CAR-T in oncology
Combining translational research, clinical trials and GMP manufacturing to accelerate personalized immunotherapies for cancer patients with high medical need.
Different cell types are being used, including alpha beta T-cells, NK cells, gamma delta T-cells, and even macrophages and other innate immune cells. The choice between an autologous or allogeneic product must be made early in product development.
The choice of antigen is crucial. The antigen should be selected correctly considering the targeted malignancy, its secondary effects and its specificity for cancer vs healthy tissues.
Selection of CAR structure and gene construct is critical. This includes choosing between single or dual CAR systems, selecting the appropriate scFv, costimulatory domain(s), linker and transmembrane domain. Special attention must also be given to the promoter driving CAR expression, as it impacts the level and regulation of CAR expression, which can influence T cell activation, persistence and safety.
Choice of the best vector (e.g. lentiviral vector or other) and optimization of the construct and transduction conditions to ensure safety and efficacy.
Experiment to optimize the construct and transduction in cell lines of specific tumor type to show preliminary efficacy and quantify cytokine secretion. Other experiments using patient tumor cells, tumor biopsies, organoids or organelles may also be useful.
Animal models, patient derived xenografts (PDX), humanized mice grafted with a human immune system, can provide additional data on the biodistribution, persistence and immunogenicity of the CAR-T product alongside efficacy data. The cancer xenograft cells can be implanted subcutaneously or orthotopically within the appropriate organ.
Safety of the CAR product to be tested during construct optimization and proof of concept steps. This includes vector integration copy number (VCN) analysis to help optimize transduction methodology based on current regulatory guidelines and on target/off tumor as well as off target binding and toxicity.
Expression of the target antigen must be evaluated in healthy tissues. If the antigen is expressed on the membrane of cells in tissues that are potentially accessible to the CAR-Ts in the product, it may be necessary to perform cytotoxicity assays to evaluate whether the level of expression can lead to on target/off tumor CAR-T cytotoxicity. Data published in the literature and databases can also provide some guidance regarding expression.
Toxicity of the binder on the CAR molecule against nonspecific targets can be evaluated by using membrane protein expression panels and can be assessed using either the binder or the CAR-T product. The former will evaluate binding, while the latter will directly demonstrate cytotoxicity.
Tests are needed to ensure that the engineering process does not transform the cells, which may cause secondary malignancies or other toxicities. Tests include vector integration copy number (VCN) analysis, based on current regulatory guidelines, and, if necessary, integration site analysis.
Development of the manufacturing process under GMP conditions. The final drug product (DP) must undergo a set of quality controls before release, and each set of analytical QC tests must be validated based on GMP requirements. The specifications of the DP must be determined based on its identity and mode of action and QC tests must be developed to ensure each batch complies with the specifications. Another consideration is whether a “classic” or rapid manufacturing process will be used.
The lentiviral (or other) vector must be manufactured and released under GMP conditions. The QC tests must also be developed and validated.
Stability analysis: the shelf life of the lentiviral vector must be determined using a stability analysis, whereby the vector is QC tested for specific periods of time over several years to ensure that it remains potent over time.
Final manufacturing process and QC assays (see below) must be developed and validated prior to manufacturing the DP. The manufacturing process, along with all the QC assays, is part of the Investigative Medicinal Product Dosier (IMPD) and must be approved by the regulatory authorities prior to beginning patient recruitment in a clinical trial. If the product is allogeneic, the manufacturing will take place before the clinical trial begins and be cryopreserved. A stability analysis will also be needed.
QC tests must be developed based on the product specifications to ensure the product identity and safety. Tests like sterility, purity, cell number and viability are important. When the DP is manufactured, a qualified person will compare the results of the tests with the product specifications, before releasing the product for human use. Sometimes a potency assay is used to demonstrate functionality.
GMP has been addressed in other steps. This section focuses on GCP and interactions with the regulatory agencies during the application process and during the trial. All the information relating to the product, its mode of action, its manufacturing, the target patient population, the clinical trial design, how the patients will be treated and followed up, mitigation of toxicities, data collection and analysis must be included in the IMPD that will be submitted to the regulatory agencies.
It is crucial that developers build a solid understanding of the target population in their chosen disease area, through discussions with clinical investigators locally and abroad. In some cases, patient advocacy groups or disease specific organizations are also interested in getting involved and can offer advice and data, sometimes of very high quality. It is important to understand the patient demographics, previous treatments they may have received and other options that may be available to them. The trial design should determine the target patient population.
The design of the first trial should determine the safety of the product, the optimal dose and ideally provide early signs of efficacy. Appropriate primary and secondary endpoints must therefore be determined. It is recommended to also include endpoints like the persistence of the CAR-T cells in the patient and, if possible, the antigen expression of cancer cells before and during treatment, and after a relapse. Subsequent trials will further evaluate efficacy and may include a control group receiving the standard of care. The final (Pivotal) trial will generate the necessary data for marketing authorization.
The execution of a clinical trial is a complex task that requires expertise and input on multiple fronts. It is also expensive and time consuming. Multiple stakeholders are needed at every step, as is precise coordination.
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