Genomic assault in the form of DNA double-strand breaks represents the most efficient mechanism by which radiotherapy mediates killing of cancer cells. In order to preserve cellular integrity and mitigate the adverse effects and complications arising from insult to healthy tissues, a fractionated dosing regimen, where low doses of ionizing radiation are administered over a course of several days, is the principal mode of radiation therapy in clinical settings. Additionally, it has been demonstrated that fractionated dosing has a greater capacity to elicit anti-tumor activity and immune mediated abscopal response, compared to bolus delivery. However, in most preclinical animal models of cancer that employ the use of focal beam radiation, administration of a single bolus dose is the standard practice. To establish a more translationally relevant approach to radiation therapy, we utilized computerized tomography (CT) guided Small Animal Radiation Research Platform (SARRP) in three murine subcutaneous tumor models, MB-49 (bladder), B16F10 (melanoma) and MC38 (colon). Each treatment cohort was subjected to a fractionated dosing regimen, and response was assessed by caliper measurements of tumor volume. Therapy was well tolerated across all models and dosing regimens, as evidenced by no significant body weight loss or presentation of adverse clinical symptoms compared to controls. Fractionated focal radiation at 2Gy delivered over a course of five consecutive days (QDx5) induced a modest anti-tumor response in the MB-49 model, resulting in a 66.7% increase in time to progression (ITP) and a 64.1% median ΔT/ΔC on day 16 post implant. Mice bearing MC38 tumors exhibited meaningful response with incidences of 55% ITIP and a median ΔT/ΔC of 29% on day 26, following treatment with radiation at 2Gy, QDx5. Additionally, fractionated doses at 10Gy and 5Gy both delivered Q5Dx2 produced robust anti-tumor response in the B16F10 model, with incidences of 5% and 21% median ΔT/ΔC on Day 15, respectively. Though notable responses were observed across all treatment cohorts, there were no incidences of tumor free survivors or complete regressions, thus allowing for additional therapeutic intervention and synergistic approaches. Further assessment of fractionated dosing regimens in additional mouse models, as well as potential impact on immunomodulation, are ongoing. Over 50% of cancer patients are treated with radiotherapy, with a significant portion requiring integration with other therapeutic modalities to enhance survival and curative benefits. It is therefore imperative to identify appropriate fractionated dose levels and schedules across preclinical cancer models, in order to more accurately reflect the clinical landscape and provide a more viable framework for interrogating rational combination strategies. Citation Format: Kerry-Ann Bright, Derrik Germain, Erin Trachet, Sheri Barnes. Fractionated dosing: A more clinically relevant approach to radiotherapy in preclinical tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2402.
Mouse syngeneic tumor models are widely used tools to demonstrate activity of novel anti-cancer immunotherapies. However, advanced prostate cancer is difficult to treat due to a lack of effective approaches for disrupting immune tolerance. RM-1 is a murine prostate cancer cell line derived from a Ras/Myc-induced prostate cancer. RM-1 is of particular interest because it is an aggressive, nonimmunogenic, potentially metastatic prostate line that is androgen independent. Subcutaneous growth kinetics were evaluated for RM-1 cells implanted at two cell inocula (1.0E+06 and 5.0E+05 cells/implant). RM-1 grew aggressively across both implant conditions, producing 100% take rate, a median tumor volume doubling time of ~2 days, and a median time to euthanasia criteria (2000 mm3) of ~11 days. Bioanalysis of tumor-infiltrating lymphocyte (TIL) composition via flow cytometry confirms that the RM-1 tumor model has characteristics of a nonimmunogenic or a “cool” tumor. CD4+ T cell and CD8+ T cell populations ranged from 0.7 to 1.9% of the CD45+ cells. Comparatively, G-MDSC and M-MDSC populations were much higher at 8.3 and 63.8% of CD45+ cells. This type of TIL profile is ideal for combination therapies, providing a clear opportunity to turn a “cool” tumor “warm” and therefore potentially more responsive to treatment. To further characterize the RM-1 model, we evaluated anti-mPD-1, anti-mPD-L1, and anti-mCTLA-4. As expected, based on the TIL profile, single agent treatment with checkpoint inhibitors produced minimal anticancer activity, with no tumor regressions, and <56% punitive responders. We also evaluated the anticancer activity of focal beam radiation treatment via small animal radiation research platform (SARRP). Single agent radiation treatment was highly effective, with 20, 10 and 5 Gy producing, 100, 78 and 89% incidence of putative responders. Radiation treatment at 5 Gy was selected as an ideal candidate for combination therapy. Combination therapy of radiation treatment at 5 Gy paired with anti-mPD-1, was evaluated to determine if there was an increase in activity with a two-prong approach. Historically, radiation treatment has been used to increase tumor infiltration and reduce the immunosuppressive tumor microenvironment, allowing for increased efficacy. Single agent treatment of anti-mPD-1 or radiation treatment (5 Gy) resulted in 20 and 30% incidence of putative responders. Combination therapy of anti-mPD-1 and radiation (5 Gy) resulted in 100% putative responders. Overall, the RM-1 model has proven to be a reliable and reproducible syngeneic tumor model for prostate cancer. Its presumed immunosuppressive microenvironment is clinically relevant and can provide a direct correlation to patient prostate cancer. Future studies will include orthotopic implant optimization with checkpoint inhibitor efficacy evaluation, including TIL analysis. Citation Format: Justin Snider, Derrik Germain, Patrick Allison, Alden Wong, Sylvie Kossodo. Model characterization and tumor immune profile assessment for syngeneic RM-1 murine prostate cancer in male C57BL/6 mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1664.
Triple immunodeficient mouse models, that lack functional T cells, B cells, and natural killer cells, are necessary when evaluating human cancer cell growth and cell-based therapy activity, allowing for assessment without innate mouse immune system involvement. Due to limited colony sizes at any given vendor, study timelines can sometimes become compromised unless a sufficient alternative is validated. Site location can also be a factor, as licensing restrictions may be an issue for international corporations. In an effort to prove overall similarities across four strains of triple immunodeficient mice, the growth characteristics of BT-474 were compared in female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice, NOD.CB17-Prkdcscid IL2rgtm1/BcgenHsd (B-NDG) mice, NOD-Prkdcem26Cd52Il2rgem26Cd22/NjuCrl (NCG) mice, and NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac (CIEA NOG) mice. An immune cell profile was also performed across these four mouse strains and evaluated via flow cytometry to observe any immune cell subset differences in blood, spleen, and tumor samples. Samples analyzed post implant were collected when subcutaneous tumors were ~500 mm3. Model development studies were completed previously to optimize tumor growth kinetics. Historic growth conditions were BT-474 cells implanted at 1.0E+07 cells/implant in the presence of an extracellular matrix into the high right axilla of female NSG mice. Using the same optimized growth conditions, NSG, B-NDG, NCG, and CIEA NOG mice were implanted with BT-474 cells. BT-474 grew well in all the tested mouse strains, producing 100% take rate. Compared to NSG mice, median tumor volume doubling times differed by 1-5 days, median times to an evaluation size of 150 mm3 (typical study enrollment tumor volume) differed by +/- ~4 days and median time to an evaluation size of 1250 mm3 (tumor volume at the end of life) differed by +/- ~10 days. BT-474 did not induce body weight loss in any of the tested mouse strains and all clinical observations were similar. Flow cytometry was performed to quantify lymphocyte and myeloid immune subsets in blood, spleen, and tumor. Overall, compared to NSG mice, the absolute counts were similar for all immune subsets measured except for regulatory T cells (Tregs). B-NDG, NCG, and CIEA NOG mice had reduced Treg numbers in the spleen of tumor-bearing mice. In the tumor, Treg numbers were lower in the B-NDG and CIEA NOG mice. No differences were observed for any immune subset in the blood. Overall, the tumor growth kinetics of BT-474 in B-NDG mice was most similar to historical data using NSG mice. The flow data also confirmed that each strain’s immune cell population generally looked very similar, with limited differences between the four strains of triple immunodeficient mice. Citation Format: Justin Snider, Derrik Germain, Lauren Kucharczyk, Anita Zaitouna, Erin Trachet, Scott Wise. Subcutaneous growth and immune cell profiling of BT-474 human breast carcinoma in four strains of triple immunodeficient mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 41.
Preclinical in vivo models are used to profile or refine CAR T therapies before advancing to human studies. Establishing long-term CAR T persistence and efficacy continues to challenge progress in the CAR T space, thus development of robust platforms that can provide longitudinal assessments of CAR T persistence and functionality is paramount. To this end, we used the NALM6-Luc ALL model to develop a flow cytometry platform that provides quantitative analysis of CAR T cells over time as well as surface markers that are documented to correlate with sustained T cell persistence and activation in vivo. Tumored NSG mice were enrolled into treatment groups based on tumor burden calculated from bioluminescence imaging (BLI) data. T cells transduced to express a CD19 CAR (or left untransduced (UTD)) were injected intravenously at 1.0E+07 cells/mouse. Tumor burden was monitored by BLI and flow cytometry was performed weekly on survival bleeds to measure CAR T persistence and examine phenotype. Treatment with CD19 CAR T cells delayed tumor growth resulting in an increase in time to progression of 107.1% compared to UTD controls. However, no regressions were observed. On day 1 post-transfer, CD3+ T cells were detectable in mice that received both CAR T and UTD cells (140+/-26 and 162+/-56 cells/uL blood respectively). T cells declined to near undetectable levels by Day 20, a point when all animals in the UTD treatment group had reached euthanasia criteria. After Day 20, T cells expanded in circulation in the CD19 CAR T treatment group reaching 695+/-327 cells/uL blood by day 40. T cell expansion coincided with exponential tumor outgrowth in the treatment group. To assess T cell functionality, flow cytometry was used to measure the expression of biomarkers for T cell activation (CD25, 4-1BB, and ICOS) and exhaustion markers (TIM-3, PD-1, and LAG-3). CD25, 4-1BB, and ICOS expression did not exceed positivity on more than 15% of CD8+ T cells and peaked by day 30 before downregulation was observed. Notably, PD-1 and LAG-3 expression levels continued to increase throughout the study, suggesting T cells were taking on an exhausted phenotype. Similar trends were observed on CD4+ T cells. To investigate whether the late phase T cell expansion was a graft vs. host response, CAR T cell measurements were compared to non-tumored animals. Expansion as well as PD-1/LAG-3 expression was only observed in tumor-bearing mice indicating the responses were tumor-specific. Taken together, these data demonstrate that CD19 CAR T cells can inhibit NALM6-Luc tumor growth in vivo and expand in circulation in an antigen-specific manner. Furthermore, CAR T cell failure to control tumor growth may be due to onset of an exhausted phenotype. Finally, we demonstrate that flow cytometry can be used to characterize T cell persistence and functionality in murine xenograft tumor models. Citation Format: David W. Draper, Derrik Germain, Stacey Roys, Olivia Nelson, Scott Wise. Preclinical assessment of chimeric antigen receptor (CAR) T persistence and functionality in the disseminated NALM6-Luc human B cell acute lymphoblastic leukemia (ALL) model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2749.
Cancer immunotherapies reprogram the patient’s immune system to mount a coordinated response against a malignant target. T cells engineered to express Chimeric Antigen Receptors (CARs) through transduction with a lentiviral vector represent an effective strategy to specifically eliminate cancerous cells from a patient. Currently, five CAR T cell therapies are approved by the FDA for the treatment of hematological malignancies. With the recent clinical and regulatory success of CAR T cell therapies, the next generation of CAR T cells are undergoing preclinical development. Labcorp Drug Development produces CAR T cells to support the development of new preclinical strategies. Here, the CAR T Cell Generation Service is demonstrated using an anti-CD19 CAR T cell as an example. Using peripheral blood mononuclear cells from healthy, human donors as a T-cell source, CAR T cells were produced by lentivirus transduction. Flow cytometry featuring a CAR-specific monoclonal antibody was used to determine transduction efficiency. To assess in vitro activity, co-cultures of T cells and CD19-expressing NALM6 cells were used to measure cytotoxicity. Proinflammatory cytokine production was analyzed using a Meso Scale Discovery V-PLEX assay. For evaluation of in vivo efficacy, a disseminated NALM6-Luc-mCh-Puro human acute B cell lymphoblastic leukemia model in female NSG mice was conducted. After T cells were treated with lentivirus, approximately 25% of total cells were CD3+/CAR+. Greater than 95% of all target NALM6 cells were killed by a high dose of anti-CD19 CAR T cells. In contrast, only 33% of target cells were killed by untransduced (UTD) T cells at the highest concentration tested. Cytotoxicity against the negative control MV-4-11 cells was equal for a high dose of UTD and a high dose of anti-CD19 CAR T cells. When co-cultured with CD19-expressing NALM6 cells, anti-CD19 CAR T cells produced proinflammatory cytokines including IFN-γ. Relative to mock-treated control mice, treatment with anti-CD19 CAR T cells at high, medium, and low doses increased the time of in vivo disease progression by 175%, 125%, and 16%, respectively. Anti-CD19 CAR T cells generated in our lab were specifically active against CD19-expressing target cells. As a premier contract research organization, Labcorp Drug Development is experienced in producing, handling and culturing T cells and CAR T cells for in vitro and in vivo early discovery studies. Preclinical screening of multiple CAR T cell candidates, alone or in combination with other agents, would facilitate the identification and selection of CARs with the most favorable activity profile for progression through the drug development pipeline. By providing a reliable source of CAR T cells, we are supporting early discovery studies and the development of therapeutic strategies in oncology. Citation Format: Andrew Karalewitz, Natalie Czeryba, Yewei Xing, Derrik Germain, Philip Lapinski, Sheri Barnes, Mark Cameron, Daniel Saims, Amber Rowse, Heidi Nielsen, Scott Wise. Chimeric antigen receptor (CAR) T-cell generation for therapeutic testing in the disseminated NALM6 human B-cell acute lymphoblastic leukemia mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2835.
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