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Due to the paucity of targetable antigens, triple-negative breast cancer (TNBC) remains a challenging subtype of breast cancer to treat. In this study, we developed and evaluated a chimeric antigen receptor (CAR) T cell-based treatment modality for TNBC by targeting stage-specific embryonic antigen 4 (SSEA-4), a glycolipid whose overexpression in TNBC has been correlated with metastasis and chemoresistance. To delineate the optimal CAR configuration, a panel of SSEA-4-specific CARs containing alternative extracellular spacer domains was constructed. The different CAR constructs mediated antigen-specific T cell activation characterized by degranulation of T cells, secretion of inflammatory cytokines, and killing of SSEA-4-expressing target cells, but the extent of this activation differed depending on the length of the spacer region. Adoptive transfer of the CAR-engineered T cells into mice with subcutaneous TNBC xenografts mediated a limited antitumor effect but induced severe toxicity symptoms in the cohort receiving the most bioactive CAR variant. We found that progenitor cells in the lung and bone marrow express SSEA-4 and are likely co-targeted by the CAR T cells. Thus, this study has revealed serious adverse effects that raise safety concerns for SSEA-4-directed CAR therapies because of the risk of eliminating vital cells with stem cell properties.

A major roadblock prohibiting effective cellular immunotherapy of pancreatic ductal adenocarcinoma (PDAC) is the lack of suitable tumor-specific antigens. To address this challenge, here we combine flow cytometry screenings, bioinformatic expression analyses and a cyclic immunofluorescence platform. We identify CLA, CD66c, CD318 and TSPAN8 as target candidates among 371 antigens and generate 32 CARs specific for these molecules. CAR T cell activity is evaluated in vitro based on target cell lysis, T cell activation and cytokine release. Promising constructs are evaluated in vivo. CAR T cells specific for CD66c, CD318 and TSPAN8 demonstrate efficacies ranging from stabilized disease to complete tumor eradication with CD318 followed by TSPAN8 being the most promising candidates for clinical translation based on functionality and predicted safety profiles. This study reveals potential target candidates for CAR T cell based immunotherapy of PDAC together with a functional set of CAR constructs specific for these molecules., There is an unmet clinical need to identify therapeutic options for the treatment of pancreatic cancer (PDAC). Here the authors present a systematic screening approach for the identification of potential PDAC cell surface target candidates for CAR-T cell based immunotherapy, followed by their functional validation in preclinical models.

A domain that is often neglected in the assessment of chimeric antigen receptor (CAR) functionality is the extracellular spacer module. However, several studies have elucidated that membrane proximal epitopes are best targeted through CARs comprising long spacers, while short spacer CARs exhibit highest activity on distal epitopes. This finding can be explained by the requirement to have an optimal distance between the effector T cell and target cell. Commonly used long spacer domains are the CH2-CH3 domains of IgG molecules. However, CARs containing these spacers generally show inferior in vivo efficacy in mouse models compared to their observed in vitro activity, which is linked to unspecific Fcγ-Receptor binding and can be abolished by mutating the respective regions. Here, we first assessed a CAR therapy targeting membrane proximal CD20 using such a modified long IgG1 spacer. However, despite these mutations, this construct failed to unfold its observed in vitro cytotoxic potential in an in vivo model, while a shorter but less structured CD8α spacer CAR showed complete tumor clearance. Given the shortage of well-described long spacer domains with a favorable functionality profile, we designed a novel class of CAR spacers with similar attributes to IgG spacers but without unspecific off-target binding, derived from the Sialic acid-binding immunoglobulin-type lectins (Siglecs). Of five constructs tested, a Siglec-4 derived spacer showed highest cytotoxic potential and similar performance to a CD8α spacer in a CD20 specific CAR setting. In a pancreatic ductal adenocarcinoma model, a Siglec-4 spacer CAR targeting a membrane proximal (TSPAN8) epitope was efficiently engaged in vitro, while a membrane distal (CD66c) epitope did not activate the T cell. Transfer of the TSPAN8 specific Siglec-4 spacer CAR to an in vivo setting maintained the excellent tumor killing characteristics being indistinguishable from a TSPAN8 CD8α spacer CAR while outperforming an IgG4 long spacer CAR and, at the same time, showing an advantageous central memory CAR T cell phenotype with lower release of inflammatory cytokines. In summary, we developed a novel spacer that combines cytotoxic potential with an advantageous T cell and cytokine release phenotype, which make this an interesting candidate for future clinical applications.

The in-depth characterization of tumor-infiltrating leukocytes (TILs) is crucial to further improve cancer immunotherapies. Since TILs generally constitute a small subpopulation of solid tumors, they can be lost in the background noise of downstream analyses such as flow cytometry or single cell sequencing. Therefore, improving pre-enrichment methods for TILs is necessary to increase the sensitivity of such studies and save resources spent on the analysis of contaminating cell populations. We have established a reliable workflow combining automated tissue dissociation with positive selection of CD45+ TILs from human tumors as well as humanized mouse models. Human tumor samples were stored and shipped in MACS® Tissue Storage Solution, maintaining cell viability and phenotype up to 48 hours after collection. Tumor dissociation was automated using the gentleMACS™Octo Dissociator and optimized for epitope conservation to overcome bias in immune-phenotyping caused by dissociation with aggressive or impure enzymes. The direct isolation of TILs by MACS® Technology was optimized for the characteristics of solid tumors, such as the presence of debris and dead cells, based on a CD45-specific enrichment reagent. As an example, TIL isolation from a human colorectal carcinoma led to purities of target cells >92% and yields >80%. To allow for the isolation of label free TILs, we developed an isolation strategy based on the REAlease™ Technology. Again, this method is independent of the tumor entity and even tumors with low frequencies of TIL infiltration, such as an ovarian carcinoma with a frequency of 4%, could be enriched to >90% purity. Upon Bead release, >99% of the cells showed no residual magnetic labeling, hence TILs could be subsequently used for a second round of magnetic isolation for the isolation of multiple subpopulations. Additionally, we demonstrated that the REAlease Biotin Complex was effectively removed from the target cells as well, making them non-distinguishable from non-labeled cells before separation. Due to the removal of all labels, epitopes used for isolation are completely available for re-labeling and can be further used for any downstream application. Taken together, we have developed a reliable workflow for the dissociation of solid tumors followed by the isolation of TILs in less than 90 minutes. We could show efficient separation performance using the standard MACS® as well as REAlease™ Technology for positive isolation of TILs. The REAlease™ Technology allows for the efficient removal of MicroBeads and the entire labeling complex, resulting in cells that are suited for a second magnetic labeling step or any kind of downstream analysis. Citation Format: Janina Brauner, Wa'el Al Rawashdeh, Andreas Bosio, Olaf Thorsten Hardt. A complete workflow for the isolation of tumor-infiltrating leukocytes from human tumors and humanized mouse models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1592.

The clinical success of gene-engineered T cells expressing a chimeric antigen receptor (CAR), as manifested in several clinical trials for the treatment of B cell malignancies, warrants the development of a simple and robust manufacturing procedure capable of reducing to a minimum the challenges associated with its complexity. Conventional protocols comprise many open handling steps, are labor intensive, and are difficult to upscale for large numbers of patients. Furthermore, extensive training of personnel is required to avoid operator variations. An automated current Good Manufacturing Practice-compliant process has therefore been developed for the generation of gene-engineered T cells. Upon installation of the closed, single-use tubing set on the CliniMACS Prodigy™, sterile welding of the starting cell product, and sterile connection of the required reagents, T cells are magnetically enriched, stimulated, transduced using lentiviral vectors, expanded, and formulated. Starting from healthy donor (HD) or lymphoma or melanoma patient material (PM), the robustness and reproducibility of the manufacturing of anti-CD20 specific CAR T cells were verified. Independent of the starting material, operator, or device, the process consistently yielded a therapeutic dose of highly viable CAR T cells. Interestingly, the formulated product obtained with PM was comparable to that of HD with respect to cell composition, phenotype, and function, even though the starting material differed significantly. Potent antitumor reactivity of the produced anti-CD20 CAR T cells was shown in vitro as well as in vivo. In summary, the automated T cell transduction process meets the requirements for clinical manufacturing that the authors intend to use in two separate clinical trials for the treatment of melanoma and B cell lymphoma.

Immunotherapies engaging T cells have proven clinical efficacy and tremendous potential. However, responses are often suboptimal. Further research is required to understand tumor-infiltrating leukocytes (TILs) biology and enhance outcomes. TIL analysis is technically challenging and labor intensive. Their number can be very low and small subpopulations may escape analysis as they get lost in the background noise. Importantly, tumor-infiltrating (TI) T cells are embedded in a highly immunomodulatory environment such that unbiased cell-intrinsic functional characterization is hindered. When working with large cohort sizes, even immunophenotyping TILs by flow cytometry is time consuming and data processing is laborious. Pre-enrichment of TI T cells is highly desirable to reduce hands-on time and generate high quality and reliable data. We developed state-of-the-art CD4, CD8, CD3 and CD4/CD8 T cell specific enrichment reagents for magnetic cell sorting incorporating novel technology enabling the removal of both superparamagnetic beads and antibody fragments (REAlease®). Single cell suspension of human ovarian, pancreas or colon tumors was obtained by automated dissociation. TI T cell frequencies ranged from 0,5–14%, were isolated to high purities and with high yield. CD69 was not upregulated after 18h culture, and cells were functional for downstream applications. Importantly, REAlease technology allowed secondary magnetic isolation of sub-populations of T cells, such as gamma-delta T cells. Our data demonstrates that magnetic enrichment using REAlease technology reduces time and increases quality of analysis, and is essential for the isolation of important T cell sub-populations within the tumor microenvironment.

Immunotherapy approaches that engage T cells to attack tumors have proven clinical efficacy and tremendous potential in multiple cancers. Syngeneic mouse tumor models represent the gold standard to develop and analyze effects of immunotherapy, as they possess a fully competent immune repertoire. However, the phenotypic and functional analysis of tumor-infiltrating leukocytes (TILs) is technically challenging and labor intensive. The number of tumor infiltrating T cells can be very low and small subpopulations might escape analysis as they get lost in the background noise. Importantly, tumor infiltrating T cells are embedded in a cellular environment where antigen is abundant and surrounding cells express highly immunomodulatory molecules, such that unbiased cell-intrinsic functional characterization is hindered. When working with large cohort sizes, even immunophenotyping of TILs by flow cytometry is usually time consuming and data processing can be laborious. Therefore, it is fundamental to use effective innovative tools to streamline the workflow and to generate reliable data. We established complete workflows combining tissue storage, dissociation, T cell isolation and flow cytometric phenotyping. These workflows were validated in four different syngeneic mouse tumor models (B16-F10, B16-OVA, 4T1 and CT26.WT). Tissues were either processed immediately or stored in a solution that was shown to maintain cell viability and phenotype up to 48h after collection (Tissue Storage Solution™). Tumor dissociation was automated and optimized for epitope preservation using a tissue dissociator (gentleMACS™ Octo). Phenotypic analysis revealed that optimal enzymatic dissociation was essential for analysis of critical tumor-specific sub-populations, such as PD1hiTim3+Lag3+CD39+CD8+ T cells present in B16-F10 tumors. We developed new T cell-specific enrichment reagents for manual and (semi-) automated magnetic cell sorting, based on MACS® Technology, which enrich for rare tumor infiltrating T cells by up to 500-fold, while maintaining activation status and phenotype. Importantly, we compared labeling of TILs using conventional hybridoma-derived antibodies with recombinant antibodies engineered to eliminate Fc receptor-mediated background (REAfinity™). Use of REAfinity antibodies significantly diminished non-specific labeling of cells present in the tumor microenvironment. Finally, flow cytometric analysis was performed using an automated analyzer (MACSQuant X™). This instrument decreased hands-on as well as total acquisition time by facilitating fast and fully automated sample processing, including sample mixing and absolute cell counting. In conclusion, we have optimized workflows that include standardized processing of tumor samples, newly developed tools for (semi-) automated magnetic isolation of tumor infiltrating T cells and automated flow cytometric analysis. These workflows greatly reduce experimental time and allow the performance of more complex experimental setups. We believe the use of these innovative tools and workflows can significantly increase the quality of the data obtained in immuno-oncology and immunotherapy research. Citation Format: Anne Richter, Ramona Siemer, Carolin Steinbrueck, Manuela Herber, David Agorku, Janina Brauner, Olaf Hardt, Christian Dose, Cesar Evaristo. Tumor infiltrating T cells: complete workflows allow faster and improved flow cytometric analysis of syngeneic mouse tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-351.

Introduction Syngeneic mouse tumour models are widely used to analyse tumour immunology due to their fully competent immune-repertoire and have paved the way for novel immunotherapy agents in multiple tumour entities. However, the amount and composition of tumour infiltrating leukocytes (TIL) is highly variable. This complicates targeted analysis, in particular for small leukocyte subpopulations that may not be analysed properly or lost in the background noise. When working with large cohort sizes, immune-phenotyping by flow cytometry is time consuming and highly work intensive. We have developed improved workflows combining automated tissue dissociation with novel TIL specific isolation reagents to allow for more accurate and faster analysis of TILs and TIL subpopulations. Material and methods We have developed novel TIL specific enrichment reagents for the magnetic cell sorting (MACS) based isolation directly from dissociated tumour tissue. The composition of these populations before and after separation was analysed by flow cytometry. The developed tools were combined with optimised tissue dissociation and flow panels to establish comprehensive workflows. Results and discussions We have established workflows combining optimised and automated tissue dissociation using the gentleMACS platform with TIL specific isolation to improve and accelerate downstream analysis. Isolation of TIL was improved by developing new CD45, CD4+, CD8+, and pan T cell enrichment reagents for MACS-based isolation directly from dissociated tumour tissue. Applying this workflow, CD45 +TIL were enriched to purities above 80%–90% with high yields (>70%) from divers syngeneic mouse tumours. This allowed for rapid and reliable analysis even of small TIL subpopulations. Moreover, by using the newly developed T cell reagents, T cells were enriched to purities higher than 90%, allowing for a reliable analysis of rare T cell subpopulations. Importantly, while TIL or T cell enrichment significantly reduced analysis time and reagent costs in immune subset analysis, the composition of infiltrating cells was not affected, excluding the risk of introducing a bias by this method. Conclusion We have developed improved workflows for the isolation of generic TIL and T cells from mouse tumours reducing time and costs of downstream analysis while standardising and enhancing the detection and quantification of immune cell subpopulations. CD45 +TILs, pan-, CD4 + - or CD8 + -T cells can directly be isolated from dissociated mouse tumours and analysed for subpopulations.

Tumor-Infiltrating (TI) T cells play an important role in anti-tumor immunity, highlighted by multiple immunotherapy strategies approved by the FDA. However, the benefit of current immunotherapies is still limited, such that additional research is needed to improve efficacy. Preclinical tumor models are essential to advance immunotherapy research. However, tumor composition is highly variable and T cell numbers can be very limited. Even flow cytometry can be very time consuming. Importantly, TI T cells are embedded in a cellular environment where antigen is abundant and surrounding cells express immunomodulatory molecules, such that unbiased cell-intrinsic functional characterization is impossible. Pre-enrichment of TI T cells is highly desirable to reduce hands-on time, and generate high quality and reliable data. We developed new CD4, CD8 and Pan T cell specific enrichment reagents for magnetic cell sorting from mouse tumors. Single cell suspension was obtained by automated dissociation. T cell isolation was done either manually, or using semi or full automation. TI T cell frequencies ranged from 0.2–9%, were isolated to purities above 80% with yields ranging from 60–95%. Pan T cell isolation maintained the original CD4:CD8 ratio. CD69 was not upregulated after 18h culture, demonstrating that isolation did not result in T cell activation. Importantly, functional analysis of bulk vs. purified cells revealed significant influence of tumor and stromal environment in cytokine expression by TI T cells, which was not due to the isolation protocol. Our data, demonstrates that magnetic enrichment reduces time of analysis, increases quality of analysis and is essential for the functional characterization of TI T cells.

T cells can eliminate tumors, but tumor infiltrating (TI) T cells can become intrinsically anergic or extrinsically suppressed. Identifying the mechanisms of T cell dysfunction is necessary to improve immunotherapy efficacy. Mouse tumor models are useful tools in tumor T cell research, but analysis of TI T cells is technically challenging, time consuming and labor intensive. Their frequency can be very low and small populations may escape analysis as they get lost in the background. Importantly, TI T cells are in an antigen-rich and highly immunomodulatory cell environment, such that cell intrinsic functional analysis is hindered. It is fundamental to use new tools to streamline the workflow and generate reliable data. We established workflows combining tissue dissociation, T cell isolation and phenotyping. Tumor dissociation was automated using the gentle MACS™ and optimized for epitope preservation. Enzymatic dissociation was essential for analysis of PD1hiTim3+Lag3+CD39+ CD8 T cells in tumors. Using new T cell-specific microbeads for magnetic cell sorting we increased TI T cell purity by up to 500-fold, while maintaining activation status and phenotype. Importantly T cell isolation from the tumor’s highly immunomodulatory cellular environment was essential to assess cell-intrinsic functional properties. Finally, our data show the use of recombinant RE Afinity antibodies eliminated non-specific labeling of cells in the tumor microenvironment. We conclude that standardized processing of tumor samples and magnetic isolation of tumor infiltrating T cells greatly reduces time and cost of total workflow and downstream analysis while significantly increasing reproducibility and the quality of data obtained from TIL analysis.

Immunotherapy against cancer has proven clinical efficacy and tremendous potential in multiple tumor entities. Syngeneic mouse tumor models represent the gold standard to analyze effects of immunotherapy due to their fully competent immune-repertoire. However, the amount and composition of tumor infiltrating leukocytes (TIL) is highly variable, complicating the targeted analysis of subpopulations. In particular, small subpopulations cannot be analyzed properly but may be lost in the background noise. When working with large cohort sizes, even the immune-phenotyping of TIL by flow cytometry is time consuming and data processing highly work intensive making pre-enrichment methods for sample debulking attractive. We have established an automated workflow combining tissue dissociation with TIL specific isolation to improve and accelerate downstream analysis. Tumor dissociation was automated using the gentleMACS™Octo Dissociator and optimized for epitope conservation to overcome bias in immune-phenotyping caused by dissociation with aggressive of impure enzymes. Next, isolation of TIL was improved by developing a new CD45-specific enrichment reagent for the magnetic cell sorting (MACS) based isolation directly from dissociated tumor tissue. The whole workflow takes only about 90 min. To validate this method on starting material showing variable frequencies of TIL infiltration, we used syngeneic mouse tumors derived by injection three independent tumor cell lines. Tumors derived from injection of B16.F10 melanoma showed TIL frequencies of 2-4%, CT26.WT colon carcinoma tumors 15-21%, and 4T1 breast carcinoma tumors 32-37% among total viable cells after dissociation. Using a manual separation system, TIL were enriched to purities above 90% at yields above 70% for CT26.WT and 4T1 tumors, and purities above 80% at high yields above 95% for B16.F10 tumors. To address the need of parallelization and automation for sample processing in large cohort sizes used in in vivo studies, a fully automated version of the MultiMACS™ Cell24 Separator was developed by integrating the instrument in a liquid handling robotic platform. This new system, the MultiMACS™ X, can process 24 cell separations in parallel with minimal hands-on time. When compared to the manual system, equal purities were achieved whereas the overall yield of target cells was increased from 70% to 90% as shown in the case of CT26.WT tumors. Importantly, while the TIL enrichment significantly reduced the time and reagent costs in immune subset analysis, the composition of infiltrating immune cells was not affected, excluding the risk of introducing a bias by this method. Taken together, we have developed an automated workflow for the isolation of TIL from mouse tumors reducing time and costs of downstream analysis while standardizing and enhancing the detection and quantification of immune cell subpopulations. Citation Format: Janina Brauner, David Agorku, Anne Langhammer, Lukas Pohlig, Jan Drewes, Thomas Dino Rockel, Oliver Schilling, Stephan Mehlhose, Wa'el Al Rawashdeh, Andreas Bosio, Olaf Hardt. Workflow automation and parallelization improves the isolation and analysis of tumor-infiltrating immune subpopulations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1672. doi:10.1158/1538-7445.AM2017-1672