Summary Alemtuzumab is a humanized monoclonal antibody against CD52, an antigen found on the surface of normal and malignant lymphocytes. It is approved for the treatment of B‐cell chronic lymphocytic leukaemia and is undergoing Phase III clinical trials for the treatment of multiple sclerosis. The exact mechanism by which alemtuzumab mediates its biological effects in vivo is not clearly defined and mechanism of action studies have been hampered by the lack of cross‐reactivity between human and mouse CD52. To address this issue, a transgenic mouse expressing human CD52 (hCD52) was created. Transgenic mice did not display any phenotypic abnormalities and were able to mount normal immune responses. The tissue distribution of hCD52 and the level of expression by various immune cell populations were comparable to those seen in humans. Treatment with alemtuzumab replicated the transient increase in serum cytokines and depletion of peripheral blood lymphocytes observed in humans. Lymphocyte depletion was not as profound in lymphoid organs, providing a possible explanation for the relatively low incidence of infection in alemtuzumab‐treated patients. Interestingly, both lymphocyte depletion and cytokine induction by alemtuzumab were largely independent of complement and appeared to be mediated by neutrophils and natural killer cells because removal of these populations with antibodies to Gr‐1 or asialo‐GM‐1, respectively, strongly inhibited the activity of alemtuzumab whereas removal of complement by treatment with cobra venom factor had no impact. The hCD52 transgenic mouse appears to be a useful model and has provided evidence for the previously uncharacterized involvement of neutrophils in the activity of alemtuzumab.
The interaction between CXCR4 on the surface of tumor cells and CXCL12 in the stroma is believed to contribute to tumor cell survival and protection against drug treatment. Inhibition of stromal survival signals by CXCR4 antagonists has been reported to render tumor cells more sensitive to chemotherapy, but little is known about potential synergy with monoclonal antibodies. In this study, administration of the small molecule CXCR4 antagonists plerixafor and GENZ-644494 was found to enhance the anti-tumor activity of the monoclonal antibodies alemtuzumab and rituximab in disseminated lymphoma models. The observed enhancement in therapeutic efficacy by CXCR4 antagonists appeared to involve several factors, including interference with the tumor-promoting signals delivered by CXCL12, disruption of the tumor/stroma interaction and mobilization of effector neutrophils capable of mediating antibody-dependent cell-mediated cytotoxicity. The involvement of neutrophils was further supported by the observed reversal in therapeutic benefit upon neutrophil depletion.
Cytokine-mobilized peripheral blood cells have been shown to participate in hematopoietic recovery after bone marrow (BM) transplantation, and are proposed to be useful targets for retrovirus- mediated gene transfer protocols. We treated mice with granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) to mobilize hematopoietic progenitor cells into the peripheral blood. These cells were analyzed for the number and frequency of pluripotent hematopoietic stem cells (PHSC). We found that splenectomized animals treated for 5 days with G-CSF and SCF showed a threefold increase in the absolute number of PHSC over normal mice. The number of peripheral- blood PHSC increased 250-fold from 29 per untreated mouse to 7,200 in peripheral-blood PHSC in splenectomized animals treated for 5 days with G-CSF and SCF. Peripheral blood PHSC mobilized by treatment with G-CSF and SCF were analyzed for their ability to be transduced by retroviral vectors. Peripheral-blood PHSC from splenectomized animals G-CSF and SCF were transduced with a recombinant retrovirus containing the human MDR-1 gene. The frequency of gene transfer into peripheral blood PHSC from animals treated for 5 and 7 days was two-fold and threefold higher than gene transfer into PHSC from the BM of 5-fluorouracil-treated mice (P < .01). We conclude that peripheral blood stem cells mobilized by treatment with G-CSF and SCF are excellent targets for retrovirus- mediated gene transfer.
2717 Poster Board II-693 Interactions between chemokines and their receptors play an important role in cancer biology and have been shown to influence several processes such as tumor growth and the development of metastatic lesions. Stromal cell derived factor-1 (SDF-1) is a chemokine that binds to the CXCR4 chemokine receptor and stimulates B cell growth. CXCR4 is frequently over-expressed on tumor cells and the SDF-1/CXCR4 axis has been reported to play a role in promoting survival, angiogenesis and metastasis. The role of the stroma, in particular SDF-1/CXCR4 interactions, in providing a “survival niche” for tumor cells is well described for both hematological and solid tumors. We hypothesized that disruption of the protective microenvironment of tumor cells through antagonism of the SDF-1/CXCR4 axis may enhance the sensitivity of the tumor cells to treatment with anti-cancer agents such as monoclonal antibodies. Mozobil (AMD3100) or its structurally related analog, AMD3465, were used in combination with either Campath (anti-CD52) or Rituxan (anti-CD20) in lymphoma xenograft models to determine whether enhanced tumor protection could be observed. Flow cytometry analysis of lymphoma cell lines confirmed the cell surface expression of high levels of CXCR4 as well as the CD20 and CD52 target antigens. Lymphoma cells were seeded by intravenous injection and tumor-bearing mice were treated with multiple injections of the CXCR4 antagonists AMD3465 or AMD3100 alone or in combination with monoclonal antibodies. Treatment with AMD3465 as a single agent was sufficient to increase survival of the animals compared to untreated controls. In addition, the combination of AMD3465 and Campath showed a synergistic effect with a prolongation of survival greater than that obtained with either agent alone (p<0.01). Similarly, combination of AMD3100 and Rituxan also resulted in a significantly prolonged survival compared to either agent alone (p=0.0340). In vivo imaging using a Raji-luciferase cell line also confirmed the impact of treatment on tumor burden. These results suggest that there is a potential role for CXCR4 antagonists in combination with a B-cell targeted therapy in the treatment of B-cell malignancies in the clinic. Disclosures: Siders: Genzyme Corporation: Employment. Hu:Genzyme: Employment. Gale:Genzyme: Employment. Jacques:Genzyme: Employment. Fogle:Genzyme: Employment. Shields:Genzyme: Employment. Garron:Genzyme: Employment. Kaplan:Genzyme: Employment.
The tumor microenvironment is an important aspect of cancer biology that contributes to tumor initiation, progression and responses to therapy. Cells and molecules of the immune system are fundamental components of the tumor microenvironment which have been known to vary widely and are important in determining the anti-tumor immune response. The approval of immune checkpoint inhibitors like PD-1 and CTLA-4 antibodies has resulted in a lot of research activity in immune-oncology. Syngeneic mouse models have been utilized to understand the preclinical immune response as a surrogate to the clinical response. The timing of immune checkpoint inhibitor dosing is supposed to play an important role on the tumor growth in mouse models. In order to understand the correlation of tumor regression, survival and immune response in a preclinical mouse model, we evaluated the effect of different times of treatment initiation with Program Death Ligand-1 (PD-L1) on tumor volume, survival and tumor infiltrating leukocytes (TIL’s). Various dosing regimens evaluated included initiation of PD-L1 therapy on the day of inoculation, 6 and 12 days post inoculation. Tumors were collected from three different syngeneic mouse models (CT26, RENCA and B16F10) and profiled for the presence of tumor infiltrating leukocytes (TIL’s) using flow cytometry. Major sub-population of TIL's examined included T-regulatory cells (Treg’s), Tumor Associated Macrophages (TAM’s) and Myeloid Derived Suppressor Cells (MDSC’s). Finally, an attempt was made to identify any correlation or lack thereof between tumor growth and tumor immune microenvironment. Citation Format: Geeta Sharma, Susan Wu, Robert Mihalek, Ann Fiore, Matthew Gale, Christa Dias, Wendy Grant, Nidia Hernandez. Characterization of tumor immune microenvironment in response to PD-L1 therapy initiated at different time points post inoculation in syngeneic mouse models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5128.
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