escape immune control. Consequently, an effective preventive vaccine against HIV must work early to block HIV infection and quickly kill HIV-infected cells, or both. To date, only antibodies to the HIV envelope glycoprotein (Env) fit this requirement. Available evidence suggests that such antibodies must recognize highly conserved domains and could inhibit infection by direct neutralization or by Fc receptor-dependent effector mechanisms including antibody-dependent cellular cytotoxicity (ADCC) (1, 2). The ideal result would be sterilizing immunity or, at a minimum, a major restriction of the infection (3). Another challenge stems from evolutionary pressures that abrogate the immunogenicity of conserved, functional epitopes on the envelope spike that are potential targets for cross-reactive antibodies. Large areas are masked by a “glycan shield” of carbohydrate molecules and extensive conformational flexibility (sometimes termed “conformational masking”) that dampen immunogenicity of the conserved functional domains (4, 5). The remaining immunogenic domains (“variable” or “V” loops) tolerate a high degree of sequence variability and generate “type-specific” neutralizing antibodies that are not cross-reactive and that limit the efficacy of vaccines that use conventional gp120 monomeric protein.An emerging concern for HIV vaccine development centers on the quantitative and qualitative aspects of T-cell activation elicited by various immunization regimens (6). Although HIV-specific T cells might potentially combat infection, certain patterns of T-cell activation (e.g., involving CD4+ CCR5+ T cells) have the potential to promote HIV replication. The latter possibility is emphasized by the HIV vaccine-associated increased risk of infection seen in two large human clinical trials that selectively generated HIV-specific T-cell responses (7). Similar associations between increased risk of infection and T-cell responses of various sorts have been reported in the nonhuman primate model (8–10). Thus, the ideal HIV vaccine strategy is likely to be one that generates antiviral humoral responses without incurring T-cell activation profiles that promote infection and/or overcome the protective benefits of antibodies. Insights for such an approach can be gained by comparative analyses of nonhuman primate models of HIV infection.The vaccine concept that we have been testing is designed to overcome some of these challenges by stably expressing a highly conserved transition state structure that is exposed on gp120 during a key step in viral entry, exposure of the coreceptor-binding domain consequent to CD4 binding. The prototype immunogen [full-length single chain (FLSC)] is a chimeric protein composed of gp120 from the HIV-1_Ba-L isolate fused to the N terminus of the two outer domains of CD4 by a flexible polypeptide linker (11). For studies of rhesus macaques, the construct is modified to contain “self” rhesus macaque CD4 sequences (rhFLSC) to avoid anti-CD4 responses. The rhFLSC elicits antibody responses to highly conserved epitopes, including the coreceptor-binding domain epitopes (CoRBS) and the C1 regions implicated as a potent ADCC target (12). In an earlier study (12), we showed that rhesus macaques vaccinated with rhFLSC formulated with QS21, a saponin adjuvant derived from the soap-bark tree Q. saponaria, exhibited accelerated clearance of plasma viremia and an absence of long-term tissue viral loads compared with unvaccinated controls after a single high-dose rectal challenge with heterologous SHIV162P3. Postinfection control correlated with stronger responses to CD4i epitopes in the rhFLSC-vaccinated animals (CD4i titers > 1:100), compared with macaques that received control immunogens including gp120, soluble CD4, or chemically cross-linked gp120-CD4. Postinfection control did not correlate with anti-CD4 responses, overall anti-gp120–binding titers, or neutralizing activity measured in conventional assays (12), although it did correlate with neutralizing titers in the soluble CD4-triggered assay using HIV-2_7312A/V434M that selectively detects responses to highly conserved epitopes in the coreceptor-binding site (13). Taken together, this study showed that rhFLSC elicits antibody responses to highly conserved CD4i epitopes that correlate with postinfection control of viremia after a high-dose rectal challenge with SHIV162P3, but it left open the question of whether rhFLSC can elicit antibodies that block acquisition. Acquisition is typically blocked only in high-dose challenge studies when the vaccine and challenge stock are matched (14), which is not the case for rhFLSC and SHIV162P3. For this reason, we performed three independent studies using different rhFLSC immunization schemes and a repeat low-dose rectal challenge model that is thought to be more reflective of sexual HIV transmission (15). These studies were designed in part as a hypothesis-generating exercise with respect to protective immunity. We consistently found (i) inverse correlations between acquisition of infection and certain aspects of humoral immunity and (ii) direct relationships between acquisition of infection and vaccine-elicited T-cell responses. Importantly, in certain test groups the apparent protective benefit of humoral responses is absent when T-cell responses are comparatively high. These results strongly suggest that a successful HIV vaccine will need to elicit protective antibody responses without eliciting attenuating levels of vaccine-elicited T-cell responses.     

Antibody-dependent cell cytotoxicity against Trypanosoma cruzi is only mediated by protective antibodies

The antibody-dependent cell cytotoxicity (ADCC) to Trypanosoma cruzi blood forms (Btry) using non-adherent spleen cells is only mediated by sera from chronic chagasic patients or mice. Both display 'lytic antibodies' (LA), which are immunoglobulins directed against epitopes only present in living BTry, and 'conventional serology antibodies' (CSA), which are responsible for the positive diagnostic tests in Chagas disease. Sera from mice immunized with different T. cruzi antigens or from treated patients (displaying only CSA but not LA) are unable to mediate ADCC. These data confirm the central role played by LA in the host resistance against T. cruzi. Moreover, they probably explain why most immunizing agents used as vaccines in Chagas' disease and which elicit CSA but not LA, do not display significant protection against T. cruzi. We also demonstrate that trypsinization of BTry increases significantly the rate of parasite destruction by ADCC, suggesting that enzyme sensitive membrane components may help BTry to evade from this immune effector mechanism.