[] PD-L1 Checkpoint MAb. 1. Several anti-PD1/PD-L1 monoclonal antibodies (MAbs) are currently providing evidence of clinical benefit in subsets of cancer patients. The mode of action of these MAbs is to inhibit PD1 on immune cells interacting with PD-L1 on tumor cells. These MAbs are either designed or engineered to eliminate antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC, however, has been implicated as an important mechanism in several highly effective MAb-mediated cancer therapies. A fully human anti-PD-L1 MAb would potentially be able to block PD-L1/PD1 interactions and also mediate the ADCC lysis of tumor cells. MSB0010718C (designated avelumab) is a fully human IgG1 anti-PD-L1 MAb. The studies demonstrate (a) the ability of avelumab to lyse a range of human tumor cells in the presence of PBMC or NK effectors; (b) IFN-gamma can enhance tumor cell PD-L1 expression and in some cases enhance ADCC tumor cell lysis; (c) purified NK cells are potent effectors for avelumab; (d) similar levels of avelumab-mediated ADCC lysis of tumor cells are seen using purified NK as effectors from either healthy donors or cancer patients; (e) very low levels of avelumab-mediated lysis are seen using whole PBMC as targets; this finding complements results seen in analyses of PBMC subsets of patients receiving avelumab; and (f) the addition of IL-12 to NK cells greatly enhances avelumab-mediated ADCC. These studies thus provide an additional mode of action for an anti-PD-L1 MAb and provide the rationale for further studies to enhance avelumab-mediated ADCC activity. 2. A study was designed to investigate the effect on immune cell subsets in the peripheral blood of cancer patients prior to and following multiple administrations of avelumab. One hundred twenty-three distinct immune cell subsets in the peripheral blood of cancer patients were analyzed by flow cytometry prior to and following one, three, and nine cycles of avelumab. No statistically significant changes in any of the 123 immune cell subsets analyzed were observed at any dose level, or number of doses, of avelumab. Controlled in vitro studies also showed lysis of tumor cells by avelumab versus no lysis of PBMC from five donors. These studies demonstrate the lack of any significant effect on multiple immune cell subsets, even those expressing PD-L1, following multiple cycles of avelumab. These results complement prior studies showing anti-tumor effects of avelumab and comparable levels of adverse events with avelumab versus other anti-PD-1/PD-L1 MAbs. These studies provide the rationale to further exploit the potential ADCC mechanism of action of avelumab as well as other human IgG1 checkpoint inhibitors. 3. Studies have shown for the first time that the addition of avelumab to an antigen-specific in vitro stimulation assay (a) increased the frequency of activated antigen-specific CD8+ T lymphocytes, (b) reduced CD4+ cell proliferation, and (c) induced a switch in the production of Th2 to Th1 cytokines. These findings provide the rationale for the use of avelumab anti-PD-L1 in in vitro assays to monitor patient immune responses to immunotherapies. [] Colorectal cancer. The first-line standard of care for patients with metastatic colorectal cancer (mCRC) is FOLFIRI (irinotecan, levo-leucovorin, 5-fluorouracil (5-FU)) plus bevacizumab. With the renewed interest in cancer immunotherapy with agents such as vaccines, checkpoint inhibitors and immune modulators, the possibility exists for the use of one or more of these immunotherapeutics in the first-line setting and thus in combination with the FOLFIRI and bevacizumab regimen. Studies were undertaken to study the effects of FOLFIRI and bevacizumab therapy on peripheral T-cell subsets, and to determine if there are any associations between these subsets and response to therapy. Peripheral blood mononuclear cell subsets of patients with mCRC (n = 23) were analyzed prior to and during therapy. There was an association (p = 0.036) between a decrease in Treg frequency during FOLFIRI therapy and overall survival, and an association (p = 0.037) between the frequency of Tregs prior to therapy and progression-free survival. Responders to the chemotherapy by RECIST criteria also had a greater decrease in Tregs during therapy vs pre-therapy (p = 0.0064) as compared to non-responders. While the number of mCRC patients undergoing chemotherapy in this study is relatively small, it provides the rationale for the use of immunotherapeutics in this first-line metastatic setting. [] Analyses of pre-therapy peripheral immunoscore and response to vaccine therapy. Tumor immunoscore analyses, especially for primary colorectal cancer and melanoma lesions, provide valuable prognostic information. Metastatic lesions of many carcinoma types, however, are often not easily accessible. We hypothesized that immune cells in peripheral blood may differ among individual patients with metastatic disease, which, in turn, may influence their response to immunotherapy. We thus analyzed immune cell subsets within peripheral blood mononuclear cells to determine if a peripheral immunoscore could have any prognostic significance for patients before receiving immunotherapy. Patients with metastatic breast cancer were randomly assigned to receive docetaxel +/- PANVAC vaccine. In another trial, prostate cancer patients with metastatic bone lesions were randomly assigned to receive a bone-seeking radionuclide +/- PROSTVAC vaccine. Predefined analyses of classic immune cell types (CD4, CD8, natural killer cells, regulatory T cells, myeloid-derived suppressor cells, and ratios) revealed no differences in progression-free survival (PFS) for either arm in both trials. Predefined analyses of refined immune cell subsets for which a biologic function had been previously reported also showed no significant prognostic value in PFS for patients receiving either docetaxel or radionuclide alone; however, in patients receiving these agents in combination with vaccine, the peripheral immunoscore of refined subsets revealed statistically significant differences in PFS (P 0.001) for breast cancer patients receiving docetaxel plus vaccine, and in prostate cancer patients receiving radionuclide plus vaccine (P = 0.004). These studies, however, provide the rationale for the evaluation of refined immune cell subsets to help determine which patients may benefit most from immunotherapy. [] 123 immune cell subsets. Recent advances in human immunology have led to the identification of novel immune cell subsets and the biological function of many of these subsets has now been identified. The results of ongoing immunotherapy clinical studies requires a more thorough interrogation of the immune system. We report the use of flow cytometry-based analyses to identify 123 immune cell subsets of peripheral blood mononuclear cells. The use of these panels defines multiple differences in younger (less than 40 years) vs. older (greater than or equal to 40 years) individuals and between aged-matched apparently healthy individuals and metastatic cancer patients, aspects not seen in the analysis of the following standard immune cell types: CD8, CD4, natural killer, natural killer-T, regulatory T, myeloid derived suppressor cells, conventional dendritic cells (DCs), plasmacytoid DCs and B cells. The use of these panels identifying 123 immune cell subsets may aid in the identification of patients who may benefit from immunotherapy, either prior to therapy or early in the immunotherapeutic regimen, for the treatment of cancer or other chronic or infectious diseases.
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