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NA LRA Watchdog - June 2022


North America Legal and Regulatory Affairs
Watchdog Update
June 2022

Kaitlin Morrison, PhD

Director of UNC Lineberger Sponsored Clinical Research, UNC Lineberger Comprehensive Cancer Center
Assistant Professor of Medicine- Hematology, University of North Carolina- Chapel Hill

Joseph (Yossi) Schwartz, MD, MPH
Professor of Pathology, Molecular and Cell Based Medicine, Mount Sinai

The Watchdog Focus: The FDA recently released a draft guidance on Considerations for the Development of Chimeric Antigen Receptor (CAR) T Cell Products. It outlines the general considerations for CAR T cell design and development and recommendations for Chemistry, Manufacturing and Control (CMC), pharmacology and toxicology, and clinical study design.

For those of us who have worked in the CAR T cell field for years, FDA issuing this guidance and the contents within it were far from surprising. We have received many of these comments from the Agency for years and have witnessed the field move in this direction.


General Notes: For those who are less familiar with chimeric antigen receptor (CAR) T cell products, FDA defines them as “human gene therapy products in which the T cell specificity is genetically modified to enable recognition of a desired target antigen for therapeutic purposes.” They may be developed as an autologous or allogenic product. Although there are many companies now in the field manufacturing CAR T cells and sponsoring clinical trials, CAR T cells have long been manufactured by academic centers for trials sponsored by these institutions making them unique from other products which typically undergo clinical development/manufacturing within pharmaceutical companies. As a result, FDA starts by specifically noting that this guidance is intended to assist sponsors “including industry and academic sponsors” in the development of their CAR T cell products. The Agency also notes that although the focus of the guidance is on CAR T cells that the advice may be applicable to studies of similar products including CAR Natural Killer (NK) cells or T cell receptor (TCR)-modified T cells.


General Considerations: FDA stresses the complexities of CAR T cell development and design and how that impacts all aspects of development from manufacturing to preclinical work to clinical design. The Agency stresses “the nature of…functional elements, how the functional elements are introduced into the cells (i.e., vector type), the cellular starting material, and the final drug product formulation are all critical to product safety, specificity, and function.” The guidance focuses on FDA’s recommendations for the design of the CMC, preclinical and clinical strategies because of these key complexities.


CMC: Of note,  the “gold standard” guidelines for manufacturing CAR T cells were released by FDA back in January 2020 in a guidance entitled “Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs): Guidance for Industry”. FDA notes the importance of this guidance and frequently refers to it as they outline CAR T cell-specific recommendations building upon this past guidance.

Some key CMC considerations include:

  • Defining the vector as a drug substance (DS) and the CAR T cell product as both a separate DS and a drug product (DP)
  • Being aware that vector manufacturing facilities are subject to FDA inspection during Biologics License Application (BLA) review
  • Providing engineering runs demonstrating the ability to manufacture the product as described within the IND. A sponsor may cross reference manufacturing information from similar INDs such as those manufacturing with the same process but using a different CAR construct at the same facility to support their process development.
  • Understanding that key differences in manufacturing, starting all the way back at the time that blood is collected from the participant to manufacture the CAR T cells, can affect the safety of efficacy of the products. This requires additional levels of consideration when the participant has previously received another CAR T cell product.
  • Performing risk assessments for proposed changes to the manufacturing process and anticipating changes needed to establish a scalable and robust manufacturing process prior to performing the primary BLA efficacy studies.
  • Understanding that in the case of multicenter trials, differences between manufacturing facilities may contribute to product variability and studies must be performed to demonstrate that a comparable product is manufactured at each location. Furthermore, when possible, the same processes, SOPs, training, reagents, and equipment should be used to minimize variability.
  • Considering all transgene elements, if there are multiple, when assessing the product (e.g., if the CAR T cell includes a cytokine transgene a potency assay should be performed to assess its activity in addition to any potency assays performed to assess the activity of the CAR).
  • Assaying any impacts that additional elements (such as safety switches) may have on CAR T cell specificity, functionality, immunogenicity, or safety.
  • Limiting variability where possible (e.g., normalizing and optimizing the amount of vector used for transduction of CAR-T cells during manufacturing)


In short, the CMC guidance is all about consistency and reducing the variability between products by having good controls and starting with as consistent as possible materials. If there are elements of variability, the guidance recommends running validation to ensure products are comparable.


Preclinical: Preclinical testing for CAR T is overall complex as there are limitations in the utility of animal models because of the species specificity of the CAR T cells and the tumor target(s). Preclinical studies often are performed in immune-incompetent mouse models that cannot model human immune responses such as cytokine release syndrome which is a frequent and serious adverse event observed in clinical studies. Animal models can be useful in alternative ways such as studying trafficking and proliferation of CAR T cells in mice engrafted with human tumor xenografts and then infused with CAR T cells. If a relevant surrogate product is available for infusion into a syngeneic tumor animal model, then this product must be shown to be suitable for such experiments. These models can then be used to study how the product behaves in an intact immune system and if there are any potential on-target off-tumor toxicities.

The guidance draws attention to the fact that the antigen recognition domain and its ability to confer target specificity are major players in determining the safety and efficacy of the CAR T cell product. As such, they recommend studies to evaluate this domain and the potential of on-target off-tumor toxicity including:

  • Tissue cross-reactivity studies using a monoclonal antibody or fusion protein with the same antigen recognition domain
  • Cytotoxicity testing on panels of human primary cells, cell lines, induced pluripotent stem cell-derived test systems, etc., for various organs/tissues
  • Protein arrays
  • Relevant animal models

Furthermore, the Agency recommends using data from previous human experience with a CAR or monoclonal antibody with an identical antigen recognition domain, if available, to avoid repetition of unnecessary animal studies.

This guidance also highlights all the parameters of the CAR T cell product that may affect the safety/efficacy of the product and thus should be considered during preclinical study design to support and inform clinical protocol design. These include:

  • Design of the vector construct (e.g., antigen recognition domain, signaling domains, transmembrane and hinge domains)
  • Vector delivery method
  • Cell source
  • Manipulation processes (e.g., activation, cell selection)
  • Biological activities (e.g., cytokine expression profiles, cytotoxicity, proliferation)
  • Addition of novel components (e.g., suicide gene, immunomodulatory elements)

The guidance highlights why some of these differences may contribute to different safety and efficacy profiles such as how certain combinations of co-stimulatory domains can lead to unique cytokine secretion profiles impacting in vivo cell expansion and persistence as well as activation of other immune cell types. Other examples include a transmembrane domain or hinge region impacting on-target activity by affecting the flexibility of the antigen recognition domain.

Finally, the preclinical section of the guidance indicates the dose levels for the clinical studies are not always based off the preclinical work as the CAR T cells are expected to expand in vivo and thus previous human experience in clinical trials with similar CAR T cell products can inform the starting dose level and dose escalation schema.


Clinical: The agency notes that in addition to first-in-human clinical studies providing the standard assessments of safety, risk/benefit profiles and the recommended phase 2 dose, in CAR T an additional important question to address is the feasibility of manufacturing.

Key recommendations for clinical studies include:

  • Separate cohorts by disease types with evaluation of dose-response and severity of toxicity through parallel dose-escalations to address challenges in evaluating efficacy and severity of toxicity across disease types.
  • Development of in vitro companion diagnostics to evaluate subject eligibility based on antigen expression (unless the antigen is expressed in nearly all tumor cells OR can be detected by a commercially available, marketed test)
  • Use of a 3+3 design for dose escalation
  • Development of eligibility criteria that will increase the likelihood of a subject receiving the CAR T cell infusion, possibly by development of separate eligibility periods.
  • Developing assays to detect humoral and cellular immune responses against the CAR T cells.
  • Consider giving a single CAR T cell dose as multiple doses are potentially unnecessary and represent an unacceptable risk until there is a preliminary understanding the product’s duration of activity and toxicity and may be accompanied by repeated lymphodepleting chemotherapy which may also pose a life-threatening risk of myeloablation. If multiple doses or split dosing is used, then this should be justified and a strategy to mitigate the risk should be developed.
  • Considering not using bridging therapy as it may confound the efficacy analysis. FDA understands that bridging therapy may be required, so if it is used then the impact on the interpretation of the overall study results should be analyzed (e.g., analyzing all subjects vs. subjects who received prior bridging therapy vs. all subjects who did not receive prior bridging therapy).
  • Evaluating failure-to-treat as a trial endpoint as part of the feasibility evaluation looking at factors that may predict which subjects may not receive the CAR T cell product and evaluate the consequences to subjects that are unable to be treated.
  • Considering using FDA’s recommended dose limiting toxicity (DLT) definitions. As DLTs are often difficult to attribute consider including them independent of attribution to CAR T cells.
  • Including stopping rules
  • Staggering enrollment within a dose level and between dose levels using monitoring periods long enough to detect acute and subacute adverse events, considering animal studies and previous related human experience, the expected duration of the product activity, and the practical timeframes required for development.


Conclusion: Overall, this guidance highlights the complexities of working with CAR T cell products and provides recommendations from the Agency on how to facilitate a success FDA review.


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