XGFR*, a novel affinity-matured bispecific antibody targeting IGF-1R and EGFR with combined signaling inhibition and enhanced immune activation for the treatment of pancreatic cancer
Juergen SchanzerKatharina WarthaEkkehard MoessnerRalf J. HosseSamuel MoserRebecca CroasdaleHalina TrochanowskaCuiying ShaoPeng WangLei ShiTina WeinzierlNatascha RiederMarina BacacCarola H. RiesHubert KettenbergerTilman SchlothauerThomas FriessPablo UmañaChristian Klein
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The epidermal growth factor receptor (EGFR) and the insulin-like growth factor-1 receptor (IGF-1R) play critical roles in tumor growth, providing a strong rationale for the combined inhibition of IGF-1R and EGFR signaling in cancer therapy. We describe the design, affinity maturation, in vitro and in vivo characterization of the bispecific anti-IGF-1R/EGFR antibody XGFR*. XGFR* is based on the bispecific IgG antibody XGFR, which enabled heterodimerization of an IGF-1R binding scFab heavy chain with an EGFR-binding light and heavy chain by the "knobs-into-holes" technology. XGFR* is optimized for monovalent binding of human EGFR and IGF-1R with increased binding affinity for IGF-1R due to affinity maturation and highly improved protein stability to oxidative and thermal stress. It bears an afucosylated Fc-portion for optimal induction of antibody-dependent cell-mediated cytotoxicity (ADCC). Stable Chinese hamster ovary cell clones with production yields of 2–3 g/L were generated, allowing for large scale production of the bispecific antibody. XGFR* potently inhibits EGFR- and IGF-1R-dependent receptor phosphorylation, reduces tumor cell proliferation in cells with heterogeneous levels of IGF-1R and EGFR receptor expression and induces strong ADCC in vitro. A comparison of pancreatic and colorectal cancer lines demonstrated superior responsiveness to XGFR*-mediated signaling and tumor growth inhibition in pancreatic cancers that frequently show a high degree of IGF-1R/EGFR co-expression. XGFR* showed potent anti-tumoral efficacy in the orthotopic MiaPaCa-2 pancreatic xenograft model, resulting in nearly complete tumor growth inhibition with significant number of tumor remissions. In summary, the bispecific anti-IGF-1R/EGFR antibody XGFR* combines potent signaling and tumor growth inhibition with enhanced ADCC induction and represents a clinical development candidate for the treatment of pancreatic cancer.Keywords:
Growth inhibition
Pancreatic tumor
Effective immunity to HIV is poorly understood. In particular, a role for antibody-dependent cellular cytotoxicity (ADCC) in controlling HIV is controversial. We hypothesized that significant pressure from HIV-specific ADCC would result in immune-escape variants. A series of ADCC epitopes in HIV-infected subjects to specific consensus strain HIV peptides were mapped using a flow cytometric assay for natural killer cell activation. We then compared the ADCC responses to the same peptide epitope derived from the concurrent HIV sequence(s) expressed in circulating virus. In 9 of 13 epitopes studied, ADCC antibodies were unable to recognize the concurrent HIV sequence. Our studies suggest ADCC responses apply significant immune pressure on the virus. This result has implications for the induction of ADCC responses by HIV vaccines.
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In addition to direct effects on virus infectivity, antibodies mediate antibody-dependent cellular cytotoxicity (ADCC), the killing of an antibody-coated virus-infected cell by cytotoxic effector cells. Although ADCC has been suggested to protect against HIV, the relationship between HIV-specific ADCC antibodies at the time of HIV exposure and infection outcome in humans remains to be assessed. We evaluated the ADCC activity of passively acquired antibodies in infants born to HIV-infected mothers. ADCC levels were higher in uninfected than infected infants, although not significantly. Increase in ADCC antibody activity in infected infants was associated with reduced mortality risk. Infant ADCC positively correlated with the magnitude of IgG1 binding, and IgG1 levels were associated with survival in infected infants. Infant IgG3-binding antibodies were not associated with infected infant survival. These data suggest a therapeutic benefit of pre-existing HIV-specific ADCC antibodies and support a role for eliciting ADCC-mediating IgG1 in HIV vaccines.
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Peripheral blood lymphocytes from patients with systemic lupus erythematosus were compared with those of normal donors for their ability to perform antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC activity was significantly decreased (p<0.001) in active SLE. Correlations appear to exist between either disease activity, CH50 or lymphocyte counts, and ADCC activity. There was no apparent relationship between ADCC activity and the level of corticosteroid administered. Also, no significant correlation was demonstrated between ADCC activity and the levels of circulating immune complex (CIC).Sera from patients with SLE inhibited ADCC activity of the normal lymphocytes. A close relationship was observed between the inhibitory effect of SLE sera on the ADCC of the normal lymphocytes and CIC levels. Sera containing CIC inhibited ADCC of the autologous lymphocytes from the patients. No correlation was observed between ADCC activity and the serum-mediated inhibition of ADCC of normal lymphocytes. These results suggest that the decreased ADCC activity in SLE patients is partly due to the killer cell dysfunction as well as to the suppressive effect of CIC on the ADCC.
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To explore the role of antibody-dependent cellular cytotoxicity (ADCC) in controlling HIV-1 infection, ADCC was compared with plasma RNA and CD4+ cell count in 40 patients not receiving antiretroviral therapy and in seven patients after the initiation of treatment. Among untreated patients, ADCC effector cell function of peripheral blood mononuclear cells, measured by 51Cr release assay, correlated inversely with viral load (R = -0.42, p = 0.007) and directly with CD4+ cell count (R = 0.52, p = 0.001). On the other hand, HIV-1-specific ADCC antibody level correlated directly with viral load, but only among patients with high CD4+ cell counts. Therapy-induced changes in ADCC effector cell function correlated strongly with changes in CD4+ cell count (R = 0.86, p = 0.014), whereas there was no consistent pattern of change in ADCC antibody with therapy. In a novel assay, ADCC reduced virus yield from CD4+ lymphocytes infected with a primary HIV isolate. ADCC may contribute to control of viremia, and CD4+ lymphocytes likely play a role in ADCC effector and antibody functions.
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In humans, pre-existing anti-HIV-1 neutralizing antibodies (nAbs) have not been associated with decreased HIV-1 acquisition. Here, we evaluate antibody-dependent cellular cytotoxicity (ADCC) present in pre-transmission infant and maternal plasma and breast milk (BM) against the contemporaneous maternal HIV-1 variants. HIV-1-exposed uninfected compared with HIV-1-exposed infected infants have higher ADCC and a combination of ADCC and nAb responses against their corresponding mother's strains. ADCC does not correlate with nAbs, suggesting they are independent activities. The infected infants with high ADCC compared with low ADCC, but not those with higher ADCC plus nAbs, have lower morbidity up to 1 year after birth. A higher IgA to IgG ratio, observed in BM supernatants and in a higher proportion of the infected compared with the uninfected infants, associates with lower ADCC. Against the exposure strains, ADCC, more than nAbs, associates with both lower mother-to-child transmission and decreased post-infection infant morbidity.
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The effects of in vitro and in vivo treatment with glutaurine, a newly discovered parathyroid hormone possessing immunostimulative activity, on human antibody-dependent cell-mediated cytotoxicity (ADCC) were studied in 15 tumor patients with healthy subjects in a xenogeneic test system using chicken erythrocytes as target cells. A marked increase in "K" cell activity was observed in 8 tumor patients with originally low cytotoxic capacity, while originally normal ADCC activity of other tumor patients and healthy subjects was not significantly influenced by glutaurine treatment. The changes in cytotoxicity were not accompanied by changes in lymphocyte populations. Incubation of effector cells with glutaurine in vitro caused no change in ADCC activity in lymphocyte populations. Some similarities between the effects of glutaurine treatment of ADCC and that of dialyzable leukocyte extracts are discussed.
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Monocyte-mediated-antibody-dependent cellular cytotoxicity (MO-ADCC) was studied in 21 patients with Hodgkin's disease (HD), 15 patients with a long-lasting remission of HD, 11 patients with non-Hodgkin's lymphoma (NHL), 11 patients with solid tumors, and 15 normal controls. Lymphocyte ADCC (LY-ADCC) was evaluated in 12 patients with HD and 9 normal controls. Monocytes and lymphocytes were isolated with cell-scatter monitored counterflow centrifugation providing high purity and yield. Antibody-dependent cellular cytotoxicity was evaluated by means of DNA flowcytometry, using antibody-coated chicken erythrocyte targets (CRBC). in comparison with normal controls MO-ADCC was significantly increased in HD (P <0.0005), NHL (P < 0.005), and solid tumors (P < 0.005). in patients in a long-lasting complete remission of HD, MO-ADCC was in the normal range. Lymphocyte-ADCC of 12 patients with HD was similar to that of 9 normal controls. in all experiments LY-ADCC was invariably lower than MO-ADCC of the same donor, indicating the monocyte as the most potent effector cell towards CRBC targets. Results indicate the following: (1) purified cell suspensions of both lymphocytes and monocytes are essential to unravel their role as effector cells; (2) LY-ADCC in HD is similar to normal controls; (3) MO-ADCC enhancement is not uncommon in malignant lymphoma and several solid tumors; (4) normal MO-ADCC in a group of successfully treated patients with HD suggests a disease-related induction of enhanced MO-ADCC.
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