Author: Zurr, Daniel ; Metzger, Philipp ; Weber, Holger ; Hummel, Jonas F. ; Mussolino, Claudio ; Antony, Dinu ; Castro, Carla N. ; Adamsky, Swetlana ; Azcona, Maria Silvia Roman

AbstractThe recent advances in cellular immunotherapies have revolutionized the treatment options for hematological malignancies. Among others, genetically engineered T cells to express chimeric antigen receptors targeting CD19 have shown clinical success in patients with B cell malignancies. However, depending on the study design only one to two thirds of the patients experience a complete response upon CD19 CAR T cells treatment. Antigen escape, immune suppressive microenvironment, CAR T cell dysfunction as well as lack of CAR T cell persistence have been discussed to be reasons for the lack of sustained therapy response. Well established models for the preclinical evaluation of cellular therapies are needed for the evaluation of next generation CAR T cells as well as novel combination therapies.Here, we describe a xenograft animal model of human acute lymphoblastic leukemia (NALM-6 cells) with a suboptimal treatment response to human CD19 CAR T cells from three different donors. First, the activation status and memory phenotype of the CAR T cells from the different donors was characterized by flow cytometry and the anti-tumoral efficacy of the CD19 CAR T cells was evaluated in an in vitro co-culture killing assay using luciferase expressing NALM-6 cells. Next, CD19 CAR T cells from three donors were injected in NALM-6_luc tumor bearing animals. The tumor growth was monitored by bioluminescence imaging and the phenotype of the transferred CAR T cells was checked by flow cytometry.The frequency of memory T cells differed between the donors whereas the T cells from all donors exhibited all low expression of activation markers. In vitro, all three donors showed high and comparable anti-tumoral killing efficacy independent of their memory status. In vivo, CD19 CAR T cells from all three donors delayed the tumor growth significantly although none of the animals had a complete remission. In line with the individual memory status of the CAR T cells prior to infusion, there were slight differences in the kinetics of the anti-tumoral response. The flow cytometric analysis revealed that the transferred CAR T cells were almost absent in the peripheral blood and spleen whereas for individual animals tumor cells and highly activated CAR T cells were detectable in the bone marrow of the animals.In summary, the NALM-6_luc xenograft model is a value tool for evaluating next generation cellular therapies and novel combination strategies to overcome the current limitations of cellular therapies.Citation Format: Philipp Metzger, Carla N. Castro, Jonas F. Hummel, Maria Silvia Roman Azcona, Dinu Antony, Claudio Mussolino, Daniel Zurr, Swetlana Adamsky, Holger Weber. Donor-dependent anti-tumoral efficacy of human CD19 CAR T cells in a leukemic xenograft mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4020.

22 Mar 2024·Cancer Research

Abstract 5242: In vitro cytotoxicity assays to support CAR T cell evaluation against solid tumors

Author: Weber, Holger ; Metzger, Philipp ; Castro, Carla N. ; Bergo, Veronica

AbstractCAR T cells are donor/patient-derived T cells genetically engineered to express a chimeric receptor against a specific tumor antigen (TA), which allows an MHC-independent recognition and targeting of tumor cells. While CART cell therapies have been tremendously successful against hematological cancers, with 6 T cell products being approved up to date from the FDA (Sengsayadeth S etal, EJHaem. 2022), the activity on solid tumors is more difficult. Solid tumors remain a unique challenge in part due to trafficking problems of CAR T cells, a hostile TME and heterogenous antigen expression, which limits CART cell capacity to find their target. Several strategies are being developed at the moment with the aim to overcome these hurdles, such as multispecific CART cells, co-therapy with checkpoint inhibitors, or inhibition of DNA methylation enzymes to prevent exhaustion. To offer an in vitro platform to assess the effectiveness of upcoming cellular therapies we developed several pipelines to allow the investigation of cytotoxic potential in 2D and 3D formats.We show here our results using a CART cell product expressing a coreceptor to enhance its activation potential upon recognition of the target. We next tested the capacity of these cells to kill solid tumor in vitro by coculturing with potential target cells cultured in a monolayer (2D). By measuring the luciferase expression of the target cells, we could track the cytotoxicity exerted by the effector cells. In vitro, the CART cells were able to kill in a dose dependent manner different solid tumor cell lines expressing the cognate antigen for the co-receptor. At Reaction Biology we have developed spheroid-based 3D killing assays in which target cells from solid tumor entities are pre-cultured to form spheroids prior to their coculture with effector cells. Using this technique, we challenged the capacity of these CART cells to recognize their antigen in a 3D format. This 3D assay system can be used as well in combination with co therapies such as PD1 inhibitors, in a sensitive and high throughput manner. Finally, we evaluated the therapeutic capacity of these CART cells in vivo in a subcutaneous THP-1 tumor model. Mice treated with these CART cells showed a marked reduction of tumor volume.In summary we show here a pipeline to characterize in vitro the effectivity of new T cell products, in particular those designed to overcome some of the hurdles against solid tumors.Citation Format: Carla N. Castro, Veronica Bergo, Holger Weber, Philipp Metzger. In vitro cytotoxicity assays to support CAR T cell evaluation against solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5242.

22 Mar 2024·Cancer Research

Abstract 91: Comprehensive 27-marker standard panel for immune monitoring of pre-clinical tumor mouse models using spectral analyzer technology

Author: Weber, Holger ; Borrero-Wolff, Dennis ; Metzger, Philipp ; Obodozie, Cynthia ; Hummel, Jonas F. ; Sonntag, Roland

AbstractWithin the last decade, major technological advances in flow cytometry and (single cell) RNA-sequencing have deepened our understanding of complex anti-tumoral immune responses. This allows a comprehensive immune monitoring of novel therapies in pre-clinical models.Conventional flow cytometry is reaching its technical limitation. The emitted fluorescence signal of the target population stained with antibodies is evaluated with simple bandpass filters, which leads to spectral overlap and thus the number of parameters that can be analyzed simultaneously is limited. The spectral analyzer technology combines both methods, providing great flexibility in evaluating different immune cell populations. This provides the opportunity to obtain target information by measuring the entire fluorescence spectrum measuring each wavelength individually and thus evaluating the true signal of each fluorochrome unaffected by autofluorescence or spillover.Our standard all-in-one flow cytometry panel uses the full capacity of a conventional BD Fortessa flow cytometer and enables the differentiation of important immune cell populations in tumors, such as T cells (CD4+, CD8+, regulatory T cells), B and NK cells as well as macrophages (M1/M2), MDSCs (granulocytes and monocytes) and dendritic cells. However, it would be of great advantage to gain further insights into activation and effector functions of the immune cells using markers like CD44 & CD62L, CD69, PD-1, Lag-3, Tim-3 and PDL-1, CD80, CD86, CD40 and Ki-67. The Sony ID7000 spectral analyzer enables the simultaneous assessment of 27 markers to evaluate immune phenotypes, with the ability to include up to 4 individual target antibodies.Flow cytometry data from various tumor models are presented, demonstrating the enormous utility and potential of the spectral analyzer, especially with limited and precious tumor sample material, by evaluating different immune cell populations simultaneously without reaching its technical limits. The spectral analyzer technology enables us to mechanistically investigate differences in tumor therapies such as PD-1 treatment or other immune cell modulators not only phenotypically but also with regard to activation and differentiation of immune cell populations.Citation Format: Jonas F. Hummel, Philipp Metzger, Roland Sonntag, Dennis Borrero-Wolff, Cynthia Obodozie, Holger Weber. Comprehensive 27-marker standard panel for immune monitoring of pre-clinical tumor mouse models using spectral analyzer technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 91.

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