Chimeric Antigen Receptor (CAR) T-cell therapy has delivered major advancements in the treatment of lymphoid malignancies. However, the FDA approval of two of these therapies, tisagenlecleucel (Novartis) and axicabtagene ciloeucel (Kite Pharma) has highlighted the many barriers to broader access and adoption. These barriers range from high price-points and the unwillingness of insurance companies to cover these treatments, to manufacturing and Quality Control challenges, and the potential for toxicities in patients. We discuss here some of the future solutions to help bring this technology to mainstream medicine, and how flow cytometry is being utilized to help ensure the safety and efficacy of this magic bullet.  

Clinical Administration of CAR-T

Post-FDA approval, the cost of CAR-T therapies ranges from $375,000 to $475,000 per dose, and many insurance companies are pushing back on coverage and reimbursement for these treatments. It is not only the cost of the therapy that is impactful, but the adoption of these cellular therapies also requires experienced treatment centers and expert teams to support their administration and follow up on patients after treatment. The success rates for remission are ~65% or higher for Acute Lymphoblastic Leukemia patients, however toxicity or cytokine release syndrome occurs in ~50% and ~15% of patients, respectively.  

The process of patient treatment is summarized in Table 1. Any breakdown in the integrity of the apheresis, manufacturing or infusion processes can lead to serious patient harm, and compromise patient outcomes, and so close co-ordination of the treatment teams, cell processing and manufacturing labs and the intensive care units is essential. Only treatment centers with Foundation for the Accreditation of Cellular Therapy (FACT) accreditation may administer immune effector cell therapies.

Table 1. The process of patient referral and acceptance for CAR-T therapy. The high cost, and complex nature of this autologous treatment, means that multiple stakeholders and decision makers are involved along the entire process.  Many factors influence the successful implementation of this therapeutic strategy and addressing each of these factors individually is helpful in overcoming barriers to more widespread adoption. 

Scaling Up CAR-T Manufacturing 

The manufacturing of CAR-T cells for clinical and commercial applications is highly complex, and variability in the final product is influenced by a range of factors. The application of GMP principals for drug manufacturing to this production has enabled the development of safe and effective therapeutic CAR-T cells.  Process characterization was used to identify parameters influencing CAR-T quality attributes and this enabled engineers to implement control measures and help to standardize the manufacturing process for scale up (Wang & Riviere, 2016). 

CD19-specific CAR-T cells typically comprise of an extracellular scFv specific for CD19, coupled with the signally domains of CD3ζ and CD28 (Kochenderfer et. al. 2009). Initial production methods developed at the NCI were manual, open-system processes that employed human serum to support T-cell expansion, but these were highly limiting. Streamlining the production process was critical for adoption of this as a viable therapeutic platform. Firstly, the use of human serum was eliminated to reduce the risk of viral contamination, and the entire workflow was adapted to a closed system to minimize the risk of bacterial contamination.  Standardization of the process improved the consistency of the CAR-T and enabled the process to be transferred across multiple sites to meet the demands of commercial manufacturing. The final process was optimized down to 12 key steps that could be completed within a 17-day window in the US or 26-day window in Europe.

Fig. 1. Process flow for a Cellular Therapy Manufacture and Distribution, from the apheresis of the patient, to the infusion of the CAR-T product, monitoring and coordination of key steps is critical to the production of a safe and effective therapeutic product.

From Lab to Patient Bedside 

Lymphapheresis, the selected removal of lymphocytes from the blood is the first step for high-quality CAR-T production. The patients are typically taken off all treatment for several days before apheresis in order to maximize the yield of CD3 T-cells. Roughly 10-20L of blood is processed to yield a final volume of apheresis of 100-500mL containing roughly 5-10x10⁹ mononuclear cells.  Patient blood composition has a significant influence on the success of the therapy since low CD3+ T cell yields can result in impaired CAR-T cell expansion in vitro, and although the percentage of T-cell in the apheresis can vary significantly, robust processes help to ensure CD3 enrichment is optimized. 

Clinics employed to collect the apheresis are typically located close to airports to ensure the logistical challenges of sample shipment can be met. The apheresis material is transported in a validated shipping container at the collection site for low temperature (1-10˚C) transport to the processing facility. GMP chain of custody guidelines are critical when handling autologous cell therapy products. All processing steps are conducted in an International Organization for Standardization (ISO) 7 cell culture suite containing an ISO 5 biological safety cabinet or clean room, with a comprehensive Quality Management System to support GMP compliance.   

The ex vivo expansion of T-cells requires sustained activation; an initial primary signal via the TcR, as well as a co-stimulatory signal such as CD28, 4-1BB or OX40. To achieve this, T-cells from the mononuclear cell fraction are transferred to a closed system and cultured in serum-free media and activated for example using anti-CD3 antibody and recombinant human IL-2.  Bead-based activation solutions are also available such as Invitrogen CTS DynaBeads CD3/28 and Miltenyi MACS GMP ExpAct Treg Beads. These reagents have greatly improved the efficiency and ease of T-cell activation on a manufacturing scale. 

After T-cell activation the modified CAR gene is introduced into the cells using either retroviral vector transduction or nonviral gene transfer. The efficiency of retroviral vector transduction is highly enhanced using RetroNectin™ to coat the closed system bags in which this process takes place.  As a result of this process, the acceptable range of (multiplicity of infection) MOI for the viral preparation is very broad, and this is significant since it implies that lot-to-lot vector titer variations do not have a marked impact on the final CAR-T cell yields.  In contrast, the total viable cell concentration has a great impact on the efficiency of transduction, and therefore must be carefully controlled. The transduced T-cells are then expanded into order to achieve a critical threshold for dosing the patient. Common, GMP compliant scale up systems include the GE Wave system and G-Rex bioreactors.

Quality Control of CAR-T

Incorporating into the CAR-T manufacturing process are several Quality Checkpoints. Rigorous QC testing is performed in the form of batch release tests for T-cell viability and sample purity, T-cell potency in the form of effector function and activation, and microbiological safety. 

The robustness of the product quality control process is clearly demonstrated through quality control of the CAR-T product, as well as the preventative/corrective action, change control and review protocols in place. Release testing frequently involves flow cytometry analysis of the cells to determine the purity of the therapeutic preparation (% CD3+ T cell, % CD8+ T-cells, % CAR-T cells, % residual AAPCs) as well as potency testing by in vitro CTL or IFN- σ secretion using ELISPOT.

Fig. 2. The use of flow cytometry to examine CAR-T treatment. Pre-purification, the culture contains a mixture of transfected and un-transfected cell population. Although transfection efficiencies are very high, enrichment of the CAR-expressing T-cells is important prior to re-infusion. Flow cytometry supports both the assessment of the transfection process, as well as characterization of the final CAR-T product. One interesting observation from flow cytometry analysis of CAR-T manufacturing processes is that often T cell immunophenotype of the final product has a more naïve phenotype (CCR7, CD45RA) that the incoming apheresis material.  Interestingly, the CD4+/CD8+ ratio does not appear to have any effect on the product’s efficacy. 

Innovations in this area include the Isoplexis, Isolight technology which may soon be able to support a simple and powerful release assay for cellular therapies through functional proteomic analysis. The Isoplexis Single Cell Adaptive Immune System enables the analysis of single-cell protein secretion data to capture two key features: the percentage of polyfunctional cells (i.e. cells secreting two or more cytokines) and the intensity of secreted cytokine profiles from these cells. Polyfunctionality is associated with effector cell function and for CAR-T cells provides a measure of potency. The system assigns a polyfunctional strength index (PSI) to a sample to enable characterization of samples and a means of direct correlation with in vivo responses.  Recently, PSI was used characterize an anti-CD19 CAR-T cell pre-infusion therapeutic product for patients with Non-Hodgkin Lymphoma (NHL). The study demonstrated a statistically significant association of PSI with target activity in vitro. A functional proteomic comparison of two CAR-T preparations manufactured in different systems (namely M1 and M2) correlated higher PSIs driven by effector and stimulatory cytokine secretion with superior CAR-T activity (Fig. 3).

Fig. 3. Recovered CAR-T cells were separated into CD4 and CD8-enriched populations using microbead enrichment, and co-cultured with CD19 or CD22 expressing target cells for 24hr at 37˚C, 5% CO₂ before Single-Cell Adaptive Immune Analysis on the Isolight system. Top panel: PSI of CD4 (left) and CD8 (right) CAR-T. Lower panel: Polyfunctionality of CD4 (left) and CD8 (right). CD19 and CD22 antigen stimulation elicits highly polyfunctional activation, whereas NGFR control stimulation has limited activation effect on the CAR-T. 

CAR-T Infusion and Patient Monitoring

A single dose of therapeutic CAR-T contains a target does of 2x10⁶ CAR-positive viable T cells per kg of body weight in a volume of 68mL. Prior to administration of the CAR-T cell infusion, the patients undergo lymphodepletion using fludarabine and cyclophosphamide over three days. This is essential for the depletion of endogenous lymphocytes and for the elevation of the homeostatic cytokines IL-15 and IL-7.  Two days later, the patient is ready to receive the CAR-T infusion and the patient remains under surveillance for the next 12 days during which Tocilizumab must be immediately available in case of CRS. In addition to monitoring for signs of infection, Cytokine Release Syndrome or neural toxicity, daily hematology and chemical lab tests are performed to track levels of CRP and ferritin, and since atypical lymphocytes that mimic blasts are often associated with the peak of CAR-T expansion, flow cytometry is used to exclude relapse and monitor blood cell counts. Trillium Diagnostics developed a flow cytometry kit, Leuko64 to measure neutrophil CD64 expression as an indicator of infection/sepsis or predictor of severe CRS. The kit can be used as a laboratory developed test (LDT) within CLIA-certified laboratories. Many other clinical labs have developed flow cytometry-based LDTs to monitor phenotypic changes in leukemia patients following antigen-directed therapies. Such flow cytometry panels can help provide early detection of antigen-escape and Minimal Residual Disease (MRD) in patients, and current recommendation include such testing every 3-6 months for up to 15 years after treatment (Yakoub-Agha et. a. 2020).   

Allogenic CAR-T cells.

Ultimately, one of the most significant breakthroughs for CAR therapy will be the development of allogenic CAR-T cells- proving an off-the-shelf product that is gene edited to minimize risk of rejection or Graft Versus Host Responses (GVHR). The manufacturing process for allogenic CAR-T cell utilizes healthy T lymphocytes harvested by leukapheresis. Gene editing enables the permanent insertion of recombinant DNA coding the CAR and supportive functions such as co-stimulation receptors or suicide functions, as well as the elimination of TcR and CD52 expression. The genetically modified T-cell are then expanded using anti-CD3/anti-CD28 beads and cytokines and the expanded cells stored for patient infusion as needed. The stratification of allogenic CAR-T based on HLA type may prove important for recipient patient outcome. Alterative proposed source materials for allogenic CAR-Ts include umbilical cord blood and iPSCs to minimize the risks of GVHR and alloimmunization by screening for donor-specific anti-HLA antibodies.  An alternative candidate is γδ T cells that are readily expanded ex vivo and may overcome solid tumor access more readily than αβ T cells. An added benefit to this approach is that the TcRs on γδ T cells are not MHC restricted. 

A Faster CAR on the Horizon? 

The development of CAR-Natural Killer (NK) cells is another attractive strategy, and in vitro studies have proved this to be a viable concept (Han et. al. 2015). NK cells are designed to patrol our bodies for abnormal cells, such as cancer cell, and destroy them. So, it seems obvious to build on this natural ability by programming these cells to seek out and destroy cancer cells that have adopted mechanisms to elude the immune system. The frequency of NK cells in peripheral blood is low, the adoption of the NK92 cells line for this application represents a renewable source for CAR-NK production using IL-2. However, at MD Anderson, they have taken the approach of extracting NK cells from the blood of donated umbilical cords and transfecting these cells with the CAR-CD19 construct. Several hundreds of doses can be made from one donation of umbilical cord blood, and frozen for use on multiple patients. Initial trials proved that the NK cells indeed work very quickly to target and destroy the cancer cells and displayed considerable stability in patients, with CAR-NK cells still detectable 12 months post treatment (Kawalekar et. al. 2016; Karadimitris, 2020). 

Another advantage of CAR-NK is their potential to eliminate cancer cells independently of CAR, via their natural cytotoxic anti-tumor functions that can be activated by NCRs, NKG2D, CD226 and certain KIRs. Furthermore, NK cells can eliminate cancer cells through CD16-mediated ADCC. Therefore, a combination of CD-16 CAR-NK cells together with a monoclonal antibody therapy may represent powerful therapeutic solutions to tumors containing cells with and without CAR target antigen expression and provide a means of addressing patient relapse. 

CAR-NK cells appear to take us one step closer to an off the shelf product, and with reported lower side effects than CAR-T treatments and prolonged efficacy in patients, the hope is that CAR-NK can transition rapidly into the clinic to quickly benefit patients. 

Final Thoughts

The design of CAR-T cells has evolved drastically over the past decade, and tumor targets for CAR-T therapies have expanded from CD19 to diverse range of targets discussed in previous blogs in this series (e.g. CD20, CD22, CD30, CD33, CD138, CD171, CEA, EFGRvIII, Erb, FAP, GD2, Her2, NKG2D). Now the transfer of this principal into CAR-NK opens new possibilities for source materials, production methods and therapeutic applications. 

Hurdles in production of CAR-T cells are being overcome with innovative designs in the manufacturing process and streamlined with the adoption of single-use closed systems for production. Point-of care CAR-T manufacturing using benchtop systems such as the Miltenyi Biotec CliniMACS Prodigy T cell Transduction Process are aiming to democratize CAR-T cell production. However, alternative strategies including implementing vehicle mediated T cell-targeted transgene technology to directly reprogram T-cell in vivo, have been proposed, although the safety and control barriers to this might seem insurmountable. 

Improving access to cellular therapies will involve several moving parts. The intense interest from the various healthcare stakeholders has significantly accelerated the development of novel CAR solutions, and the hope is for improved manufacturing platforms to soon deliver safer and more cost effective cell therapies that can be adopted more readily into mainstream medicine.  

Dr. Julie Bick is a medicinal biochemist who has spent close to 7 years with FlowMetric Life Sciences. After receiving her doctorate in Biochemistry at Southampton University in the UK, she began her career as Associate Professor at Rutgers University, NJ, before moving to the west coast to perform biomedical research with Syngenta and Novartis at the Torrey Mesa Research Institute in San Diego. Dr. Bick specializes in biomedical engineering of cells and proteins in order to provide innovative therapeutic and diagnostic solutions. She brings to FlowMetric a clinical expertise across a wide range of therapeutic areas from autoimmunity to oncology and chronic inflammatory conditions, acquired over 25 years of research experience in academic, biotechnology and pharmaceutical laboratories. In leading FlowMetric Life Sciences’ innovation initiatives, Dr. Bick has been collaborating with BurstIQ to implement Block Chain solutions into the company’s Contract Research Organization division, with a focus on enhanced big data analytics and process control solutions in the regulated clinical environment. Dr. Bick is committed to working with local Community Colleges to support STEM programs for the next generation of scientists.

References

Wang, W. & Riviere, I. (2016) Clinical manufacturing of CAR T cells: foundation of a promising therapy. Molecular Therapy Oncolytics vol. 3, 16015.

Kochenderfer JN, Dudley ME, Kassim SH et al. (2015) Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor. J. Clin. Oncol. 33: 540–549.

Kawaleskar et. al. (2016) Distinct Signaling of Coreceptors Regulates Specific Metabolism Pathways and Impacts Memory Development in CAR-T cells. Immunity 44, 380-390. 
http://dx.doi.org/10.1016/j.immuni.2016.01.021

Yakoub-Agha et. al. (2020) Management of adults and children undergoing chimeric antigen receptor T-cell therapy: best practice recommendations of the European Society for Blood and Marrow Transplantation (EBMT) and the Joint Accreditation Committee of ISCT and EBMT (JACIE). Haematologica 105(2) 297-316.
DOI: 10.3324/haematol.2019.229781

Karadimitris, A. (2020) Cord Blood CAR-NK Cells: Favorable Initial Efficacy and Toxicity but Durability of Clinical Responses Not Yet Clear. Cancer Cell vol. 37 (4) 426-7.
DOI:https://doi.org/10.1016/j.ccell.2020.03.018

Han, J. et al. (2015) CAR-engineered NK cells targeting wild-type EGFR and EGFRvIII enhance killing of glioblastoma and patient-derived glioblastoma stem cells. Sci. Rep. 5, 11483