CAR-T Cell Therapy for Solid Tumors: In Vitro Efficacy and Safety

Balancing cell therapy efficacy and safety during early lead optimization and development

T cell therapies are a relatively novel form of cancer of cancer treatment. They have shown great potential so far, but they also present some formidable limitations. Follow our three-part series, Driving the CAR-T, and see how we take a CAR-T cell therapy for solid tumors all the way from early-stage development to IND filing and lot release.

Engineered T cell therapies such as Chimeric Antigen Receptor T cells (CAR-T) and T Cell Receptor T cells (TCR-T) have emerged as a promising cancer therapy— witness these recent findings of the persistent curative potential of CAR-T —and are moving into the clinic at a high rate. At the same time, the whole pipeline from CAR design to IND-filing can be a long and complicated process. This blog series aims to highlight different cell therapy development stages: from lead optimization to cell engineering and production, followed by in vitro and in vivo efficacy and safety assessments, and lastly with lot release testing. A HER2-targeting CAR-T cell therapy for solid tumors has been generated and used at Charles River Labs to demonstrate the assays required in all those stages of the cell therapy development pipeline.

To date, two anti-BCMA CAR-T and four anti-CD19 CAR-T products have been approved by the US Food and Drug Administration (FDA) for the treatment of lymphoid malignancies, and one TCR T-cell therapy has been granted fast track designation. Building on these successes, many more T cell therapy products are now in in either early-stage research or clinical trials. So far, most success with CAR-T cells has been booked in liquid tumors. More challenges arise for CAR-T cell therapies for solid tumors, such as CAR-T cells penetrating and surviving in the tumor micro-environment or losing target antigen expression. In addition, even though CAR-T products can have high specificity and sensitivity, solid tumor target antigens are often expressed at low levels in healthy tissue as well, which can have a serious impact on the safety of the product due to on-target, off-tumor activity and create major challenges for developers of CAR-T cell therapies for solid tumors. Excitingly, the latest developments of CAR-T cells are not only directed towards liquid and solid tumors but other therapeutic areas are currently under exploration, such as autoimmune diseases, infectious diseases, cardiac fibrosis, and cellular senescence.

In preclinical research of engineered T cell therapies the use of in vivo animal models has shown its challenges in translatability to humans. Therefore, establishing high-quality in vitro efficacy studies complementary to in vivo research is critical to facilitate the development of such therapies. Here we show several in vitro efficacy experiments aimed at determining CAR-T cell therapy for solid tumor for efficacy, specificity, persistence, and safety.

How to assess the efficacy, potency, and persistence of your CAR-T cell therapy in vitro

The HER2 CAR-T cells’ efficacy/potency was first determined in a cytotoxicity co-culture assay using cancer cell lines. One of the cell lines highly expressed HER2, while the other had no HER2 expression. In this assay, multiple different effector-to-target cell ratios were tested to assess efficacy and potency, to understand the lower and upper limits of the cell therapy activity. After 24 hours the co-cultured cells were collected and analyzed for cytotoxicity by flow cytometry. (Figure 1)

Figure 1 . Efficacy and potency of CAR-T cells.

CAR-T and non-transduced T (NTD-T) cells were co-cultured with cell lines expressing high levels of the HER2 (ZR-75-30) or lacking the expression of HER2 (MDA-MB-468), at four different Effector:Target (E:T) ratios. After 24h the co-cultured cells were collected and stained with viability/cell death dyes (Hoechst/PI) and analyzed by flow cytometry. Clear efficacy of the CAR-T cells was observed against HER2+ cells confirming their efficacy. In addition, the clear E:T ratio-dependent effect gives an indication of potency

Next, we determined the persistence of the CAR-T cells using the Repeated Antigen Stimulation (RAS) assay. In this assay CAR-T cells from two donors were co-cultured with the HER2 positive target cells to compare their persistent killing ability over serial rounds of tumor exposure. Every 3 to 4 days following co-culture, the T cells were harvested, counted and re-exposed to a fresh tumor cell monolayer. After several rounds one of the donors showed a less persistent cytotoxic and cell expansion profile indicating faster cellular exhaustion. The principle of this assay can be applied to different CAR-T cell therapies for solid tumors already in the lead-optimization phase. (Figure 2)

Figure 2 . Targeted depletion of HER2+ target cells and CAR-T cell expansion over 4 serial rounds of antigen exposure. CAR-T cells and NTD- T cells were co-cultured with the HER2 positive target cells (ZR-75-30). Every 3- or 4-days following co-culture, the CAR-T and NTD-T cells were harvested, counted and re-exposed to a fresh tumor cell monolayer at original E:T ratio. Cytotoxic ability (A) and expansion potential (B) were assessed over a 2-week period. In (A) Images were taken after CAR-T were harvested and washed off. Donor 2 showed less persistent cytotoxicity and expansion abilities compared to Donor 4, reflecting a faster exhaustion profile.

Is your CAR-T cell targeting the right tissue?

Another important aspect of CAR-T cell therapy for solid tumors is the specificity of the CAR-T cells, which can be determined using a 3-way co-culture aimed at assessing the CAR-T bystander effect. Here, CAR-T cells were co-cultured with HER2 positive and HER2 negative cells within one single well in which each cell type represented one third of the total population. After 24 hours we observed that the CAR-T cells specifically killed HER2 positive cells with no bystander effect on the HER2 negative cells. (Figure 3)

Figure 3 . Assessment of CAR-T bystander effect as measure of specificity through 3-way co-culture . CAR-T cells and NTD-T cells were co-cultured with HER2 positive and HER2 negative cells within one single well; each cell type represented ~33% of the total population. No effect on viability was observed in response to NTD- T cells, whereas CAR-T cells specifically killed HER2+ cells with no bystander effect on the HER2 negative cells (similar ratio of CAR-T cells and HER2 negative cells post co-culture).

So far, our studies have generated an efficacy data package that defines and describes the activity, specificity, and persistence of our CAR-T cells therapies for solid tumors. One lesson learnt the hard way is that the most active and potent drug is not always the safest; for all cell therapies, efficacy and safety will need to be balanced and taken into consideration during early lead optimization and development. We have therefore also generated an in vitro safety profile for CAR-T cells targeting HER2 using primary and iPSC-derived cells.

To that end, we turned to online databases containing protein expression data and performed an in silico assessment of the HER2 gene expression in healthy tissues. Based on these in silico HER2 expression data, tissues at risk were selected to assess HER2 expression at the protein level by flow cytometry using human primary and iPSC-derived cells. Relevant tissues were evaluated in a cytotoxicity co-culture assay with HER2 CAR-T cells. Cytotoxicity was assessed by flow cytometry or xCELLigence, a real-time impedance assessment as a measure of cell attachment and health. Subsequently, IFNγ production in these co-cultures was assessed as a measure of CAR-T cell functionality using the MSD platform. With this assay we could identify specific tissues that are a potential safety risk. (Figure 4)

Figure 4 . Safety assessment of CAR-T cells on HER2-expressing human healthy cardiomyocytes and hepatocytes. CAR-T and NTD-T cells were co-cultured with healthy iPSC-derived cardiomyocytes and primary hepatocytes at two E:T ratios. Impedance was measured using xCELLigence. Cell viability was calculated by normalizing impedance to target cells only (100%) and target cells + toxicity control (0%). A clear cytotoxic effect of the CAR-T cells on both healthy cell types could be observed compared to the NTD-T cells.

Does genetic modification of T cells lead to unwanted cell proliferation and survival?

Lastly, in our study of CAR-T cell therapy for solid tumors, the random integration of the CAR-encoded DNA cassettes in the host cell genome has the potential risk of causing insertional mutagenesis (i.e. mutations in the DNA) which may contribute to oncogenic transformation of the T cells. To determine whether the genomic editing of our HER2 targeting T cells affected their cytokine-dependency, an oncogenicity assay was developed using flow cytometry quantifying the survival and proliferation of the HER2 CAR-T cells in the absence and presence of cytokines. If CAR-T cells survive and proliferate much longer without cytokines compared to their unedited counterparts, then we can consider using next-generation sequencing (NGS) to identify the DNA cassette insertional sites. We would also recommend reviewing the CAR-T cell generation procedure if you are generating CAR-T cell therapies for solid tumors. (Figure 5)

Figure 5 . Quantification of viability and proliferation of CAR-T or NTD-T cells in presence or absence of cytokines . Cells were labeled with CellTrace Violet and acquired by flow cytometry. Event counts were normalized to precision counting beads to determine absolute cell counts. A clear increase in viability and proliferation was observed for both CAR-T and NTD-T cells with cytokines compared to without on day 3 and day 6, as expected. Interestingly, increased viability and proliferation were also observed for CAR-T cells compared to NTD-T cells in the absence of cytokines.

We have developed several in vitro assays for the preclinical assessment of a CAR T cell for efficacy, specificity, persistence and safety to aid early-stage lead discovery, optimization, and development, and to support Investigational New Drug (IND) applications (Figure 6).

Figure 6 . Summary of in vitro assays used for activity, specificity, and safety profiling of CAR-T cells

This in vitro efficacy and safety assessment is only a fraction of the end-to-end solution Charles River Laboratories offers for advancing CAR-T cell therapies for solid tumors into the clinic. The next two parts of this blog series, which you can access here, will highlight other aspects like in vivo efficacy and lot release testing.

This article was written by Sophie Vermond and Dr. Gemma Moiset, scientists at Charles River Laboratories' Early Discovery site in Leiden, The Netherlands. Sophie is a Senior Scientist whose research focuses on efficacy and toxicity testing of novel drug therapies in human primary cell-based models. Her main focus is assay development for T cell therapy safety studies. Furthermore, she is head of the flow cytometry group.

Dr. Moiset is Group Leader at the Leiden lab. She contributes to early-stage research and discovery programs for novel drug therapies such as large molecules and cell therapies. Formerly, she worked in Biotech contributing to the development of monoclonal antibodies against cancer. Prior to that, she was a post-doctoral fellow at the Netherlands Cancer Institute. Dr. Moiset has a combined background in immunology, cellular biology and medicinal chemistry. She holds a PhD in Life Sciences from the University of Groningen and Zernike Institute for Advanced Materials.

Tune in tomorrow for Part II of our series, Driving the CAR-T, when we look at in vivo pharmacology studies done on the CAR-T cells. 

And check out this webinar presented by Sophie and colleague Jezrom Self-Fordham about Mastering CAR Principles to Accelerate Your Therapeutic.

Series logo designed by Chelsea Bates, Charles River Laboratories