GENE THERAPY – Developing Affordable Point of Care CAR-T Therapies: Expanding Efficacy & Impact


The hypothesis that engineered T cells could recognize and eliminate hematologic malignancies has proven true. The most recent headlines in which two of three chronic lymphocytic leukemia (CLL) patients treated with an anti-CD19 CAR-T cell product remain disease free 10 years after treatment continue to affirm the durable efficacy of CAR-T cell therapy.1 At the most re­cent annual meeting of the American Society for Hematology (De­cember 2021), major sessions were devoted to the question as to when CAR-T cell therapy could be moved up to be a second line therapy in adults with B cell leukemia and lymphoma.2 This would significantly expand the pool of patients eligible for this therapy.


A CAR (Chimeric Antigen Receptor) is a cell surface protein expressed on the surface of a cell, introduced by a gene vector. CARs can be expressed on T lymphocytes (T cells), NK (natural killer) cells, and macrophages. T cells exhibit a broad range of activity.

The T cells engineered by means of a gene vector to express a CAR (CAR-T) were initially intended to exert immune effects de­signed to eliminate cancer cells. However, the field of use has broadened to include the engineering of T-regulatory cells and T cell populations that can remodel tissue. T-regulatory cells can reverse inflammation or auto-immunity in serious human dis­eases, such as diabetes and rheumatoid arthritis, and also facil­itate organ engraftment. Thus, a number of companies focused on CAR-Treg have been founded or acquired CAR-Treg assets, in­cluding Sangamo, Sonoma Biotherapeutics, GentiBio, and Abata Therapeutics.

In a remarkable new application, CAR-T cells specific for FAP (fibroblast activation protein, expressed on activated fibroblasts) were shown to reduce cardiac fibrosis in an animal model.3 This represents a third field of use for CAR engineered T cells, wherein the therapeutic goal is to alter the cellular composition of a tar­geted tissue.

The primary domain of activity for CAR-T remains hemato­logic malignancies. All of the currently approved products target either the CD19 protein expressed on leukemia and lymphoma or the BCMA protein, expressed on multiple myeloma. These are: Abecma (idecabtagene vicleucel for multiple myeloma), Breyanzi (lisocabtagene maraleucel, for diffuse large cell B cell lym­phopma, DLBCL), Carvykti (ciltacabtagene autoleucel, for multi­ple myeloma), Kymriah (tisagenlecleucel, DLBCL and pediatric acute lymphocytic leukemia, ALL), and Tecartus (brexucabtagene autoleucel, mantle cell lymphoma)/Yescarta (axicabtagene ciloleucel, DLBCL and follicular lymphoma). The final two listed are essentially the same anti-CD19 CAR-T, but with different ap­plications and slightly different manufacturing, one of which fea­tures a T cell enrichment step.


One of the major critiques of the currently approved CAR-T cell products is their cost. Drug acquisition costs range from $373,000 to $475,000 and represent the largest component cost of the therapy. A real-world estimate of total costs places the av­erage at $700,000 and can exceed $1 million in some cases (Aislinn Antrim, Pharmacy Times, 2021).

How did we arrive at this astronomical price tag? In past ap­proaches, complex therapeutics in which cells were removed from the body and selected or genetically engineered, the work was carried out in academic medical centers using reagents and devices that were clinically safe, but not specifically designed as an all-encompass­ing GMP product. The many different ap­proaches to bone marrow transplantation, implemented at numerous cancer centers, had been the working model for cell and gene therapy to this point. As the process of creating a CAR-T cell generation be­came more refined and reproducible, pharmaceutical companies stepped in and created a pathway to generate autologous cell-based therapeutic products.

While a boost to the field with regard to interest from investors, there are draw­backs. By adopting a large-scale central manufacturing model, new innovations are potentially stifled, as corporations have to recover the extraordinary costs de­voted to creating what is essentially a first-generation product. Moreover, these first-generation products are not as effi­cient or effective as the T cells bearing the CAR are over-activated and differentiated, leading to decreased efficacy in the body.


The following presents three pillars by which efficacy and impact can be in­creased, and by which costs can be significantly lowered, leading to greater availability of CAR-T products for all those who would benefit. The first is using a place-of-care method for CAR-T produc­tion, the second is the advent of extremely short CAR-T generation protocols, and the third, while still on the horizon, has the ability to upend the entire field. Genera­tion of CAR-T by direct injection of gene vector particles into the body, allowing for transduction, activation, and expansion CAR-T, would entirely alter how we ap­proach cell and gene therapy.


The current approved therapies for CAR T cell production require extensive lo­gistical and regulatory support. When pa­tients are approved to receive CAR-T cell therapy, they first undergo apheresis (leukocytapheresis/leukopheresis), a pro­cedure whereby a patient is connected to a medical device that receives whole blood from a vein, the white cell fraction of the blood (leukocytes) is harvested by means of differential centrifugation, and the red cell fraction returned to the patient. Apheresis is a costly step requiring dedi­cated personnel and devices. The advan­tage is that far more white blood cells (lymphocytes) can be collected than from a normal blood draw. Once collected, the leukocytes are processed, frozen, and then shipped to a central manufacturing facility. There, the cells are thawed, processed, and transduced with a retroviral or lentivi­ral gene vector. Once transduction has taken place, and the engineered cells pass release criteria (such as sterility and evi­dence of CAR expression), they are repackaged and shipped to the clinical site for infusion into the patient.

Caring Cross proposes that the eco­nomics of place-of-care manufacturing will continue to grow in importance, both in the current US market, and across the world. The argument or challenge against place-of-care manufacturing usually cen­ters on reproducibility. In order to demon­strate that place-of-care manufacturing can be robust and reproducible, we tested CAR-T production in two geographically disparate sites using identical manufactur­ing platforms, reagents, and lentiviral vec­tor (LV).

Very similar CAR-T were manufac­tured and used to treat patients at both Case Western Reserve/University Hospitals Medical Centers in Cleveland, OH, and the Dimitry Rogachev Pediatric Cancer Hospital in Moscow, Russia. These place-of care products were generated in a much shorter time than commercial prod­ucts and were used to treat patients who had more severe disease (and could not wait the weeks required for central manu­facturing of CAR-T).4 Our experience with other sites employing place-of-care man­ufacturing show similar high-quality prod­ucts being produced with exceptional outcomes.5 Using a closed system, the cell manufacturing steps in these trials were carried out in the bone marrow transplant laboratories, and thus in a controlled en­vironment, but outside of a traditional clean room. This has great implication for cost savings. Using the closed manufactur­ing devices, currently available on the market, allows a center to operate with minimal reliance on a clean room suite, Figure 1.

Combining technology, distributive manufacturing, and place-of-care production significantly reduces cost of cell therapy products. On the left side, the current high-cost process with central manufacturing is illustrated. On the right side, a distributed model with local production that does not require the complex custody, regulatory, and shipping logisitics required for a centrally manufactured product. Manufacturing could occur at a regional center serving a number of local institutions or be housed directly in the hospital.

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The use of place-of-care manufactur­ing has been essential in the manufactur­ing of CAR-T cell products outside the US. Not waiting for costs to drop on their own, Spanish investigators formed a national network, generated their own LV, and a employed a commercial cell production platform (CliniMCAS Prodigy, Miltenyi Biotec). Hospitals were able to produce CAR-T products for B cell malignancies at a greatly reduced cost that had similar clinical benefit as current commercial products.6 National healthcare systems across the globe are taking note of this ap­proach, and it will surely expand. The UK government has collected comments and will soon issue guidance for place-of-care manufacturing.7 This will establish the needed regulatory framework and potentially place the UK in the lead for both CAR-T production and innovation.


The creation of CAR-T cell products outside the body is a complex and lengthy process. Putting the logistics of where the CAR-T cells are produced aside, the man­ufacturing process can be 8 days or longer. At the production facility, the CAR-T cell production process starts with the isolation of either total leukocytes (PBMC) or selection for CD4 and CD8 T cells by immunomagnetic bead selection. Isolated T cells are then activated with reagents de­signed to cause cell activation and expan­sion (ie, Dynabeads CD3/CD28 CTS™, Invitrogen; T Cell TransACT™, Miltenyi Biotec; GMP Cloudz T Cell Activation Kit, R&D Systems). This set-up process usually takes a single day, followed by transduc­tion with a lentiviral or retroviral gene vec­tor that encodes the CAR. This adds an­other 2-3 days. The subsequent 5-9 days of culture allows for CAR-T to expand to sufficient numbers for formulation for ei­ther infusion or cryopreservation. The media and supplements (including cy­tokines like IL-2, IL-7, IL-15, or IL-21) must also be clinical grade and add to the costs.

Regulatory agencies require some ev­idence that the T cells treated with a gene vector do not carry along with them suffi­cient amounts of plasmid used during pro­duction of the gene vector (usually a PCR-based test) to be expressed in the host. Also the product is tested for the ab­sence of replication-competent virions (termed RCL, replication competent lentivirus) arising from recombination of genetic elements. While RCL has never been observed in a clinical process, sam­ples must at least be retained in order to allow for subsequent testing.

Investigators have been working to decrease the culture time following trans­duction with LV. The barriers have been both regulatory and methodological. The regulatory hurdle seeks to quantify the amount of residual elements used during CAR-T manufacture that are carried for­ward into the infusion product. The methodological challenge is to prove that the CAR is indeed now being expressed by the transduced T cell population. In addi­tion to these concerns, it is well recognized the longer T cells are cultured the more “differentiated” they become. A differenti­ated T cell has decreased ability to expand and exert activity against diseased tissue.

A recent publication from Dr. Milone’s lab at the University of Pennsylvania took all of these challenges straight on and cre­ated a single-day CAR-T generation process that is not dependent on the addi­tion of a T cell stimulation reagent.8 Al­though, a regulatory path forward has yet to be determined, the methodological bar­riers to ultra-rapid CAR-T production are falling rapidly. The ability to create a CAR-T product in a single day renders the com­plexities of a central manufacturing approach essentially a moot question. It is no longer worth the trouble.


Given the onset of ultra-rapid gener­ation of CAR-T outside the body, the next logical step is to do away with cell process­ing outside the body entirely, and to di­rectly inject gene vectors (Figure 2). For such a vector to be successful, it would need to have both selective targeting/transduction of the intended T cell population, as well as some mecha­nism to expand or select for transduced cells once the gene vector has been ad­ministered.

Moving to a low-cost cure platform with the advancement of new improved technologies. Illustrated is the increasing risk (left to right) as we move from the current known ex vivo process, and toward a direct injectable, that has yet to be accomplished but is the current focus of intense research and development activity. The advent of a directly injectable vector would entirely upend the current processes that are highly dependent of cell manipulation ex vivo.

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A recent entrant to the field, Umoja Biopharma, is proposing to generate CAR-T with a vector particle that can be directly injected to the body, and which binds to target T cells by virtue of a specific receptor in the particle outer membrane. In addi­tion to a CAR, the payload within the gene vector includes a rapamycin-activated sig­naling protein that initiates IL-2 like signal­ing in the transduced T cells. Rather than encoding a “driver” like signaling protein, companies like Enochian BioSciences endow their gene vector with a drug-resis­tance gene, in this case to the drug cy­clophosphamide. Cyclophosphamide is used in CAR-T therapy to reduce endoge­nous immune cells and allow for CAR-T ex­pansion. Enochian will take advantage of this process by using the drug for in vivo selection of transduced cells.

While targeting the LV to the right T cells and providing for a method of selec­tion or expansion is essential, one more barrier may yet remain. It appears the most common envelope protein (the pro­tein on the lentiviral vector surface that allow for entry into the targeted cell) VSV-G, can be inactivated by serum comple­ment. Fortunately, researchers at the Fred Hutchinson Cancer Research Center have developed a new envelope protein that can be used instead of VSV-G, derived from the Cocal virus.9 Thus, use of Cocal envelope protein in lentiviral vector pro­duction, combined with preferential target­ing and selection, will soon bring the era of direct LV transduction in the body to pass.

Given the rapid development of the field, other instantaneous methods of gene transduction, such as electroporation with Crispr-based gene modification, will also play a role but will likely remain a process carried out on cells outside the body.


The commercialization of CAR-T man­ufacturing was a watershed moment in cell and gene therapy. What was once a process limited to highly skilled and resource-rich academic medical cells was now made available to any medical center with cell therapy experience, essential a center THAT has a track history in bone marrow transplantation. However, the high price for central manufacture of CAR-T has important limitations to its long-term use. The high price for CAR-T products makes them unavailable in low- and mid­dle-income countries, and for individuals with inadequate healthcare coverage in high-income countries.

This represents a serious justice issue. These therapies were developed using many public resources (such as NIH-funded comprehensive cancer centers, and IP developed in public institutions). If the benefit of these therapies are available only for the economically elite, public trust in medical science will be undermined. In addition, while the learning required to create a commercial product featuring ge­netically engineered cells was impressive, the costs to setting up these systems cre­ates a competitive landscape against new innovative technologies that could drive down cost. And finally, the technology and the regulatory framework required to safely oversee the development of CAR-T therapy continues to evolve.

The future belongs to those who will be able to innovate rapidly, maintain reg­ulatory confidence, and drive down costs in order to make CAR-T cell and other en­gineered cell therapies available to all who would benefit.


  1. Melenhorst JJ, Chen GM, Wang M et al., Decade-long leukemia remissions with persistence of CD4+ CAR T cells. Nature 602:503-509 (2022). 10.1038/s41586-021-04390-6. PMID: 35110735.
  2. Locke FL, Miklos DB, Jacobson C, et al. Pri­mary analysis of ZUMA-7: a phase 3 ran­domized trial of axicabtagene ciloleucel (axi-cel) versus standard-of-care therapy in patients with relapsed/refractory large B-cell lymphoma. Abstract #2. Presented at the 2021 American Society of Hematology An­nual Meeting, December 12, 2021.
  3. Rurik JH, Tombacz I, Yadegari A, et al., CAR R cells produced in vivo to treat cardiac in­jury. Science 375:91-96 (2022). PMID: 35990237.
  4. Maschan M, Caimi PF, Reese-Koc J, et al., Multiple site place-of-care manufacturied anti-CD19 CAR-T cells induce high remis­sion rates in B-cell malignancy patients. Na­ture Communications 12:7200 (2021). PMID: 34893603.
  5. Hah N, Johnson BD, Schneider D, et al. Bis­pecific anti-CD20, anti-CD19 CAR T cells for relapsed B cell malignancies: a phase I dose escalation and expansion trial. Nature Medicine 26:1569-1575 (2020). PMID: 33020647.
  6. Ortiz-Maldonado V, Frigola G, Espanol-Rego M, et al. Results of ARI-0001 CASRT19 Cells in patients with chronic lymphocytic leukemia and Richter’s transformation. Frontiers in Oncology 12:828471 (2022). PMID: 35174095.
  7. Medicines and Healthcare Products Regula­tory Agnecy, United Kingdom. Consultation on Point of Care Manufacturing (2021).­tions/point-of-care-consultation/consultation-on-point-of-care-manufacturing.
  8. Ghassemi S, Durgin JS, Nunez-Cruz S, et al. Rapid manufacturing of non-activated po­tent CAR T cells. Nature Biomedical Engi­neering 6:118-128 (2022). PMID: 35190680.
  9. Rajawat YS, Humbert O, Cook SM, et al. In Vivo gene therapy for canine SCID-X1 using Cocal-pseudotyped lentiviral vector. Human Gene Therapy 32:113-127 (2021). PMID: 32741228.

Dr. Rimas Orentas is the Co-founder and Scientific Director of Caring Cross. He earned his PhD from The Johns Hopkins University School of Medicine. After 12 years in academia, he worked for 5 years at the Pediatric Oncology Branch of the NCI, and for a total of 5 years at Lentigen Corporation, where he served as Scientific Director. He currently is an investigator at the Ben Towne Center for Childhood Cancer Research, Seattle Children’s Research Institute, Director for Scientific Integration at CureWorks, and Professor of Pediatrics and Laboratory Medicine and Pathology at the University of Washington School of Medicine.

Dr. Boro Dropulić is the Co-founder and Executive Director of Caring Cross, an organization focused on improving business models to support the affordability and accessibility of gene therapy products. Dr. Dropulić earned his PhD from the University of Western Australia and his MBA from the Johns Hopkins University (JHU). He has been in the gene therapy field since the late 1980s.