A decade ago, two patients received an innovative therapy for leukemia that revolutionized immunotherapy research. Today, chimeric antigen receptor (CAR) T cell therapy continues to evolve with advances in T cell and receptor engineering as a promising treatment for blood malignancies, solid tumors, and autoimmune diseases.
Explore this milestone article from Drug Discovery News to learn how scientists harnessed the body's defenses to develop CAR T cell therapy.
BY DANIELLE GERHARD, PHD
In 2022, researchers at the University of Pennsylvania published a paper in Nature reporting long-term remission in two patients with leukemia who received an innovative cell therapy treatment more than a decade ago (1). The patients were among the first to be treated with their own genetically engineered cells in clinical trials that paved the way for an FDA-approved treatment called CAR T cell therapy.
1980s-1990s
Multiplying patient cells in the lab
In the 1980s, Steven Rosenberg at the National Cancer Institute (NCI) used adoptive cell transfer to treat patients with metastatic melanoma (2,3). Rosenberg and his colleagues extracted cancer-fighting lymphocytes from the patient’s tumors and multiplied them in the laboratory. Rosenberg hoped that reintroducing an army of the patient’s lymphocytes instead of a few lone soldiers would provide enough force to kill the cancer.
Unfortunately, studies like Rosenberg’s using unmodified lymphocytes demonstrated limited clinical efficacy. However, they provided a valuable proof-of-concept for the in vitro expansion and reintroduction of a patient’s cells. Subsequent research from Carl June at the University of Pennsylvania (UPenn) introduced valuable techniques for expanding human T lymphocytes, or T cells, in the laboratory and fundamental insights into T cell signaling (4).
1992
Gene transfer in T cells
Studies in the ‘70s and ‘80s suggested that the immune system, and T cells in particular, had cancer fighting potential. Following intensive chemotherapy, cancer patients often receive bone marrow transplants to replenish blood-forming stem cells. However, donor T cells included in these transplants attacked the recipient’s healthy cells. Scientists noticed that removing T cells from the transplant prevented these attacks, but also increased cancer relapse.
In 1989, Michel Sadelain, currently an immunologist at Memorial Sloan Kettering Cancer Center (MSKCC), started a postdoctoral research position at the Massachusetts Institute of Technology to gain experience in the nascent field of cell engineering. Although his advisors discouraged him from working with T cells, Sadelain was determined to modify these cells to fight cancer. In 1992, Sadelain eagerly presented the first genetically engineered primary T cell at the World Congress in Immunology in Budapest. Unfortunately, his excitement was not matched. “The remarkable thing is that there was zero interest,” said Sadelain. “Maybe nobody thought that an engineered cell would become a medicine.”
1993
Honing the T cell attack
Despite the lack of interest at the conference, Sadelain was not alone in pursuing engineered T cells for therapy. Zelig Eshhar, an immunologist at the Weizmann Institute, was experimenting with methods for endowing T cells with greater tumor specificity to improve their efficacy in adoptive cell transfer studies like Rosenberg’s.
T cell receptors (TCRs) consist of an extracellular component that binds to different antigens and an intracellular component that activates the T cell and prepares it for battle. To redirect and bolster the T cell attack toward cancer cells, Eshhar and his colleagues engineered a new receptor (5). The researchers fused a cluster of differentiation three zeta (CD3ζ) intracellular signaling domain of a TCR known to activate T cell signaling (6) with the antigen binding region of an antibody. Introducing this new receptor to specially modified mouse T cells and redirected their attack toward cancer cells expressing the target antigen. These synthetic receptors are considered the first generation of what are today called chimeric antigen receptors (CARs).
1998-2002
The first chemotherapy patient
The first-generation CARs gave T cells an enhanced navigation system to find and kill specific cancer cells, but the modified T cells soon ran out of gas. When Sadelain started his lab at MSKCC in 1994, he continued searching for ways to boost T cell function. T cells express costimulatory receptors that bind to chemical messengers on nearby cells. Costimulatory receptors such as cluster of differentiation 28 (CD28) are necessary to fully activate the T cell and mount an immune response.
In 1998, Sadelain’s team found that replacing the CD3ζ component with the intracellular domain of CD28 gave engineered human primary T cells the stamina to maintain their attack (7). Next, the scientists demonstrated that T cells engineered with a CAR expressing CD28 in tandem with CD3ζ and directed to an antigen expressed on prostate cancer cells had a turbocharged immune response (8). “Now a T cell could find a tumor cell and not just kill it but continue to divide,” said Sadelain.
The addition of a costimulatory domain established the second-generation CARs used in all six currently approved CAR T cell therapies.
2003
Giving CARs a new destination
The prospect of an effective therapy was inching closer, but the CARs needed a good target. Sadelain’s team considered a number of different cancer models and landed on B cell malignancies. Leukemia originates in the bone marrow where T cells easily infiltrate. However, efficiently transporting the CAR T cells to B cells required an updated antigen-sensing navigation system. On intuition, Sadelain chose the cluster of differentiation 19 (CD19) protein to navigate his CAR T cells (9). This idea was influenced by the very high expression of CD19 on B cells and in leukemias, but his thinking was clear: The more cells that express the target, the more CAR parking spots. In the end, Sadelain’s intuition paid off. Although other targets are under investigation, 4 of the 6 currently approved CAR T cell therapies target CD19.
These newly engineered T cells proved effective at killing cancer cells in a dish, but the team lacked evidence on their in vivo efficacy. Renier Brentjens, an oncologist at Roswell Park Comprehensive Cancer Center, joined Sadelain’s research team during his oncology fellowship at MSKCC and set out to test CARs in mouse models. Brentjens vividly recalled this first CAR T experiment. When he injected mice with Raji Burkitt lymphoma cells, a B cell malignancy, they developed hindlimb paralysis within weeks and died. Before he left on a family trip to Ireland, he injected half of these mice with human CD19 CAR T cells. Having promised his family he wouldn’t call the lab while on holiday, he rushed to the phone to get an update as soon as the plane landed on his return trip to New York City. To his amazement, all of the control mice died, while nearly all of the CAR T cell treated mice survived. “You almost didn’t have to do the statistics on this,” said Brentjens. “This was what every investigator dreams of — a homerun experiment.”
2011
Living drugs
At the American Society of Hematology conference, Sadelain and Brentjens were excited to present their findings that established CAR T cells can eradicate systemic disease in a mouse. But history repeated itself. No one came to Brentjen’s poster tucked away in the corner of the room.
Again, Sadelain pressed ahead. In pursuit of a clinical trial, he first needed to establish a facility at MSKCC to produce CAR T cells. To create these so-called living drugs, scientists needed to extract T cells from patients’ blood, engineer them with CARs in the lab, grow them into millions of cells, and infuse them back into the patient. At the time, there were no companies in this space and only a few institutions capable of producing this type of therapy, including UPenn and the NCI.
In August 2011, preliminary results across these institutions trickled in. Carl June and colleagues at UPenn presented data from patients with refractory chronic lymphocytic leukemia (CLL), while Sadelain and Brentjens shared their findings in adults with acute lymphoblastic leukemia (ALL) (10). The results were promising and exemplified the power of these super-killer CAR T cells. “We calculated that one dose of CAR T cells killed somewhere between two and a half to seven pounds of tumor cells,” said David Porter, a medical oncologist at UPenn and coinvestigator of the UPenn clinical trials.
Interest in CAR T cell therapy picked up once scientists at MSKCC, UPenn, and the NCI started releasing their findings. “The fact that the three centers reported around the same time had a lot to do with its immediate believability,” said Sadelain. “But the other big impact is that it swayed industry to look at cells as drugs.”
2017
Frist CAR T cell therapy approved
Soon after these findings surfaced, Novartis teamed up with the UPenn group to develop Kymriah, a CAR T cell therapy approved by the FDA in 2017 for treating ALL in certain pediatric and young adult patients. Children suffering from this incurable leukemia showed a greater than 80 percent complete response rate to the treatment. “There is no precedent for that kind of response,” said Porter.
As well as being the first CAR T cell therapy, Kymriah was also the first gene-transfer therapy to gain FDA approval. “At the same time, it’s a cell therapy, a gene therapy, and an immune therapy,” said Sadelain. So far, CAR T cells are only approved for blood malignancies, but efforts are underway to modify CARs for solid tumors (11) and autoimmune diseases (12).
REFERENCES
1. Melenhorst, J.J. et al. Decade-long leukaemia remissions with persistence of CD4+ CAR T cells. Nature 602, 503-509. (2022).
2. Rosenberg, S.A. et al. A new approach to the adoptive immunotherapy of cancer with tumor-infiltrating lymphocytes. Science 233, 1318-1321 (1986).
3. Rosenberg, S.A. et al. Use of tumorinfiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. NEJM 319, 1676-1680 (1988).
4. June, C.H. Toward synthetic biology with engineered T cells: A long journey just begun. Human Gene Therapy 25, 779-784 (2014).
5. Eshhar, Z. et al. Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the λ or ζ subunits of the immunoglobulin and T-cell receptors. PNAS 90, 720-724 (1993).
6. Irving, B.A. & Weiss, A. The cytoplasmic domain of the T cell receptor zeta chain is sufficient to couple to receptor-associated signal transduction pathways. Cell 64, 891- 901 (1991).
7. Krause, A. et al. Antigen-dependent CD28 Signaling selectively enhances survival and proliferation in genetically modified activated human primary T Lymphocytes. J Exp Med 188, 619-626 (1998).
8. Maher, J. et al. Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRζ /CD28 receptor. Nature Biotechnology 20, 70-75 (2002).
9. Brentjens, R.J. et al. Eradication of systemic B-cell tumors by genetically targeted human T lymphocytes co-stimulated by CD80 and interleukin-15. Nature Medicine 9, 279-286
10. June, C.H. & Sadelain, M. Chimeric antigen receptor therapy. NEJM 379, 64-73 (2018).
11. Klichinsky, M. et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nature Biotechnology 38, 947-953 (2020).
12. Mougiakakos, D. et al. CD19-Targeted CAR T Cells in Refractory Systemic Lupus Erythematosus. NEJM 385, 567-569 (2021).