CD16-158-valine chimeric receptor T cells overcome the resistance of KRAS-mutated colorectal carcinoma cells to cetuximab

KRAS mutations hinder therapeutic efficacy of epidermal growth factor receptor (EGFR)-specific monoclonal antibodies cetuximab and panitumumab-based immunotherapy of EGFR+ cancers. Although cetuximab inhibits KRAS-mutated cancer cell growth in vitro by natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), KRAS-mutated colorectal carcinoma (CRC) cells escape NK cell immunosurveillance in vivo. To overcome this limitation, we used cetuximab and panitumumab to redirect Fcγ chimeric receptor (CR) T cells against KRAS-mutated HCT116 colorectal cancer (CRC) cells. We compared four polymorphic Fcγ-CR constructs including CD16^158F-CR, CD16^158V-CR, CD32^131H-CR, and CD32^131R-CR transduced into T cells by retroviral vectors. Percentages of transduced T cells expressing CD32^131H-CR (83.5 ± 9.5) and CD32^131R-CR (77.7 ± 13.2) were significantly higher than those expressing with CD16^158F-CR (30.3 ± 10.2) and CD16^158V-CR (51.7 ± 13.7) (p < 0.003). CD32^131R-CR T cells specifically bound soluble cetuximab and panitumumab. However, only CD16^158V-CR T cells released high levels of interferon gamma (IFNγ = 1,145.5 pg/ml ±16.5 pg/ml, p < 0.001) and tumor necrosis factor alpha (TNFα = 614 pg/ml ± 21 pg/ml, p < 0.001) upon incubation with cetuximab-opsonized HCT116 cells. Moreover, only CD16^158V-CR T cells combined with cetuximab killed HCT116 cells and A549 KRAS-mutated cells in vitro. CD16^158V-CR T cells also effectively controlled subcutaneous growth of HCT116 cells in CB17-SCID mice in vivo. Thus, CD16^158V-CR T cells combined with cetuximab represent useful reagents to develop innovative EGFR+KRAS-mutated CRC immunotherapies.     

抗表皮生长因子受体单抗治疗晚期结直肠癌疗效预测因子的研究

 研究证实抗表皮生长因子受体（EGFR）单克隆抗体西妥昔单抗和帕尼单抗是晚期结直肠癌的有效治疗药物，采用免疫组织化学测定的EGFR表达强弱与临床疗效无关。介绍了目前有可能预测从西妥昔单抗或帕尼单抗治疗中受益的标志物，包括KRAS突变、EGFR拷贝数、EGFR配体（EGF、表皮调节素和双调蛋白）、细胞周期蛋白D1、IgG FcγR（FCGR2A-H131R和FCGR3A-V158F）和核因子κB。这些标志物将在避免抗EGFR治疗毒性、减少治疗费用方面起到重要作用。     

Tumour antigen targeted monoclonal antibodies incorporating a novel multimerisation domain significantly enhance antibody dependent cellular cytotoxicity against colon cancer

Tumour antigen targeted antibodies (mAbs) can induce natural killer (NK) cells to kill tumours through antibody dependent cellular cytotoxicity (ADCC) upon engagement of NK cell expressed FcγRIIIa. FcγRIIIa polymorphisms partially dictate the potency of the ADCC response. The high affinity FcγRIIIa-158-valine (V) polymorphism is associated with more potent ADCC response than the low affinity FcγRIIIa-158-phenylalanine (F) polymorphism. Because approximately 45% of patients are homozygous for the FcγRIIIa-158-F polymorphism (FF genotype), their ability to mount ADCC is impaired. We investigated whether a novel mAb capable of binding multiple antigen specific targets and engaging multiple low affinity FcγRIIIa receptors could further enhance ADCC against colon cancer in vitro. Specifically, we generated a novel anti-epidermal growth factor receptor (EGFR) antibody (termed a stradobody?) consisting of an unmodified Fab sequence and two Immunoglobulin G, subclass 1 (IgG1) Fc domains separated by an isoleucine zipper domain and the 12 amino-acid IgG2 hinge. The stradobody? framework induced multimerisation and was associated with increased binding to the EGFR and FcγRIIIa. From a functional perspective, when compared to an unmodified anti-EGFR mAb with a sequence identical to cetuximab (a commercially available anti-EGFR mAb), stradobodies? significantly enhanced ADCC. These effects were observed using both KRAS wild type HT29 and KRAS mutant SW480 colon cancer cells as targets, and by NK cells obtained from healthy donors and a cohort of patients with colon cancer. These data suggest that high avidity cross-linking of multiple tumour surface antigens and multiple NK cell associated FcγRIIIa molecules can enhance ADCC and partially overcome impaired ADCC by FF genotype individuals in vitro.     

Immunoglobulin G fragment C receptor polymorphisms and KRAS mutations: Are they useful biomarkers of clinical outcome in advanced colorectal cancer treated with anti‐EGFR‐based therapy?

KRAS mutations have been identified as a strong predictor of resistance to anti‐epidermal growth factor receptor (EGFR) therapies. Besides inhibiting the EGFR pathway, anti‐EGFR monoclonal antibodies may exert antitumor effects through antibody‐dependent cell‐mediated cytotoxicity (ADCC). Through this mechanism, the antibody fragment C portion (Fcγ) interacts with Fc receptors (FcγRs) expressed by immune effectors cells. We investigated the association of FcγR polymorphisms and KRAS mutation with the clinical outcome of 104 refractory metastatic colorectal cancer (mCRC) patients treated with anti‐EGFR antibodies. FcγRIIa‐H131R and FcγRIIIa‐V158F polymorphisms were analyzed in genomic DNA using a 48.48 dynamic array on the BioMark system (Fluidigm, South Sanfrancisco, CA, USA). Tumor tissues from 96 cases were screened for KRAS mutations. KRAS mutation was associated with a lower response rate (RR) (P = 0.035) and a shorter progression‐free survival (PFS) (3 vs 7 months; P = 0.36). FcγRIIa‐H131R and FcγRIIIa‐V158F polymorphisms did not show statistically significant associations with response, PFS, or KRAS status. In the logistic regression analysis, KRAS status (P = 0.04) and skin toxicity (P = 0.03) were associated with RR. By multivariate analysis, the clinical risk classification (P = 0.006) and skin toxicity (P < 0.0001) were found to be independent risk factors for PFS. In conclusion, the FcγRIIa and FcγRIIIa polymorphisms are not useful as molecular markers for clinical outcome in mCRC patients. To date, the EORTC (European Organization for Research and Treatment of Cancer Classification), skin toxicity, and KRAS status are the only reliable biomarkers to identify patients that would benefit from anti‐EGFR therapy. (Cancer Sci 2010)     

KRAS mutation confers resistance to antibody‐dependent cellular cytotoxicity of cetuximab against human colorectal cancer cells

Cetuximab is a chimeric IgG1 monoclonal antibody (mAb) that targets the extracellular domain of epidermal growth factor receptor (EGFR). Oncogenic KRAS mutations in tumors have been shown to be a negative predictor of the response of colorectal cancer (CRC) to cetuximab treatment. Cetuximab exerts its therapeutic effects through several mechanisms including antibody‐dependent cellular cytotoxicity (ADCC). However, the influence of KRAS mutations on cetuximab‐mediated ADCC is not fully understood. Here, we investigated cetuximab‐mediated ADCC in two pairs of isogenic CRC cells with or without a KRAS mutation. Peripheral blood mononuclear cells (PBMCs) from healthy volunteers and NK92, a natural killer (NK) cell line that exogenously expresses FcγRIIIa (CD16a), were used as effector cells. In an ADCC assay, perforin‐dependent target cell lysis was not affected by the KRAS mutation status. On the other hand, perforin‐independent ADCC was observed only in CRC cells with wild‐type KRAS, but not in cells with mutant KRAS. Neutralizing experiments revealed that the Fas‐Fas ligand (FasL) interaction was responsible for the induction of apoptosis and perforin‐independent ADCC. Furthermore, the presence of effector cells clearly enhanced the growth‐inhibitory effect of cetuximab only in CRC cells with wild‐type KRAS, but not in those with mutant KRAS. These findings suggest that ADCC is an important mode of action of cetuximab and that KRAS mutation impairs the therapeutic effect exerted by cetuximab‐mediated ADCC.     

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.     

血清抗体及补体对利妥昔单抗介导的NK 细胞杀伤Raji细胞作用的影响

 【摘要】 目的 体外检测血清抗体及补体对利妥昔单抗介导的NK细胞对Raji细胞杀伤作用的影响。方法 通过巢式反转录聚合酶链反应（nest-PCR）检测NK细胞上FcγRⅢa（CD16a）基因型;流式细胞术检测加入血清抗体前后NK细胞上FcγRⅢa的表达;DIO-PI双荧光染色法检测添加血清补体及抗体后NK细胞对Raji细胞的杀伤效应强度。结果 添加血清补体组杀伤率较未添加组明显增强,而添加血清抗体组杀伤率较未添加组明显减弱,在FcγRⅢa-158V／V组内,添加血清补体组、添加血清抗体组与未添加组细胞毒性指数分别为（94.25±1.79） %、（59.79±0.66） %和（69.05±2.38） %,三组之间两两比较均P＜0.05;在FcγRⅢa-158V／F组内,添加血清补体组、添加血清抗体组与未添加组细胞毒性指数分别为（66.71±5.57） %、（18.13±2.99） %和（39.63±3.86） %,三组之间两两比较均P＜0.05。结论 血清补体可增强利妥昔单抗介导的NK细胞对Raji细胞的杀伤作用。血清抗体会减弱利妥昔单抗介导的NK细胞对Raji细胞的杀伤作用。使用肿瘤特异性单克隆抗体（MAb）治疗肿瘤患者时,同时输注新鲜冷冻血浆可提高其抗肿瘤疗效;而MAb与静脉注射用免疫球收白（IVIG）同时使用则可能降低其抗肿瘤疗效。     

Predictive biomarkers of anti-EGFR monoclonal anti-body in colorectal cancer

The epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, triggers a downstream signaling cascade through areas such as the RAS-RAF-MAPK and PI3K-AKT pathways, which are involved in cell proliferation, survival and motility.Inhibiting EGFR activation has demonstrated significant promise as a molecular targeting therapy for various solid tumors. Two monoclonal antibodies (mAbs) targeting EGFR, cetuximab and panitumumab, are established to be new treatment options for metastatic colorectal cancer (mCRC). Among activating mutations in downstream of EGFR, the KRAS mutation, which is present in 40% of mCRC patients, has shown to be a predictive biomarker for resistance to anti-EGFR antibody therapy based on Caucasian studies. However, only a small proportion of patients achieved an objective response and benefit from anti-EGFR antibody, even among those with wild-type KRAS tumors. Other downstream factors in EGFR signaling are now being explored, such as the BRAF, PIK3CA, PTENgenes. Cetuximab, a chimeric immunoglobulin 1 (IgG1) monoclonal antibody, may also exert antitumor effects through antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC is influenced by FCrR1 a-H131R and FC7RIKa-V158F polymorphisms. Additional analysis of BRAF, PIK3CA, PTEN and FcqR genes in KRAS wild-type patients could narrow down the selection of patients who are most likely to benefit from anti-EGFR antibody therapy.     

FcγR polymorphisms and clinical outcome in colorectal cancer patients receiving passive or active antibody treatment

Fcγ receptors (FcγRs) on effector cells are of importance for mediating antibody-dependent cellular cytotoxicity (ADCC). FcγRIIIa158valine (V)/phenylalanine (F) and FcγRIIa131histidine (H)/arginine(R) polymorphisms have been shown to relate to prognosis in antibody-treated patients. The aim of the present study was to analyze the polymorphisms of both FcγRIIIa and FcγRIIa in colorectal carcinoma (CRC) patients receiving either passively administered monoclonal antibodies (MAbs) or antibodies induced by carcinoembryonic antigen (CEA) vaccination. One hundred and thirty CRC patients were included. Thirty-eight patients received adjuvant treatment with an anti-EpCAM monoclonal antibody (edrecolomab) (n=17) or rCEA vaccination therapeutic cancer vaccine (TCV) (n=21) inducing anti-CEA IgG antibodies. Ninety-two patients had metastatic disease and received anti-EpCAM MAb based therapies. FcγR genotypes were analysed using genomic DNA and PCR. ADCC was tested in a standard 18 h Cr51 release assay. In all adjuvant-treated patients, FcγRIIIa158V carriers (V/V and V/F) had a significantly better overall survival compared to F/F homozygous patients (p<0.05), FcγRIIa R carriers vs. H/H (p=0.05) as well as V and R carriers combined compared to the others (p<0.05). Similar findings were obtained when antibody and TCV-treated patients were analysed separately. No impact on the prognosis of FcγR polymorphisms was noted in advanced disease. FcγRIIIa V carriers had a significantly higher ADCC activity compared to F/F patients (p=0.001). Our model study might support the notion that FcγRIIIa V carriers as well as FcγRIIa R carriers receiving adjuvant, passively or actively (TCV)-induced antibody treatment might have a better prognosis than the others. Prospective extended clinical trials are warranted to study the predictive/prognostic impact of FcγR polymorphisms in antibody-treated patients and might be a valuable biomarker to optimize antibody-based treatment strategies.     

FCGR多态性对单克隆抗体抗肿瘤治疗的疗效预测价值

抗体依赖细胞介导的细胞毒作用（ADCC）是单克隆抗体如西妥昔单抗,利妥昔单抗,曲妥珠单抗发挥抗肿瘤作用的主要机制。FCG受体（FCGR）主要在人白细胞上,在ADCC中发挥重要作用。单克隆抗体的疗效与FCGR多态性密切相关,其中FCGR2A（H131R）和FCGR3A（V158F）是主要的基因型。这篇文章对FCGR多态性对单克隆抗体在肿瘤患者中的疗效预测价值做一综述。     

Two immunoglobulin G fragment C receptor polymorphisms independently predict response to rituximab in patients with follicular lymphoma.

PURPOSE: Although rituximab is now routinely used in the treatment of B-cell non-Hodgkin's lymphoma, the mechanism of its antitumor effect is not clear. One potential mechanism of action involves antibody-dependent cellular cytotoxicity (ADCC). Two aspects of ADCC influence the effectiveness of this process: the susceptibility of tumor cells and the activation of effector cells via their immunoglobulin G fragment C receptors (Fc gamma Rs). Several Fc gamma R polymorphisms have been identified that may affect the killing function of natural killer cells and macrophages. PATIENTS AND METHODS: The pretreatment tumor cells from 43 patients with follicular lymphoma were tested for their intrinsic susceptibility to rituximab-mediated ADCC. In addition, the Fc gamma RIIIa (CD16) and Fc gamma RIIa (CD32) polymorphisms were determined in an expanded group of 87 patients. The results were then correlated with clinical outcome of these patients. RESULTS: No difference was found between the susceptibility of tumors from patients who clinically responded to rituximab versus those who did not respond. Conversely, both the Fc gamma RIIIa 158 valine/valine and the Fc gamma RIIa 131 histidine/histidine genotypes were found to be independently associated with the response rate and freedom from progression. CONCLUSION: These data support the hypothesis that ADCC plays an important role in the clinical effect of rituximab at the level of the effector cell. It will be important to include information on Fc receptor polymorphisms in future trials of rituximab therapy.     

Gene status of head and neck squamous cell carcinoma cell lines and cetuximab-mediated biological activities

Cetuximab is a chimeric IgG1 monoclonal antibody that targets epidermal growth factor receptor (EGFR). Cetuximab binds to EGFR and prevents phosphorylation of EGFR. Moreover, preclinical results have shown the ability of cetuximab to induce either complement-mediated tumor cell killing (CDC) or antibody-dependent cell-mediated-cytotoxicity (ADCC). We previously reported mutation in EGFR regarding head and neck squamous cell carcinoma (HNSCC) cell lines. In the present study, we analyzed the same 16 HNSCC cell lines for mutations in KRAS, PIK3CA, BRAF and PTEN. Furthermore, we evaluated cetuximab-mediated biological activities (antiproliferative effect by the MTT assay and ADCC) regarding these cell lines. Mutations in PIK3CA and PTEN were observed in two cell lines (2/16, 12.5%), but no mutation was observed in KRAS and BRAF. The antiproliferative effect of cetuximab by the MTT assay was not strong, and no correlation was observed between the antiproliferative effect of cetuximab and mutations in EGFR, KRAS, PIK3CA, BRAF and PTEN in these cell lines. Therefore, the mutation status of EGFR and downstream molecules were not useful for predicting the antitumor effects of cetuximab on HNSCC. Cetuximab-mediated ADCC was observed in these cell lines and might have been influenced by the expression of EGFR. Therefore, cetuximab-mediated ADCC seems to be an important part of the antitumor mechanisms of cetuximab and the expression levels of EGFR might influence the antitumor activity of cetuximab. Therefore, besides the antiproliferative effect of cetuximab by the MTT assay, it appeared important to evaluate cetuximab-mediated ADCC as well as EGFR expression in HNSCC cells.     

Anti-tumor activity and toxicokinetics analysis of MGAH22, an anti-HER2 monoclonal antibody with enhanced Fcγ receptor binding properties

Introduction

Response to trastuzumab in metastatic breast cancer correlates with expression of the high binding variant (158V) of the activating Fcγ receptor IIIA (CD16A). We engineered MGAH22, a chimeric anti-HER2 monoclonal antibody with specificity and affinity similar to trastuzumab, with an Fc domain engineered for increased binding to both alleles of human CD16A.

Methods

MGAH22 was compared to an identical anti-HER2 mAb except for a wild type Fc domain. Antibody-dependent cell cytotoxicity (ADCC) assays were performed with HER2-expressing cancer cells as targets and human PBMC or purified NK cells as effectors. Xenograft studies were conducted in mice with wild type murine FcγRs; in mice lacking murine CD16; or in mice lacking murine CD16 but transgenic for human CD16A-158F, the low-binding variant. The latter model reproduces the differential binding between wild type and the Fc-optimized mAb for human CD16A. The JIMT-1 human breast tumor line, derived from a patient that progressed on trastuzumab therapy, was used in these studies. Single and repeat dose toxicology studies with MGAH22 administered intravenously at high dose were conducted in cynomolgus monkeys.

Results

The optimized Fc domain confers enhanced ADCC against all HER2-positive tumor cells tested, including cells resistant to trastuzumab's anti-proliferative activity or expressing low HER2 levels. The greatest improvement occurs with effector cells isolated from donors homozygous or heterozygous for CD16A-158F, the low-binding allele. MGAH22 demonstrates increased activity against HER2-expressing tumors in mice transgenic for human CD16A-158F. In single and repeat-dose toxicology studies in cynomolgus monkeys, a species with a HER2 expression pattern comparable to that in humans and Fcγ receptors that exhibit enhanced binding to the optimized Fc domain, MGAH22 was well tolerated at all doses tested (15-150 mg/kg) and exhibited pharmacokinetic parameters similar to that of other anti-HER2 antibodies. Induction of cytokine release by MGAH22 in vivo or in vitro was similar to that induced by the corresponding wild type mAb or trastuzumab.

Conclusions

The data support the clinical development of MGAH22, which may have utility in patients with low HER2 expressing tumors or carrying the CD16A low-binding allele.     

Antibody-dependent cellular cytotoxicity of cetuximab against tumor cells with wild-type or mutant epidermal growth factor receptor

Cetuximab (Erbitux, IMC-C225) is a monoclonal antibody targeted to the epidermal growth factor receptor (EGFR). To clarify the mode of antitumor action of cetuximab, we examined antibody-dependent cellular cytotoxicity (ADCC) activity against several tumor cell lines expressing wild-type or mutant EGFR. ADCC activity and complement-dependent cytolysis activity were analyzed using the CytoTox 96 assay. ADCC activities correlated with the EGFR expression value (R = 0.924). ADCC activities were detected against all tumor cell lines, except K562 cells in a manner dependent on the cellular EGFR expression level, whereas complement-dependent cytolysis activity was not detected in any of the cell lines. The ADCC activity mediated by cetuximab was examined in HEK293 cells transfected with wild-type EGFR (293W) and a deletional mutant of EGFR (293D) in comparison with the mock transfectant (293M). ADCC activity was detected in 293W and 293D cells, in a cetuximab dose-dependent manner, but not in 293M cells (<10%). These results indicate that ADCC-dependent antitumor activity results from the degree of affinity of cetuximab for the extracellular domain of EGFR, independent of EGFR mutation status. These results suggest ADCC activity to be one of the modes of therapeutic action of cetuximab and to depend on EGFR expression on the tumor cell surface.     

Acquired resistance to cetuximab is mediated by increased PTEN instability and leads cross-resistance to gefitinib in HCC827 NSCLC cells

EGFR inhibitors, including the small-molecule tyrosine kinase inhibitors such as gefitinib, and the monoclonal antibodies directed at the receptor such as cetuximab, have demonstrated promising effects in non-small cell lung cancer (NSCLC). In this study, we generated cetuximab-resistant cell lines (HCC827-CR) from HCC827 NSCLC cells to investigate acquired resistance mechanisms to cetuximab. In HCC827-CR cells, Akt was hyperactivated and its activity was persistent upon cetuximab treatment. Blockade of PI3K/Akt activity restored cetuximab sensitivity in HCC827-CR cells. Further investigation revealed that increased PTEN instability mediates constitutive Akt activation. By 1 μM proteosomal inhibitor, MG-132, PTEN protein levels were restored and Akt activity was dramatically reduced. Overexpression of PTEN by transfection could not restore cetuximab sensitivity in HCC827-CR because overexpressed PTEN was degraded rapidly (∼72 h). The increased PTEN instability was confirmed by the treatment of HCC827-CR with a protein synthesis inhibitor, cycloheximide. In the presence of cycloheximide, overexpressed PTEN was degraded more rapidly (∼12 h) in HCC827-CR cells. Interestingly, HCC827-CR cells also revealed de novo resistance to gefitinib. Inhibition of PI3K/Akt signaling pathway restored sensitivity to gefitinib in HCC827-CR cells. Taken together, these data show that PTEN instability-mediated constitutive Akt activation is involved in acquired resistance mechanisms to cetuximab and also induces de novo resistance to gefitinib. Importantly, these findings suggest emergence of cross-resistance between two agents as a potential serious problem in the clinical setting.     

FcγR2A and 3A polymorphisms predict clinical outcome of trastuzumab in both neoadjuvant and metastatic settings in patients with HER2-positive breast cancer

BackgroundAntibody-dependent-mediated cytotoxicity (ADCC) is one of the modes of action for trastuzumab. Recent data have suggested that fragment C γ receptor (FcγR) polymorphisms have an effect on ADCC. This prospective phase II trial aimed to evaluate whether these polymorphisms are associated with clinical efficacies in patients who received trastuzumab.Patients and methodsPatients in a neoadjuvant (N) setting received Adriamycin and cyclophosphamide followed by weekly paclitaxel/trastuzumab. Patients in a metastatic (M) setting received single trastuzumab until progression. In total, 384 distinct single nucleotide polymorphisms of different FcγR, HER2, and fucosyltransferase loci were assessed.ResultsFifteen operable and 35 metastatic HER2-positive breast cancer patients were enrolled in each of the N and M settings, respectively. The FcγR2A-131 H/H genotype was significantly correlated with the pathologically documented response (pathological response) (P = 0.015) and the objective response (P = 0.043). The FcγR3A-158 V/V genotype was not correlated with the pathological response, but exhibited a tendency to be correlated with the objective response. Patients with the FcγR2A-131 H/H genotype had significantly longer progression-free survival in the M setting (P = 0.034).ConclusionThe FcγR2A-131 H/H polymorphism predicted the pathological response to trastuzumab-based neoadjuvant chemotherapy in early-stage breast cancer, and the objective response to trastuzumab in metastatic breast cancer.     

Fc gamma receptor polymorphisms as predictive markers of Cetuximab efficacy in epidermal growth factor receptor downstream-mutated metastatic colorectal cancer

BackgroundThe immunoglobulin G1 (IgG₁) monoclonal antibody (MoAb) Cetuximab is active in metastatic colorectal cancer (mCRC) as first or subsequent lines of therapy. Efficacy seems restricted to KRAS wild-type tumours. IgG₁ may also induce antibody dependent cell mediated citotoxicity (ADCC) by recruitment of immune effector cells. ADCC is influenced by Fc gamma receptor (FcγR) polymorphisms. We investigated the association of FcγR polymorphisms and disease control rate (DCR) in mCRC patients treated with chemotherapy plus Cetuximab.Patients and methodsTumour tissues from 106 patients were screened for KRAS codon 12 and 13 mutations using a sensitive multiplex assay (DxS, Manchester, United Kingdom). NRAS (codons: 12, 13 and 61), PI3K (exon 20) and BRAF (exon 15) were analysed by direct sequencing. Fcγ RIIa and Fcγ RIIIa polymorphisms were genotyped by TaqMan assays.ResultsDCR was significantly higher in KRAS wild-type tumours (61% versus 39%, p = 0.049). In epidermal growth factor receptor (EGFR) downstream-mutated mCRC patients, those harbouring an FcγRIIa H/H genotype had a higher DCR than alternative genotypes (67% versus 33%, p = 0.017). By multivariate analysis, FcγRIIa-131H/H remained significantly correlated with DCR (p = 0.008).ConclusionFcγR polymorphisms may play a role in the clinical efficacy of Cetuximab in EGFR downstream mutated mCRC patients. Further research into Cetuximab immune-based mechanisms in KRAS-mutated patients seems warranted.     

Antibody-dependent cellular cytotoxicity mediated by cetuximab against lung cancer cell lines.

PURPOSE: Epidermal growth factor receptor (EGFR) is commonly overexpressed in lung cancer. Cetuximab is a chimeric mouse-human antibody targeted against EGFR. Compared with its inhibitory properties, its immunologic mechanisms have not been well studied. In this study, we investigated the antibody-dependent cellular cytotoxicity (ADCC) activity of cetuximab against lung cancer cell lines. EXPERIMENTAL DESIGN: We studied the correlation between EGFR expression in lung cancer cell lines and the ADCC activity of cetuximab as well as the influence of interleukin-2 and chemotherapy on the ADCC activity. EGFR expression was measured by a quantitative flow cytometric analysis and immunohistochemistry. The ADCC activity was assessed by a 4-h (51)Cr release assay. Peripheral blood mononuclear cells, purified T cells, natural killer (NK) cells, and monocytes from healthy donors or lung cancer patients were used as effector cells. RESULTS: Fresh peripheral blood mononuclear cells exhibited cetuximab-mediated ADCC activity against lung cancer cell lines at a low concentration of cetuximab (0.25 microg/mL). A logarithmic correlation was observed between the number of EGFRs and ADCC activity. Even low EGFR expression, which was weakly detectable by immunohistochemistry, was sufficient for maximum ADCC activity, and further increases in EGFR expression on the target cells had no further effect on the ADCC activity. In addition, ADCC activity was enhanced by interleukin-2 mainly through activation of NK cells and was less susceptible to immunosuppression by chemotherapy than NK activity in lung cancer patients. CONCLUSIONS: These observations suggest the importance of ADCC activity as an immunologic mechanism of cetuximab in biological therapy for lung cancer patients.     

Elements related to heterogeneity of antibody-dependent cell cytotoxicity in patients under trastuzumab therapy for primary operable breast cancer overexpressing Her2

Preliminary results from a pilot trial on trastuzumab's mechanism of action against operable breast tumors overexpressing Her2 suggested a role for antibody-dependent cell cytotoxicity (ADCC). To examine factors affecting ADCC intensity and variability, we extended this study to the phenotypic and functional analysis of circulating mononuclear cells in 18 patients. ADCC was induced by trastuzumab therapy in 15 of 18 patients (83%). Inability to develop ADCC in three patients did not depend on inadequate levels of trastuzumab because further increase in its concentration in vitro was ineffective. Rather, susceptibility to develop ADCC was fairly predicted by test with trastuzumab before therapy and was correlated to the number of lymphocytes coexpressing CD16 and CD56. Phenotypic analysis at the end of ADCC evaluating down-regulation of CD16, and up-regulation of CD69 and CD107a, confirmed that natural killer (NK) cells and CD56(+) T cells were involved in productive engagement of trastuzumab. Also, the killing efficiency of CD16(+) lymphocytes was influenced by 158 V/F polymorphism of Fc gamma RIII (CD16), whereas variations of CD247 on NK cells were consistent with trends between ADCC before and after therapy. Complete pathologic response was observed in one patient showing ADCC of outstanding intensity, whereas four cases of partial response showed intermediate ADCC; none of the three patients unable to mount ADCC had significant tumor regression. These data indicate that quantity and lytic efficiency of CD16(+) lymphocytes are major factors for ADCC induction by trastuzumab, and confirm that breast cancer responses to short-term trastuzumab monotherapy may depend on involvement of the ADCC mechanism.     

Cytotoxic drugs up-regulate epidermal growth factor receptor (EGFR) expression in colon cancer cells and enhance their susceptibility to EGFR-targeted antibody-dependent cell-mediated-cytotoxicity (ADCC)

Cetuximab is a human–murine chimeric IgG1 monoclonal antibody to epidermal growth factor-receptor (EGFR) which exerts synergistic antitumour interactions with several cytotoxic drugs. Therefore, it is presently recommended in combination with chemotherapy in the treatment of colon, head and neck and non-small cell lung cancer. Cetuximab has been designed to inhibit EGFR signalling; however, preclinical evidence suggests that its anti-cancer effects in vivo are also related to the ability of its human IgG1 backbone to trigger immunological mechanisms. Here we have investigated whether the exposure to different cytotoxic drugs may affect the susceptibility of colon cancer cells in vitro to cetuximab immuno-targeting and related lymphokine-activated killer (LAK)-mediated antibody-dependent cell cytotoxicity (ADCC).Five colon cancer cell lines expressing a different k-ras mutational status were evaluated for: (i) EGFR-expression, (ii) susceptibility to LAK cells and (iii) cetuximab-mediated ADCC, before and after exposure to 5-flurouracil (5-FU), gemcitabine (Gem), irinotecan (Iri) alone or in multiple two/three drug combinations.These drugs were able to up-regulate EGFR expression on the surface of all the colon cancer cell lines with a maximal effect observed few hours after the exposure to GILF regimen (Gem, Iri, Levofolinic acid and 5-FU). Chemotherapy was able to greatly enhance the sensitivity to either LAK cells or cetuximab-mediated ADCC in all the colon cancer cell lines with a mechanism independent from k-ras status.The results of our study suggest that chemotherapy may enhance cetuximab-mediated immuno-targeting and ADCC thus providing the rationale to design novel immuno-biochemotherapy regimens.