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Dr. Corinna Fiorotti
Posted by Dr. Corinna Fiorotti BioAgilytix Insight, Cell Therapy

Supporting CAR-T Therapies, Part 1: Innovation to Overcome Bioanalytical Challenges

Supporting CAR-T Therapies, Part 1: Innovation to Overcome Bioanalytical Challenges

At this year’s Applied Pharmaceutical Analysis (APA), I had the opportunity to participate in the conversation surrounding the current and next generations of chimeric antigen receptor (CAR) T-cell therapies and innovations. Specifically, in my presentation entitled “Bioanalytical Strategies to Support Novel Generation CAR-T Therapies” I took a closer look at how the evolution of biologics through the centuries has brought us increasingly promising but also increasingly complex therapeutic classes. I wanted to drive home the point that as scientists, we need to create more sophisticated bioanalytical assays in order to better assess these innovative types of therapies.

For example, most bioanalytical methods for canonical small or large molecule drugs utilize ligand binding or LC–MS technology for pharmacokinetic (PK) assessments. CAR-T cell therapies involve administration of ‘living drugs’ capable of proliferation after infusion. This behavior is very different from conventional drug compounds, and the term ‘cellular kinetics’ was coined for in vivo ‘PK monitoring’ of both the expansion and persistence of the genetically engineered CAR-T cells.

Bioanalytical methods used to measure the levels of these infused cells also differ from conventional methodologies and involve both molecular and cellular assays. Even within the last decade we have seen an acceleration in the evolution of CAR-T therapies, with properties altered or added to allow them to more effectively expand and persist in vivo and to enhance their antitumor properties. In this blog, I am presenting some of the main challenges involved with CAR-T therapies in order to emphasize which factors should be focused on during the development of an adequate bioanalytical strategy.

Bioanalytical Challenges
There is minimal regulatory guidance for the development and validation of assays to support CAR-T cell therapies, and these ‘living’ drugs require unique assessments which make it impracticable to follow existing guidelines, guidances, and recommendations.

There are many bioanalytical challenges when developing and validating accurate methods to evaluate CAR-T therapies and it is important to ask three main questions: 1) Are the CAR-T cells expressing the desired antibodies/transgenes, and are they able to target the desired malignant cell? 2) Are the CAR-T cells expanding and persisting in vivo? and 3) If they are expanding and persisting long enough, are they remaining safe and effective?

The development, validation, and implementation of robust, accurate methods is vital to monitor persistence of CAR-T cells and test their safety and efficacy. During CART-T cell therapy the initial dose is followed by in vivo cell expansion and decline. Bioanalytical assays need to ascertain whether the transferred cells survive, express the appropriate markers and expand in vivo. Cellular kinetics, cytokine release testing, immunogenicity, as well as specificity, selectivity, and sensitivity requirements influence the technology platforms selected.

A key challenge to keep in mind is that it can be very difficult to secure a large enough supply of well-characterized critical reagents for long-term support of a CAR-T cell therapy program. Timelines and critical reagent supplies should therefore be carefully planned for when creating a bioanalytical strategy for CAR-T cell therapies.

Bioanalytical Platforms for Cell Therapies
Technologies used for a CAR-T program include:

  • Quantitative PCR (qPCR) (real time and/or Droplet Digital™ PCR), which is a highly sensitive method to quantify the number of CAR transgenes (number of CAR vector copies /µg of sample DNA or /ml of blood) in a population of cells and is a surrogate measurement for presence of CAR and therefore of the cellular kinetics.
  • High-throughput, multi-parameter flow cytometry for cellular kinetics. This method provides direct measurement, accurate quantification and phenotypic analysis of CAR-T cells, real-time immune cell monitoring in vivo and supports qPCR data for cellular kinetic assessments. High-throughput, multi-parameter flow cytometry is also used to assess humoral immunogenicity and to perform immune cell phenotype analysis.
  • The enzyme-linked immunospot (ELISpot) technique is used to assess cellular immune responses against the CAR-T construct. Multiplex immunoassays to assess cytokines and chemokines are implemented for identification of severe responses, such as cytokine release syndrome (CRS). This assessment is crucial for patient safety monitoring and treatment.

At BioAgilytix, we understand the many bioanalytical challenges that accompany CAR-T therapies, which is why we are focused on continually refining and innovating our methods to address those complexities. In our upcoming blog, we will discuss how the next generation of CAR-T therapies can best be supported, as well as where the therapeutic field is headed next.

In the meantime, you can schedule a discussion with our scientists to talk through your CAR-T requirements and how we can support them.

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