CAR T-Cell: Cell Processing Laboratory Considerations

Introduction

Chimeric antigen receptor (CAR) T-cells are one of the most rapidly growing cancer immunotherapies, particularly for B-cell malignancies. While the manufacturing of CAR T-cells is relatively straightforward, there are many barriers associated with the manufacturing process. Additionally, most CAR T-cells are produced from a patient's own or autologous lymphocytes, which creates many unique cell production and logistical challenges. Many patients receiving CAR T-cells have life-threatening cancers, and the progression of their disease may not allow for the manufacturing of another CAR T-cell product, should the first manufacturing attempt not yield sufficient quantities of cells.

∗ Role of the Local Cytotherapy Lab contributed by David Stroncek, Sandhya Ramanathan Panch, Ping Jin & Steven L. Highfil is under Public domain as the contributors are US Government employee.

An important goal for manufacturing all cell therapies is to consistently produce high-quality products. This is especially true for CAR T-cells. Factors that can cause or contribute to manufacturing failure include inability to collect sufficient quantities of T-cells, microbial contamination, low levels of T-cell transduction, insufficient expansion of T-cells in culture, and low levels of CD3+ T-cells in the final CAR T-cell product. Occasionally, CAR T-cells do not meet lot release criteria and cannot be used. Although generally not a release criteria, skewing of CD4 and CD8 T-cells within the culture may have unintended clinical consequences and should be considered. The critical need for patients to receive CAR T-cells along with the possibility of poor manufacturing outcomes makes it important for healthcare professionals involved with cellular immunotherapy to understand the CAR T-cell manufacturing processing.

Some CAR T-cells are being manufactured commercially, and many others are in early-phase clinical trials. The commercial development of CAR T-cells has led to centralization of CAR T-cell manufacturing in large cell processing facilities. Although CAR T-cells are increasingly being manufactured in centralized current good manufacturing practice (cGMP) cell manufacturing facilities, academic hospital-based cell processing laboratories continue to manufacture CAR T-cells. In addition, academic medical centers that do not manufacture CAR T-cells may be involved with many aspects of CAR T-cell therapy. Hospital-based cell processing laboratories may ship the autologous cells used as starting material in the manufacturing process and receive, store, thaw, and issue the CAR T-cells. For commercially manufactured CAR T-cells, the details of the manufacturing process may not be available, but the processing laboratory will have access to the results of analysis of the final CAR T-cell product performed by the manufacturing facility, which the lab staff can help clinical care teams interpret. The cell processing laboratory staff can also address questions related to the CAR T-cell infusion and reactions associated with their infusion. This chapter will provide a general overview of manufacturing CAR T-cells as well as specific issues of importance to clinical care teams involved with treatment of patients with CAR T-cells.

General Considerations

Nature of the Cells Used to Produce CAR T-Cells

The manufacturing of any cellular therapy is highly dependent on the source of the cellular material used to manufacture the product. Genetically engineered cell therapies that must be HLA compatible with the recipient, such as CAR T-cells, are generally produced from the patient's own, or autologous, cells. Cell therapies that do not require HLA matching can be manufactured from any person willing to donate cells. In the latter scenario, cell therapies made from any healthy donor cells can be produced in large batches or lots, which may contain enough cells to treat hundreds of patients. In contrast, for CAR T-cells, an individual autologous cell therapy must be produced for each unique patient treated. Since each cell therapy lot manufactured must undergo in-process and lot release testing and quality review, producing autologous cell therapies is more resource-intensive and costly.

Currently, almost all CAR T-cells are being manufactured from autologous leukocytes. Efforts are underway to produce CAR T-cells using T-cells from healthy subjects, which have been engineered to no longer express T-cell receptor (TCRs) and major histocompatibility complex (MHC) molecules. CAR T-cells that lack TCRs and MHC molecules could be used to treat any patient without causing graft-versus-host disease or being subject to immune-mediated rejection. The development of such a “universal donor” CAR T-cells would have several advantages over autologous CAR T-cells ( Table 3.1 ). They could be produced in large quantities, which would allow cells from one lot to be used to treat many patients. This would eliminate many of the logistical problems related to manufacturing CAR T-cells and would allow for the distribution of the cost of in-process and lot release testing, and GMP manufacturing across many CAR T-cells doses rather than just one autologous lot. Overall, the cost of producing one dose of universal donor CAR T-cells would be much less than that of one autologous CAR T-cell product. In addition, an inventory of universal donor CAR T-cells could be developed. These cells would be available for immediate administration and hence would be an “off-the-shelf” therapy.

Table 3.1

Comparison of Autologous and Universal Donor Chimeric Antigen Receptor T-Cells.

Considerations	Autologous	Universal Donor
Lot size	A separate lot is required for each patient	One lot for many patients
Product testing	In-processing and lot release testing is performed on cells for every patient	In-process and lot release testing is performed on each lot
Consistency	High variability among products given to each patient	Same or similar product given to each patient
Time required to provide cells	Cells must be collected, manufactured, and tested	Cells can be given immediately
Manufacturing failures	Common	Rare
Consequence of manufacturing failure	Repeat manufacturing or no treatment	Provide cells from a different lot
Possible immune rejection	Low	High unless genetically modified
Possibility of causing graft-versus-host disease	Rare observance in patients with previous allogeneic hematopoietic stem cell transplantation	Possible unless genetically modified

Manufacturing Facilities and Closed-System Processing

Since the media and conditions used to culture T-cells can promote the growth of microbes and any microbial contamination of the cells at any point in the collection and manufacturing process may result in microbial growth and cause serious harm to the recipient, considerable effort is taken to maintain sterility. The manufacturing process takes place in a highly controlled facility specifically designed to maintain an environment with minimal quantities of microbes. In addition, whenever possible, cells are cultured in vessels that are closed to the environment. T-cells can be expanded in a variety of different culture systems. Classically, T-cells have been expanded in flasks. However, standard laboratory culture flasks, such as T-flasks, are open to the environment and hence are subject to microbial contamination. The simplest closed culture systems are cell culture bags. Bags that are specifically designed for cell culture are available in a variety of sizes, which allows bag culture systems to easily accommodate a wide variety of culture volumes. Culturing T-cells in bags is limited by the relatively low T-cell concentration that must be maintained. Closed-system flasks specifically designed for T-cell growth and closed-system bioreactors are also available and are being used for CAR T-cells production in several early phase clinical trials. The impact of manufacturing methodologies on CAR T-cell function will be important to study.

Cells collected in bags can be sterilely connected to cell culture bags or closed-system culture flasks and bioreactors. In addition, culture media and other reagents can be purchased in bags, which can be sterilely connected to maintain the closed nature of these culture systems. Cells and culture media can also be removed sterilely from these closed-system bags, flasks, and bioreactors.

Automation

Automation of the CAR T-cell manufacturing process is highly desirable ( Table 3.2 ). Automated instruments are available for cell separation, washing, concentration, and culture. Automation reduces the amount of staff time required in the manufacturing process. Manual processing is also associated with some variability due to differences among staff, and automation reduces this variability. In general, the time required to train staff to operate automated instruments is less than the time required to train staff to perform similar operations manually. Automated instruments developed more recently are likely to be more highly automated and to electronically document important elements of the procedure.

Table 3.2

Comparison of Manual and Automated Processing Operations.

Considerations	Manual	Automated
Labor requirements	High	Low
Training requirements	High	Low
Processing closed to the environment	Possibly	Usually
Consistency of outcomes	Variable	High
Documentation of results	Manual entry	Electronic
Scalability	High	Limited

There are some disadvantages to automation. Most instruments are designed for a specific range of cell quantities and culture media volume, and it may be difficult to work with quantities of cells and media outside this range. In addition, some automated culture systems provide limited measures of culture conditions, and it may be difficult to change these conditions. From more of a logistical perspective, many of the automated devices currently on the market require specific tubing sets and reagents that are only available from the vendor that manufactures the instrument. Therefore, manufacturing facilities with these types of instruments run the risk of halting cell production if there are issues with a particular vendor. With the growth of cancer cellular immunotherapy, it is expected that the number and types of automated instruments available will grow and their quality and capabilities will improve.

Chain of Custody

As autologous cells used to manufacture CAR T-cells move from the collection center to the manufacturing facility and back to the medical center that administers the cells, it is critical to be sure that the cells can be linked to the patient throughout this process. Typically, to maintain this “chain of custody,” the cells are labeled with a unique identification number that is assigned by the collection center and with patient identifiers such as name, date of birth, and medical record number. Whenever the cells are manipulated in a way that requires changing the container or vessel holding the cells, the unique identifiers are added to the new container or vessel. Each CAR T-cell product is labeled with patient identifiers, the product name, number of cells, and name of the manufacturing site. The label also includes the date of product manufacture and the product expiration date.

Regulatory Considerations

Medical facilities that treat patients with CAR T-cells, in general, are experienced with allogeneic and/or autologous hematopoietic stem cell (HSC) transplantation. However, the regulations concerning CAR T-cell manufacturing and HSCs processing differ greatly. All CAR T-cells are regulated much like drugs, vaccines, and other biologics and must be manufactured as a licensed biologic or under an investigational new drug (IND) application. If under an IND, they must contain the following statement, “Caution: New Drug—Limited by Federal Law to Investigational Use.” HSCs are considered minimally manipulated cell therapies and are not subject to these regulations. CAR T-cells are expected to be manufactured using cGMPs. The number of air exchanges, level of air filtration, and staff and material flow requirements for the facilities used to manufacture CAR T-cells exceed those required for processing HSCs.

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