- Email: [email protected]
Development of a novel potency assay to quantify immune cell-mediated cancer cell killing
Poster Abstracts
and function of adoptively transferred T cells which will most likely result in improved clinical benefit to more individuals. There are several challenges in T cell therapy manufacturing processes due to variation in input cell material from patients with advanced disease states such as terminally differentiated cells, low CD8+T cell numbers etc. Depending on patient disease state, some populations of T cells are lost during expansion. CD8+T cells are cytotoxic lymphocytes that kill tumor cells and virus infected cells. Some approaches explored to mitigate CD8+ T cell loss include isolating CD4+T cells and CD8+T cells from the same donor and mixing them at set ratios. This approach adds complexity and could further decrease the input cell population due to selection process loss. As an alternative to this approach, we explored whether supplementation of our serum-free prototype medium with a novel compound could increase the CD8+T cell population in pan CD3+ T cells. We added the supplement to our medium continuously or at selected time points in a CTS™ Dynabeads™ CD3/CD28 expansion process. Using flow cytometry, we observed an increase up to 2.5 fold in CD8:CD4 ratio with supplement addition. Importantly, the supplement in our serum-free medium did not inhibit cell growth, where T cells expand routinely to greater than five population doublings. The applications of these findings can enable creation of new cell culture reagents to modulate T cell populations as well potentially enabling in-process modulation of T cell populations during manufacturing processes.
82 DEVELOPMENT OF AN LMP-SPECIFIC T CELL BANK FOR THIRD PARTY USE AS A CURATIVE STRATEGY FOR POSTTRANSPLANT LYMPHOPROLIFERATIVE DISEASE (PTLD) AFTER SOLID ORGAN TRANSPLANT (SOT) L. McLaughlin1, L. Roesch1, K. Mintz1, C. Barese1, F. Hoq1, M. Keller1, J. Hochberg2, C. Cruz1, C. Allen3, B. Wistinghausen4, M.S. Cairo2, C. Bollard1 1 Children’s National Medical Center, Washington, District of Columbia, United States, 2New York Medical Center, Valhalla, New York, United States, 3 Baylor College of Medicine, Houston, Texas, United States, 4Mount Sinai School of Medicine, New York, New York, United States Post-transplant lymphoproliferative disease (PTLD) is a complication that arises after SOT as the suppression in EBV-specific T cell immunity allows for uncontrolled proliferation of EBV-infected B cells. Outcomes for PTLD are suboptimal with relapse rates approaching 50%. EBV-positive PTLD is highly immunogenic disease as proliferating B cells express the EBV antigens LMP1 and LMP2 that can be targeted with immune therapy. We hypothesize that third party “off the shelf” LMP-specific T cell products (LMP-TCs) may improve outcomes and decrease associated co-morbidities for patients with PTLD by not only targeting the proliferating EBV-infected B cells but also restoring EBVspecific immunity. To develop the third party bank, we manufactured LMP-TCs from eligible donors in our GMP facility for an upcoming Children’s Oncology Group trial ANHL1522 for patients with PTLD after SOT. Over half of the donors represent ethnic minorities and thus have a broad range of HLA types. Thus far, 23 LMP-TC products have been manufactured using autologous monocytes and lymphoblastoid cell lines transduced with an adenoviral vector expressing ΔLMP1 and LMP2 as antigen presenting cells and released for third party use. LMP-TCs were active against LMP1 (mean: 40 SFU/1x10^5 cells; range: 0–322) and LMP2 (mean: 189 SFU/1x10^5 cells; range: 9–655) as determined by IFN-γ ELISPOT assay. The LMP-TCs comprised a mean of 45% CD8+ T-cells, 35% CD4+ T-cells, and 9% NK cells. No B cells or monocytes were detected in the final products. LMP-TCs undergo comprehensive characterization by IFN-γ ELISPOT assay to determine LMP-specific epitopes, Class I and/or II response, and HLA restriction to guide selection of LMP-TC product for each patient. We have identified 3 novel LMP epitopes. Approximately 60% of the LMP-TC products have LMP-specific activity through multiple HLA alleles, and 50% have a mixed Class I and II response. Thus, LMP-specific T cell products can be expanded from healthy donors to create a third party bank. Identification of epitopes and HLA alleles with LMP activity will facilitate the selection of the most appropriate product for patients and broaden the applicability of a third party bank as products that match at only one HLA allele can be safely infused if the shared allele has LMPactivity. While LMP-specific T cells have previously demonstrated safety/ efficacy in phase I studies, ANHL1522 will be the first trial using cellular therapy within a cooperative group setting.
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83 MANUFACTURING SOLUTIONS FOR AUTOLOGOUS CELL THERAPIES S. Luther, S. Punreddy, T. Lawson, A. Verma, S. Rigby, A. Schnitzler, M. Rook, J. Murrell Cell Therapy Bioprocessing, MilliporeSigma, Bedford, Massachusetts, United States The long-term view of regenerative medicine therapies predicts an increased need for expansion solutions that are robust and are compatible with limited downstream processing steps. As more cell therapeutics progress through clinical testing, current in vitro culture methods lack efficiency and reproducibility. Moreover, single-use devices and high quality animal origin-free reagents support the future implementation of manufacturing solutions that will be required following clinical success. Here, we describe the implementation of single use bioreactors and high quality media for expansion of cell therapies. We focus on an example of autologous T cells. The presentation will review solutions addressing animal origin-free expansion of cells within the context of different upstream process development steps with good cell yield, high viability and maintenance of identity. Start to finish solutions for manufacturing, including high quality reagents, are key enabling technologies for success in commercializing cell therapies. 84 DEVELOPMENT OF A NOVEL POTENCY ASSAY TO QUANTIFY IMMUNE CELL-MEDIATED CANCER CELL KILLING A. Oumie, A. Chan, M. Baradez, D. Marshall Industrialisation, Analytical Development, Cell and Gene Therapy Catapult, London, United Kingdom Harnessing the immune system to recognize and destroy cancer cells is one of the fastest growing areas in the cell and gene therapy field. A significant number of these therapies use genetic modifications of T-cells using viral vectors to engineer their specificity or enhance their function. Examples of these products include gene modified T-cells expressing Chimeric Antigen Receptors (CAR) or engineered T-cell receptors (TCR). Various strategies are applied for the manufacture of gene modified immunotherapies but the use of patient cells as a starting material and viruses to deliver the CAR or TCR construct can lead to variability in transduction efficiency and expression of the CAR/TCR. Application of a suitable potency assay as part of product release testing is therefore essential to ensure sufficient function. Here we demonstrate a novel potency assay developed for a gene modified TCR immunotherapy product. This assay uses impedance spectroscopy to give a label free, real time measurement of cell killing and involves the anchoring of peptide loaded antigen presenting cells (APC) to an electrode array using specific capture antibodies. When an electrical current is passed across the array the attached cells create resistance (impedance) which can be directly measured. Killing of these APC’s by the gene modified T cells reduces the impedance in a dose and time dependent manner. The killing response was cross validated using flow cytometry and high content live cell imaging. This assay has many advantages over commonly used alternative methods such as the chromium51 killing assay. For example, the impedance assay is label free and does not require pre-loading of the target cells with a radioactive isotopes or other detection labels which can interfere with the assay readout. It can be performed with established cell lines, reducing the assay variability associated with primary cells. The impedance assay also provides real time data showing the kinetics of the killing response rather than just a single end-point measurement. While we demonstrate the application of this novel assay with a gene modified TCR cell therapy it is equally applicable for non-gene modified immunotherapies targeting a range of hematological malignancies. 85 HIV SPECIFIC T CELLS GENERATED FROM HIV NAIVE ADULT AND CORD BLOOD DONORS TARGET A RANGE OF NOVEL VIRAL EPITOPES—IMPLICATIONS FOR A CURE STRATEGY AFTER ALLOGENEIC HSCT AND CBT S. Patel1,2, S. Lam2, J. Sung3, R. Cruz1,2, N. Goonetilleke3, Y. Xu3, J. Kuruc3, C. Gay3, B. Jones1, E. Shpall4, D. Margolis3, R. Ambinder5, C. Bollard1,2 1 The George Washington University, Washington, District of Columbia, United States, 2Children’s National Health System, Washington, District of Columbia, United States, 3HIV Cure Center, University of North Carolina,
and function of adoptively transferred T cells which will most likely result in improved clinical benefit to more individuals. There are several challenges in T cell therapy manufacturing processes due to variation in input cell material from patients with advanced disease states such as terminally differentiated cells, low CD8+T cell numbers etc. Depending on patient disease state, some populations of T cells are lost during expansion. CD8+T cells are cytotoxic lymphocytes that kill tumor cells and virus infected cells. Some approaches explored to mitigate CD8+ T cell loss include isolating CD4+T cells and CD8+T cells from the same donor and mixing them at set ratios. This approach adds complexity and could further decrease the input cell population due to selection process loss. As an alternative to this approach, we explored whether supplementation of our serum-free prototype medium with a novel compound could increase the CD8+T cell population in pan CD3+ T cells. We added the supplement to our medium continuously or at selected time points in a CTS™ Dynabeads™ CD3/CD28 expansion process. Using flow cytometry, we observed an increase up to 2.5 fold in CD8:CD4 ratio with supplement addition. Importantly, the supplement in our serum-free medium did not inhibit cell growth, where T cells expand routinely to greater than five population doublings. The applications of these findings can enable creation of new cell culture reagents to modulate T cell populations as well potentially enabling in-process modulation of T cell populations during manufacturing processes.
82 DEVELOPMENT OF AN LMP-SPECIFIC T CELL BANK FOR THIRD PARTY USE AS A CURATIVE STRATEGY FOR POSTTRANSPLANT LYMPHOPROLIFERATIVE DISEASE (PTLD) AFTER SOLID ORGAN TRANSPLANT (SOT) L. McLaughlin1, L. Roesch1, K. Mintz1, C. Barese1, F. Hoq1, M. Keller1, J. Hochberg2, C. Cruz1, C. Allen3, B. Wistinghausen4, M.S. Cairo2, C. Bollard1 1 Children’s National Medical Center, Washington, District of Columbia, United States, 2New York Medical Center, Valhalla, New York, United States, 3 Baylor College of Medicine, Houston, Texas, United States, 4Mount Sinai School of Medicine, New York, New York, United States Post-transplant lymphoproliferative disease (PTLD) is a complication that arises after SOT as the suppression in EBV-specific T cell immunity allows for uncontrolled proliferation of EBV-infected B cells. Outcomes for PTLD are suboptimal with relapse rates approaching 50%. EBV-positive PTLD is highly immunogenic disease as proliferating B cells express the EBV antigens LMP1 and LMP2 that can be targeted with immune therapy. We hypothesize that third party “off the shelf” LMP-specific T cell products (LMP-TCs) may improve outcomes and decrease associated co-morbidities for patients with PTLD by not only targeting the proliferating EBV-infected B cells but also restoring EBVspecific immunity. To develop the third party bank, we manufactured LMP-TCs from eligible donors in our GMP facility for an upcoming Children’s Oncology Group trial ANHL1522 for patients with PTLD after SOT. Over half of the donors represent ethnic minorities and thus have a broad range of HLA types. Thus far, 23 LMP-TC products have been manufactured using autologous monocytes and lymphoblastoid cell lines transduced with an adenoviral vector expressing ΔLMP1 and LMP2 as antigen presenting cells and released for third party use. LMP-TCs were active against LMP1 (mean: 40 SFU/1x10^5 cells; range: 0–322) and LMP2 (mean: 189 SFU/1x10^5 cells; range: 9–655) as determined by IFN-γ ELISPOT assay. The LMP-TCs comprised a mean of 45% CD8+ T-cells, 35% CD4+ T-cells, and 9% NK cells. No B cells or monocytes were detected in the final products. LMP-TCs undergo comprehensive characterization by IFN-γ ELISPOT assay to determine LMP-specific epitopes, Class I and/or II response, and HLA restriction to guide selection of LMP-TC product for each patient. We have identified 3 novel LMP epitopes. Approximately 60% of the LMP-TC products have LMP-specific activity through multiple HLA alleles, and 50% have a mixed Class I and II response. Thus, LMP-specific T cell products can be expanded from healthy donors to create a third party bank. Identification of epitopes and HLA alleles with LMP activity will facilitate the selection of the most appropriate product for patients and broaden the applicability of a third party bank as products that match at only one HLA allele can be safely infused if the shared allele has LMPactivity. While LMP-specific T cells have previously demonstrated safety/ efficacy in phase I studies, ANHL1522 will be the first trial using cellular therapy within a cooperative group setting.
S41
83 MANUFACTURING SOLUTIONS FOR AUTOLOGOUS CELL THERAPIES S. Luther, S. Punreddy, T. Lawson, A. Verma, S. Rigby, A. Schnitzler, M. Rook, J. Murrell Cell Therapy Bioprocessing, MilliporeSigma, Bedford, Massachusetts, United States The long-term view of regenerative medicine therapies predicts an increased need for expansion solutions that are robust and are compatible with limited downstream processing steps. As more cell therapeutics progress through clinical testing, current in vitro culture methods lack efficiency and reproducibility. Moreover, single-use devices and high quality animal origin-free reagents support the future implementation of manufacturing solutions that will be required following clinical success. Here, we describe the implementation of single use bioreactors and high quality media for expansion of cell therapies. We focus on an example of autologous T cells. The presentation will review solutions addressing animal origin-free expansion of cells within the context of different upstream process development steps with good cell yield, high viability and maintenance of identity. Start to finish solutions for manufacturing, including high quality reagents, are key enabling technologies for success in commercializing cell therapies. 84 DEVELOPMENT OF A NOVEL POTENCY ASSAY TO QUANTIFY IMMUNE CELL-MEDIATED CANCER CELL KILLING A. Oumie, A. Chan, M. Baradez, D. Marshall Industrialisation, Analytical Development, Cell and Gene Therapy Catapult, London, United Kingdom Harnessing the immune system to recognize and destroy cancer cells is one of the fastest growing areas in the cell and gene therapy field. A significant number of these therapies use genetic modifications of T-cells using viral vectors to engineer their specificity or enhance their function. Examples of these products include gene modified T-cells expressing Chimeric Antigen Receptors (CAR) or engineered T-cell receptors (TCR). Various strategies are applied for the manufacture of gene modified immunotherapies but the use of patient cells as a starting material and viruses to deliver the CAR or TCR construct can lead to variability in transduction efficiency and expression of the CAR/TCR. Application of a suitable potency assay as part of product release testing is therefore essential to ensure sufficient function. Here we demonstrate a novel potency assay developed for a gene modified TCR immunotherapy product. This assay uses impedance spectroscopy to give a label free, real time measurement of cell killing and involves the anchoring of peptide loaded antigen presenting cells (APC) to an electrode array using specific capture antibodies. When an electrical current is passed across the array the attached cells create resistance (impedance) which can be directly measured. Killing of these APC’s by the gene modified T cells reduces the impedance in a dose and time dependent manner. The killing response was cross validated using flow cytometry and high content live cell imaging. This assay has many advantages over commonly used alternative methods such as the chromium51 killing assay. For example, the impedance assay is label free and does not require pre-loading of the target cells with a radioactive isotopes or other detection labels which can interfere with the assay readout. It can be performed with established cell lines, reducing the assay variability associated with primary cells. The impedance assay also provides real time data showing the kinetics of the killing response rather than just a single end-point measurement. While we demonstrate the application of this novel assay with a gene modified TCR cell therapy it is equally applicable for non-gene modified immunotherapies targeting a range of hematological malignancies. 85 HIV SPECIFIC T CELLS GENERATED FROM HIV NAIVE ADULT AND CORD BLOOD DONORS TARGET A RANGE OF NOVEL VIRAL EPITOPES—IMPLICATIONS FOR A CURE STRATEGY AFTER ALLOGENEIC HSCT AND CBT S. Patel1,2, S. Lam2, J. Sung3, R. Cruz1,2, N. Goonetilleke3, Y. Xu3, J. Kuruc3, C. Gay3, B. Jones1, E. Shpall4, D. Margolis3, R. Ambinder5, C. Bollard1,2 1 The George Washington University, Washington, District of Columbia, United States, 2Children’s National Health System, Washington, District of Columbia, United States, 3HIV Cure Center, University of North Carolina,