Antibody-dependent cellular cytotoxicity in patients on chronic hemodialysis.

BACKGROUND: Antibody (Ab)-dependent cellular cytotoxicity (ADCC) is considered to be a relevant mechanism of action of Ab-based tumor therapies. However, knowledge about ADCC capacity of dialysis patients (DP) is limited. The aim of our study was to investigate if ADCC capacity of effector cells obtained from DP differed from those of healthy individuals (HI). METHODS: First, we performed ADCC assays with isolated polymorphonuclear cells (PMN) and peripheral blood mononuclear cells (PBMC), mediated by the epidermal growth factor receptor Ab cetuximab or panitumumab. As cetuximab is of human IgG1 and panitumumab of human IgG2 isotype, both Abs differ in their affinity to Fcγ receptors and effector cell recruitment. RESULTS: Using PMN as effectors, ADCC levels via panitumumab proved to be higher than via cetuximab, but did not differ between DP and HI. In contrast, IgG2-mediated ADCC with PBMC from DP was significantly enhanced compared to HI. IgG2 Abs predominantly bind to FcγRIIa. Within the PBMC, monocytes are the only cytotoxic cells physiologically expressing this receptor. ADCC experiments with isolated monocytes confirmed them to be the pivotal cells for the observed effect. Analysis of monocytes' Fc receptor expression demonstrated no difference between DP and HI, but monocytes of DP proved to be numerically increased and appeared preactivated. CONCLUSION: Our studies implicate that ADCC capacity is not impaired in DP and that it might particularly be reasonable to apply human IgG2 Abs as therapeutics for these patients.

Fc engineering of antibodies and antibody derivatives by primary sequence alteration and their functional characterization.

Therapeutic antibodies used in the treatment of cancer patients are able to mediate diverse effector mechanisms. Dependent on tumor entity, localization, and tumor burden different effector mechanisms may contribute to the in vivo antitumor activity to a variable degree. Especially Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) have been suggested as being important for the in vivo activity of therapeutic antibodies like rituximab or trastuzumab. In recent years, several strategies have been pursued to further optimize the cytotoxic potential of monoclonal antibodies by modifying their Fc part (Fc engineering) with the ultimate goal to enhance antibody therapy.Since Fc engineering approaches are applicable to any Fc-containing molecule, strategies to enhance CDC or ADCC activity of full antibodies or scFv-Fc fusion proteins by altering the primary Fc sequence are described.

Increasing FcγRIIa affinity of an FcγRIII-optimized anti-EGFR antibody restores neutrophil-mediated cytotoxicity.

Antibody-dependent cell-mediated cytotoxicity (ADCC) has been suggested as an essential mechanism for the in vivo activity of cetuximab, an epidermal growth factor receptor (EGFR)-targeting therapeutic antibody. Thus, enhancing the affinity of human IgG1 antibodies to natural killer (NK) cell-expressed FcγRIIIa by glyco- or protein-engineering of their Fc portion has been demonstrated to improve NK cell-mediated ADCC and to represent a promising strategy to improve antibody therapy. However, human polymorphonuclear (PMN) effector cells express the highly homologous FcγRIIIb isoform, which is described to be ineffective in triggering ADCC. Here, non-fucosylated or protein-engineered anti-EGFR antibodies with optimized FcγRIIIa affinities demonstrated the expected benefit in NK cell-mediated ADCC, but did not mediate ADCC by PMN, which could be restored by FcγRIIIb blockade. Furthermore, eosinophils and PMN from paroxysmal nocturnal hemoglobinuria patients that expressed no or low levels of FcγRIIIb mediated effective ADCC with FcγRIII-optimized anti-EGFR antibody. Additional experiments with double FcγRIIa/FcγRIII-optimized constructs demonstrated enhanced PMN-mediated ADCC compared with single FcγRIII-optimized antibody. In conclusion, our data demonstrate that FcγRIIIb engagement impairs PMN-mediated ADCC activity of FcγRIII-optimized anti-EGFR antibodies, while further optimization of FcγRIIa binding significantly restores PMN recruitment.

Combined Fc-protein- and Fc-glyco-engineering of scFv-Fc fusion proteins synergistically enhances CD16a binding but does not further enhance NK-cell mediated ADCC.

Protein- or glyco-engineering of antibody molecules can be used to enhance Fc-mediated effector functions. ScFv-Fc fusion proteins (scFv-Fc) represent interesting antibody derivatives due to their relatively simple design and increased tissue penetration. Here, the impact of protein- and glyco-engineering on ADCC potency of a panel of human IgG1-based scFv-Fc was tested. Three matched sets of scFv-Fc variants targeting CD7, CD20 or HLA class II and optimized for CD16a binding by mutagenesis, lack of core-fucose, or their combination, were generated and functionally tested in comparison to the corresponding wild type scFv-Fc. Antigen binding activity was not compromised by altered glycosylation or Fc mutagenesis, whereas Fc binding to CD16a was significantly enhanced in the order: non-core fucosylated/Fc-mutated double-engineered≫Fc-mutated≥non-core-fucosylated>wild-type IgG1-Fc. All engineered variants triggered potent ADCC with up to 100-fold reduced EC50 values compared to non-engineered variants. Interestingly, double-engineered variants were similarly effective in triggering ADCC compared to single-engineered variants irrespective of their 1 log greater CD16a binding affinity. Thus, these data demonstrate that protein- and glyco-engineering enhances NK-cell mediated ADCC of scFv-Fc similarly and show that enhancing CD16a affinity beyond a certain threshold does not result in a further increase of NK-cell mediated ADCC.