The polymorphism of CD16 that predicts a valine (V) versus phenylalanine (F) substitution at amino acid position 158 was determined by performing allele-specific polymerase chain reaction (PCR)

The polymorphism of CD16 that predicts a valine (V) versus phenylalanine (F) substitution at amino acid position 158 was determined by performing allele-specific polymerase chain reaction (PCR). ADCC. These data demonstrate that cells coated with mAb with enhanced affinity for CD16 are more effective at activating NK cells at both low and saturating mAb concentrations irrespective of CD16 polymorphism, and they provide further evidence for the clinical development of such mAbs with the goal of improving clinical response to mAb. Introduction Monoclonal antibodies (mAbs) are an integral component of therapy for a number of cancers, but there is still much (S)-Glutamic acid we do not understand about their mechanisms of action. Laboratory and clinical correlative studies are beginning to shed some light on how mAbs can induce tumor regression. Evidence in both human in vitro systems and animal models suggests mAb-induced apoptotic signaling via CD201,2 and fixation of match3,4 can play a role in rituximab-mediated removal of CD20+ cells. In vitro and animal model studies demonstrate that numerous human effector cell populations, including natural killer (NK) Rabbit polyclonal to PCBP1 cells,5 monocytes,6 and granulocytes7 can mediate antibody-dependent cellular cytotoxicity (ADCC) under select conditions. Among the most convincing evidence that ADCC plays a role in mediating the clinically relevant antitumor response of rituximab is the demonstration that polymorphisms in CD16, also known as Fc receptor IIIa or the low-affinity Fc receptor, affect clinical response to rituximab (R). Two groups have exhibited that R is more effective in patients with follicular lymphoma homozygous for valine (VV) at CD16 amino acid position 158 compared with subjects who are heterozygotes (VF) or homozygous for phenylalanine (FF) at that position.8,9 Weng et al10 also reported a correlation between the higher-affinity CD16 polymorphism and response to active idiotype immunization. Dall’Ozzo et al11 found that NK cells from subjects with the higher-affinity polymorphism for CD16 mediate ADCC at a lower mAb concentration than do NK cells from subjects with the low-affinity CD16 polymorphism; however, there was considerable intersubject variability. Polymorphisms in CD16 did not correlate with clinical response to R or alemtuzumab in chronic lymphocytic leukemia (CLL),12,13 or in preliminary reports of patients treated with the combination of chemotherapy and R. Nevertheless, these data provide convincing evidence that response to mAb, at least with some clinical scenarios, is dependent on the conversation between CD16 and mAb-coated target cells. Much of the effort over recent years in the area of mAb engineering has focused on decreasing immunogenicity, or generating mAbs that target different antigens. We have used a directed evolution approach to produce mAb with varying affinity for Fc receptors and for antigen to investigate the functional effect of modifying mAb sequences. We also recently reported a system using peripheral blood mononuclear cells (PBMCs) and target cells to assess how mAbs impact NK-cell phenotype.14 The mAb of the IgG1 subclass induced modulation of CD16 (S)-Glutamic acid and up-regulation of CD54 on NK cells when the appropriate target cells were present. Greater concentrations of mAbs were needed to induce these changes on NK cells from subjects with the lower-affinity CD16 polymorphism. Phenotypic changes were greater in NK cells from subjects with the higher-affinity polymorphism even when saturating (S)-Glutamic acid concentrations of mAb were used, demonstrating that increased concentration of mAb can overcome some, but not all, of the influence CD16 polymorphisms have on NK activation. These studies provide a straightforward and very easily reproducible technique to measure the ability of mAb-coated tumor cells to activate NK cells in vitro. We therefore evaluated anti-CD20 mAbs with altered affinity for target antigen alone, or target antigen and CD16, for their ability to activate NK cells. These data show that tumor cells coated with mAb with enhanced affinity for CD16 are more effective at activating NK cells at both low and saturating mAb concentrations irrespective of CD16 polymorphism. These studies provide further support for the clinical development of such mAbs with the goal of improving clinical response to mAb. Patients, materials, and methods Antibodies Rituximab (R) (Biogen-Idec, Cambridge, MA; Genentech, South San Francisco, CA) was purchased commercially. AME-B and AME-D are anti-CD20 IgG mAbs with human germ line framework regions that were generated using directed development technology. Functional analyses using intact cells were used to screen and select mAbs with the most promising characteristics. For AME-B, libraries for all those 6 CDRs were synthesized using a mutagenesis process that introduced.