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Formula for Success: Preparing CAR T Cells for Infusion

Updated on Mar 1, 2024 By Jason Ellis, PhD

Scientists in a Lab Looking at Data on a Computer

As modern medicine continues to advance, the battle against cancer is increasingly being fought on a cellular level—and CAR T cell therapy is at the vanguard. Chimeric antigen receptor (CAR) T therapy is a remarkable form of immunotherapy that harnesses the body’s cellular defenses. It has revolutionized the treatment of malignancies such as lymphoma and blood cancers.

Yet, the success of this therapy relies on more than just the genetically engineered cells. The steps after T cell activation and expansion—the post-expansion cleanup and formulation of the CAR T cell infusion—are pivotal to ensuring that the treatment is safe, effective, and compatible with the patient. These processes are not merely final touches but fundamental components of CAR T cell therapy’s success.

Understanding CAR T Cell Therapy

Chimeric antigen receptor T cell therapy is a type of immunotherapy that reprograms lymphocytes to attack cancer cells. The process begins with collecting and isolating different types of T cells from the blood, which are then genetically engineered to express CARs on their surface. These receptors are specifically designed to bind to antigens on the surface of cancer cells. Once infused into the patient, these modified T cells multiply, seek out, and eradicate cancer cells.

The journey from a lab bench to clinical trials to regulatory approval has been nothing short of extraordinary. CAR T cell therapy represents a significant shift in cancer treatment paradigms, particularly for patients who have not responded to conventional treatments like chemotherapy. These living drugs have shown promise in achieving remission even in refractory malignancies, paving the way for them to become a staple in cancer therapeutics.

The Critical Role of Post-Expansion Cleanup

After the genetically modified T cells multiply in vitro, the cells undergo post-expansion cleanup. This involves a series of steps designed to isolate the desired modified T lymphocytes from unwanted materials, such as dead cells, cytokines, residual viral vectors used for gene transfer, and serum proteins. The cleanup also includes the removal of unedited T cells so that the final product is a concentrated cohort of viable, potent CAR T cells.

Moreover, the stringency of the cleanup process is governed by rigorous quality control measures. During this phase, the cells are assessed for their purity, potency, and functionality. These quality controls are critical, as they determine the safety of the infusion for patients and the potential long-term success of the treatment. In many cases, serum-free media is preferred to avoid the risk of patient immune reactions that can occur due to the presence of animal-derived serum components in the growth media.

Any compromise in the cleanup process can lead to adverse effects or decrease the immunotherapy’s efficacy. It’s a meticulous process that is indispensable for the eventual success of the treatment and the patient’s well-being.

Manufacturers can ensure that the final CAR T therapy infusion meets the Food and Drug Administration’s (FDA) regulatory standards by integrating precise cleanup protocols.

Formulation of CAR T Cells for Infusion

Formulating the CAR T therapy mixture for infusion is a critical step to create optimal conditions for the engineered T cells to fight cancer upon re-entry into the patient’s body. This phase is tailored to preserve the viability and functionality of the cells so that they remain robust and capable of mounting an effective long-term attack against malignancies.

The process involves suspending cleaned and concentrated CAR T cells in a solution that is designed to support their survival and function once infused into the patient. Four main factors impact formulation:

  1. T cell dose: The concentration of CAR T cells per unit of infusion volume is calibrated according to the individual patient’s body size and the severity of their malignancy. Adequate dosing is crucial for ensuring the immunotherapy has enough effector cells to mount a significant immune response against the cancer cells without overloading the patient’s system, which could lead to adverse effects.
  2. pH: The pH level of the infusion solution is carefully adjusted to match the physiological conditions of the human body, typically around 7.4. This ensures that the infused CAR T cells function optimally and are not damaged or impaired by an environment that is too acidic or alkaline.
  3. Osmolarity: The osmolarity of the solution is fine-tuned to prevent osmotic shock when the cells are reintroduced into the bloodstream. A solution that closely mimics the osmolarity of the patient’s blood is ideal to ensure cell integrity and function.
  4. Cryoprotectants: For CAR T cells that will be cryopreserved for future use, cryoprotectants like dimethyl sulfoxide (DMSO) are added to protect the cells during the freezing process. These agents help prevent ice crystal formation, which can damage cell membranes and compromise cell viability and function upon thawing. However, this is an area of ongoing study, given the inherent cell toxicities of DMSO once thawed.

These aspects directly impact how the cells will behave once inside the body. Careful consideration is crucial because the infusion must be tolerable and safe for the patient while also maintaining the therapeutic potency of the CAR T cells.

Quality Control Measures

Throughout the manufacturing process, CAR T cells are rigorously assessed to ensure they meet the stringent criteria for clinical trial and therapeutic use. Potency tests are conducted to verify that the T cells are active and able to proliferate.

Purity assessments ensure that there are no unwanted cells or materials that could trigger adverse reactions. Safety tests are also performed to check for the presence of any residual factors from the genetic modification process that could be harmful.

Characterization of the final product is essential, not only for meeting regulatory requirements but also for building a robust body of data that can predict the therapy’s outcome. Consistency is key; therefore, each batch of CAR T cells must pass through the same stringent quality control checkpoints. This level of scrutiny ensures that every patient receives immunotherapy that is as safe as it is potent, thereby providing a reliable and effective treatment option.

Therapy Customization and Personalization

A major benefit of CAR T cell therapy is its potential for customization. Each patient’s cancer is unique, and thus, personalization of the treatment is possible and often necessary. In some cases, the engineering of CAR T cells allows for specific targeting of antigens present on an individual patient’s cancer cells. This patient-specific customization leads to more effective targeting of cancer cells while minimizing impact on healthy cells.

Moreover, the use of CAR T cells is not a one-size-fits-all approach. Factors such as the patient’s immune system status, the type and load of malignancy, and previous treatments like chemotherapy and radiation therapy all influence the formulation of CAR T cells for clinical trials and beyond. A tailored immunotherapy ensures that the cells have the best chance of engraftment and proliferation within the patient’s body, leading to a more successful outcome.

This approach also opens doors to addressing the unique challenges posed by different malignancies. Lymphoma cells, for example, might present different antigens than leukemia cells, and thus, the CAR T cells must be formulated accordingly. With advancements in characterization and assessment technologies, there’s a continuous improvement in the ability to tailor these therapies to meet the nuanced demands of personalized medicine.

Cryopreservation and Long-Term Use

Cryopreservation is a process that allows CAR T cells to be stored for long-term use, ensuring that they are available on demand for patients. The ability to freeze cells is a critical aspect of the treatment’s logistical framework, especially for patients who may require repeated doses. The cells must maintain their potency and viability even after being thawed, a challenge that requires precise control during the freezing and storage process.

During cryopreservation, CAR T cells are mixed with cryoprotectants, such as dimethyl sulfoxide (DMSO) and human serum albumin (HSA), and cooled to very low temperatures, effectively halting all biological activity. DMSO is widely used because it effectively permeates cell membranes and protects cells during the freezing process. However, it’s not without drawbacks; DMSO can be toxic to cells at warmer temperatures and can cause adverse reactions in patients post-infusion. In addition, serum derivatives like HSA can also cause harmful immune reactions in patients.

Consequently, the field is actively researching alternatives that could mitigate these downsides. Scientists are studying amino acids and saccharides like poly-l-lysine, dextrose, and pentaisomaltose for their cryoprotective properties. These substances may offer less toxicity and better compatibility with cell and patient physiology.

The viability of cryopreserved CAR T cells upon thawing directly depends on the formulation and cleanup steps that preceded it. Any impurities or suboptimal conditions during those phases can lead to compromised cells that do not survive the freeze-thaw cycle well. Therefore, the success of cryopreservation is heavily reliant on the meticulous attention to detail in the earlier stages of CAR T cell therapy manufacturing.

Clean Up CAR T Cells Better With BACSTM Microbubble Technology

The journey of CAR T cells from a patient’s vein, through the laboratory, and back into the bloodstream is a story of precision, customization, and innovation. Post-expansion cleanup and formulation are not mere afterthoughts but critical steps that directly determine the therapy’s final impact. Clinical researchers must ensure that the treatment infused into patients is safe and primed for maximum efficacy to succeed during clinical trials.

Akadeum’s buoyancy-activated cell separation (BACSTM) technology offers a cutting-edge solution for the post-expansion cleanup of CAR T cells. Our easy-to-use product lineup includes the Human T Cell Depletion Kit, Human T Cell Depletion Kit – GMP Grade, and Dead Cell Removal Microbubble Kit. By leveraging the power of microbubbles, BACSTM gently and efficiently isolates cells within minutes, enhancing the purity and potency of the final product.

We invite you to contact us or explore our cell isolation products and see how Akadeum’s BACSTM microbubble kits can elevate your CAR T cell therapy manufacturing processes. With Akadeum, the promise of cleaner, safer, and more effective cellular therapeutics is within reach. Join us in advancing the future of cancer treatment.

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