A Review of CAR-T Generations and Applications on Hematologic Malignancies


Since the establishment of the immunotherapy by William Coley in the 19th century, great advancements have been made harnessing the capability and potential of the immune system (McCarthy, 2006). One of the current leading trends in immunotherapy is the development of the chimeric antigen receptor (CAR) fused in a T lymphocyte (T-cell). The T-cells play a key role in cell-mediated immunity through direct cellular contact or secretion of cytokine factors using antigen receptors as a mode of anomaly recognition (Actor, 2014). In the development of the chimeric antigen receptor T-cell (CAR-T), the T-cell receptor (TCR) is replaced with a CAR which is composed of an ectodomain, transmembrane domain and endodomain (Zhang et al, 2017). The extracellular domain is made of a single chain fragment (scFv) generated from a monoclonal antibody which can be designed specifically against a certain antigen of the tumor. The transmembrane domain regulates the stability of the CAR in the T-cell while the endodomain is the functional end of the receptor where signal transduction occurs.

Up to date, there are four CAR-T generations that have been developed. The first generation CAR-T uses CD3 ζ signaling and this is somewhat inefficient because the activated T cells fail to persist if there is no administration of exogenous interleukin-2 (IL-2) (Brocker, 2000). This limitation paved the way to the development of the second generation CAR-T which included an additional costimulatory molecule in the endodomain (Park and Brentjens, 2015; Zhao et al., 2018). As scientists keep on improving the CAR-T technology, third generation CAR-T was developed where two costimulatory molecule is incorporated (Daga and Davilla, 2016; Zhao et al., 2018). The addition of another component in the signal transduction domain where CAR inducible expression of a transgenic product such as cytokines can be made, led to the establishment of the fourth generation CAR-T (Zhao et al., 2018).

CAR-T cell therapy starts from the collection of the peripheral blood via phlebotomy followed by leukapheresis. The cells are then cultured without the addition of granulocyte colony stimulating factor to allow the proliferation of the T cells. Viral and non-viral vectors can be used for the transfection of the T cells. The transfected cells are then expanded, purified and tested for quality and sterility. Before the administration of the engineered CAR-T cells to the patient, conditioning treatment like lymphodepletion should be done (Zhao et al., 2018; Ninomiya et al., 2015).

Hematologic malignancies are neoplastic diseases of the blood categorized according to the lineage of the affected blood cell type (Jurlander, 2011). Classification is divided into myeloid neoplasm and lymphoid neoplasm. Treatments include chemotherapy, radiotherapy, immunotherapy and/or in combination. In the recent years, immunotherapy has been gaining popularity due to the more personalized and specific targeted approach. CAR-T cell technology is a current trend for immunotherapy in which successful clinical trials has been reported on hematologic tumors (Maude et al., 2014). Emerging possibilities on CAR-T for solid tumor targeting is now underway.

Despite aggressive treatments available for blood cancers, it is still challenging for the doctors and scientists to treat, but CD19 targeting CAR-T cells proved to be an efficient treatment (Park et al., 2016; Maude et al., 2014). In clinical trials conducted in Children’s Hospital of Philadelphia and the Hospital of the University of Pennsylvania, 90% of the patients with acute lymphoblastic leukemia (ALL) who received the 2nd generation CD19 directed treatment have complete remission (Maude et al., 2014; Zhao et al., 2018). Second generation CAR-T CD19 with 4-1BB as costimulatory had the most success in clinical trials with the rate of patients who have complete remission after treatment ranging from 75 to 93 percent. CD28 as costimulatory for CD19 had a range of 66.7 to 88 percent. 5 out of 6 patients with ALL achieved minimal residual disease (MRD)-negative remission after receiving 4th generation CAR-T treatment with CD19 as target with CD28, 4-1BB and CD27 as costimulatory and an additional inducible apoptotic caspase9 (Zhao et al., 2018).

For chronic lymphocytic leukemia (CLL), 2nd and 3rd generation CAR T with CD19 as target and 4-1BB, CD28 and combination of both for the 3rd generation as costimulatory, achieved lower rate of patients who had complete remission and partial remission after treatment (Zhao et al., 2018). Patients with chemotherapy refractory diffuse large B-cell lymphoma treated with 2nd generation CD19 targeted CAR-T with CD28 costimulatory obtained complete and partial remissions. One of the patient with DLBL had already tried five different treatments prior to the CAR-T treatment. Complete remission was observed after CAR-T CD19 directed treatment and has been ongoing after 9 months (Kochenderfer et al., 2015).

However, there are also studies that reported associated toxicities during the CD19 targeted CAR-T treatment. One of which is the cytokine release syndrome (CRS) that leads to the systemic inflammatory response (Park et al., 2016). Neurologic toxicities such as confusion, obtundation and aphasia was also reported (Kochenderfer et al. 2015). Toxicities is usually observed a few weeks after CAR-T administration.

Aside from the CD19 directed CAR-T, there are emerging potential targets for the treatment of hematologic malignancies such as CD23, ROR-1 and IgK (Pegram et al., 2015). Current target for treatment of B-cell chronic lymphocytic leukemia (B-CLL) is CD19 and CD20. However, it was reported that it also deplete the normal B cells. The receptor tyrosine kinase-like orphan receptor 1 (ROR1) was found to be highly expressed gene in B-CLL but not in normal B cells suggesting that it may not affect the normal B cells (Hudecek et al., 2010). The potential of ROR1 still needs more in vivo studies for the establishment of this novel target for B-CLL.

Despite the success of the CD19 CAR-T technology in hematologic cancers, challenges like toxicity management must be given attention. Novel targets should be explored for more advancement and efficacy of the treatment. Emerging possibilities in CAR is having multiple targets in one engineered CAR T and applications not just for blood tumors but also for targeting of solid tumors.


References:

Jurlander J. (2011). Hematological Malignancies, Leukemias and Lymphomas. In: Schwab M. (eds) Encyclopedia of Cancer. Springer, Berlin, Heidelberg

McCarthy, E. F. (2006). The Toxins of William B. Coley and the Treatment of Bone and Soft-Tissue Sarcomas. The Iowa Orthopaedic Journal, 26, 154–158.

Actor, J. K. (2014). Introductory Immunology: Chapter 4 T Lymphocytes Ringleaders of Adaptive Immune Function. Elsevier. doi.org/10.1016/B978-0-12-420030-2.00004-4

Zhang, C., Liu, J., Zhong, J. F., & Zhang, X. (2017). Engineering CAR-T cells. Biomarker Research, 5, 22. http://doi.org/10.1186/s40364-017-0102-y

Park, J. H. and Brentjens, R. J. (2015). Are All Chimeric Antigen Receptors created equal? Clinical Oncology. Vol. 33, no. 6, pp. 651-653.

Zhao, Z., Yu, C., Francisco, N. G., Zhang, Y. and Wu, M. (2018). The Application of CART-T Cell Therapy in Hematological Malignancies: Advantages and Challenges. Acta Pharmaceutica Sinica B. https://doi.org/10.1016/j.apsb.2018.03.001

Maude, M. L., Frey, N. Shaw, P. A., Aplenc, R., Barrett, D. M., Bunin, N. J., Chew, A., Gonzalez, V. E., Zheng, Z., Lacey, S. F., Mahnke, Y. D., Melenhorst, J. J., Rheingold, S. R., Shen, A., Teachey, D. T., Levine, B. L., June, C. H., Porter, D. L. and Grupp, S. A. (2014). Chimeric Antigen Receptor T Cells for Sustained Remissions in Leukemia. The New England Journal of Medicine. DOI: 10.1056/NEJMoa1407222

Brocker, T. (2000). Chimeric Fv-ζ or Fv-ϵ receptors are not sufficient to induce activation of cytokine production in peripheral T cells. The American Society for Hematology. Blood, 1 September 2000 Z Volume 96, Number 5

Park, J. H., Geyer, M. B., Brentjens, R. J. (2016). CD19-trageted CAR T-cell therapeutics for hematologic malignancies: interpreting clinical outcomes to date. The American Society of Hematology. DOI 10.1182/blood-2016-02-629063.

Ninomiya, S., Narala, N., Huye, L., Yagyu, S., Savoldo, B., Dotti, G.,Heslop, H. E., Brenner, M. K., Rooney, C. M., and Ramos, C. A. (2015). Tumor indoleamine 2,3-dioxygenase (IDO) inhibits CD19-CAR T cells and is downregulated by lymphodepleting drugs. The American Society of Hematology. Blood, 18 June 2015 X Volume 125, Number 25.

Kochenderfer, J. N., Dudley, M. E., Kassim, S. H., Somerville, R. P. T., Carpenter, R. O., Stevenson, M. A., Yang, J. C., Phan, Y. G., Hughes, M. S., Sherry, R. M., Raffel, M.,Feldman, S., Lu, L., Li, Y. F., Ngo, L. T., Goy, A.,Feldman, T., Spaner, D. E., Wang M. I., Chen, C. C., Kranick, S. M., Nath, A., Nathan, D. N., Morton, K. E., Toomey, M. A., and Rosenberg, S. A. (2015). Chemotherapy-Refractory Diffuse Large B-Cell Lymphoma and Indolent B-Cell Malignancies Can Be Effectively Treated With Autologous T Cells Expressing an Ati-CD19 Chimeric Antigen Receptor. Journal of Clinical Oncology. DOI: 10.1200/JCO.2014.56.2025

Maus, M. V., Grupp, S. A., Porter, D. L., and June, C. H. (2014). Antibody-modified T cells: CARs take the front seat for hematologic malignancies. The American Society of Hematology. DOI 10.1182/blood-2013-11-492231.

Pegram, H. J., Smith, E. L., Rafq, S., and Brentjens, R. J. (2015). CAR therapy for hematological cancers: can success seen in treatment of B-cell acute lymphoblastic leukemia be applied to other hematological malignancies? Author Manuscript. Immunotherapy. 2015; 7(5): 545–561. doi:10.2217/imt.15.6

Hudecek, M., Schmitt, T. M., Baskar, S., Stanghellini, M. T. L., Nishida, T., Yamamoto, T. N., Bleakly, M., Turtle, C. J., Chang, W. C., Greisman, H. A., Wood, B., Maloney, D. G., Jensen, M. C., Rader, C., and Riddell, S. R. (2010). The B cell tumor-associated antigen ROR1 can be targeted with T cells modified to express a ROR1-specific chimeric antigen receptor. The American Society of Hematology. DOI 10.1182/blood-2010-05-283309.