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Guest Commentary: The growing potential of CAR T-cell therapy
June 2019
by Andrea Toell of Lonza  |  Email the author
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The growing potential of CAR T-cell therapy
How CAR T cells are transforming immuno-oncology
 
Andrea Toell of Lonza Biosciences
 
Introduction
The rising global incidence of cancer[i] highlights the importance of developing more effective therapeutic strategies. Amid a wealth of innovative anticancer research, new therapeutic tools continue to emerge in the field of immuno-oncology. However, these approaches, which are focused on boosting the ability of a patient’s immune system to target and eliminate cancer cells, are not without risk, and many immunotherapies can trigger potentially harmful side effects. Given the need for greater treatment efficacy, safety and specificity, innovative immunotherapeutic strategies that more closely target cancers and minimize the risk of adverse responses are being developed using genetic engineering approaches that modify the patient’s own immune cells.
 
T lymphocytes (T cells) play a key role in cell-mediated immune responses to cancer, making them prime candidates for ex-vivo genetic modification. One such modification is the transfer of chimeric antigen receptor (CAR) transgene cassettes into a patient’s primary T cells. This produces CAR T cells that express CARs specific to the tumor of interest. Following characterization and expansion, these cells are re-infused into the patient. The ability of CAR T cells to effectively treat hematopoietic malignancies such as lymphoblastic leukemia[ii] has fueled interest in advancing this technology. Further progress in gene transfer strategies is improving the safety and efficacy of CAR T cells in the treatment of blood cancers, and a major question now is whether CAR T-cell therapy can be applied to solid tumors. This will require novel strategies to address the considerable challenges involved in both avoiding autoimmune side-effects and overcoming the hostile tumor microenvironment.
 
CAR T cells and their success against B cell malignancies
Cancer immunotherapy relies on reinforcing the body’s natural immune response to tumor-associated antigens (TAA). T cell participation in cell-mediated immune responses to cancer begins when T cells encounter B cells or dendritic cells that have digested TAAs and display antigen peptide fragments that are bound to major histocompatibility complex (MHC) molecules (antigen-MHC). T cell receptors (TCRs) recognize and bind with the antigen-MHC complex, stimulating T cell maturation to produce T cells that either regulate the immune response or become cytotoxic “killer” T cells capable of attacking cancerous cells directly. Major challenges to the effectiveness of this immune response include the ability of cancer cells to create an immunologically tolerant microenvironment and to employ a variety of immunosuppressive mechanisms.[iii],[iv] Growing tumors are also known to down-regulate specific effector T cell responses and induce resistance to T cell killing.[v]
 
Early strategies for T cell modification involved engineering the TCR. However, down-regulation of antigen presentation to the TCR in the cancer limits their efficacy. CAR T cells have advantages here, as CARs are highly specific targeting molecules that combine the binding properties of a monoclonal antibody with signaling through the TCR complex. When expressed by T cells, CARs target TAAs in their native conformation, independent of the MHC complex. This allows them to bypass human leucocyte antigen (HLA) recognition and thereby avoid some of the key mechanisms that facilitate tumor escape from TCR-mediated immunity.
 
CAR T cells are currently the subject of more than 100 clinical trials,[vi] with B-lineage antigens such as CD19 the most investigated to date. Two therapies are FDA approved: one is for the treatment of diffuse large B cell lymphoma, and the second is for certain types of adult non-Hodgkin lymphoma[vii]. The application of CAR technology in cancer therapy is currently the focus of intense study. However, the safety and efficacy of the resulting treatments largely depends on the methods used to generate the CAR T cells and, as such, understanding how to engineer them is critically important.
 
 
Enhancing gene transfer in CAR T cells to advance their therapeutic potential
To date, the introduction of CAR transgene cassettes into T cells has generally relied on either viral-mediated transduction or non-viral transfer of DNA plasmids (see Table 1). While viral transduction has proved effective,[viii] concerns have been raised over its safety, the cost of clinical-grade viral vectors, and limitations on the genetic payload.[ix] As a result, nonviral transfection of DNA plasmids is gaining traction. Methods based on liposomal formulation, nanoparticles and cell-penetrating peptides are all being used as cheaper and safer alternatives to viral transduction, yet these approaches typically exhibit low gene transfer efficiencies. Advanced electroporation techniques, which involve the application of an electrical field to increase cell membrane permeability, enable greater efficiencies as well as offering co-transfection capabilities and improved flexibility to work with various substrate types such as plasmids, mRNA and proteins. There is early evidence to suggest that CAR T cells produced in this way are effective in treating blood cancers such as Philadelphia chromosome-positive acute lymphoblastic leukemias that are resistant to tyrosine kinase inhibitors.[x] Transfection using electroporation is therefore emerging as one of the preferred methods for nonviral engineering CAR T cells.
 
 
Despite the advantages described, there are issues with nonviral methods that must be solved if they are to be adopted for routine use. Significantly, their validation in human applications has proven difficult, largely because gene transfer efficiency can be lower than with viruses and gives insufficient subsequent persistence in immune system due to low stable integration frequency. Work using enhanced electroporation techniques together with DNA transposition methods is addressing these challenges and paving the way for nonviral gene transfer in human cancer therapy. In DNA transposition, defined segments of DNA (transposons) move from one genomic location to another, facilitated by one or more proteins known as the transposase. Transposons have long been recognized as powerful genetic editing tools for mutagenesis[xi], and transposon-transposase systems have been used to transfer CAR transgene cassettes into T cells to produce CAR T cells for therapeutic purposes. Two examples are the fish-derived “Sleeping Beauty” and insect-derived “piggyBAC” human-adapted systems.[xii] The advent of advanced electroporation technologies has been transformational in enabling the application of these systems to non-viral generation of CAR T cells. As a result, gene transfer efficiency is improving, resulting in safer, more effective integration of CAR T cells into the patient’s immune system. This approach also presents the possibility of large-scale CAR T cell generation.
 
Nonviral transposition remains the subject of considerable research aimed at enhancing the technique and securing the availability of safe, low-cost and efficient CAR T-cell cancer therapy for routine use. For example, new systems involving DNA minicircles[xiii]—bacterial plasmid derivatives from which all prokaryotic vectors parts have been excised—combine the efficiency of viral transfer with the safety and low cost of nonviral methods.
 
In other areas of study, methods are being sought that reduce the unwanted off-tissue toxicity sometimes associated with genetically modified T cells. These include the use of transiently expressed mRNAs to switch off or limit normal tissue toxicity.[xiv] CRISPR/Cas9 has also been employed to introduce CAR sequences into T cells, highlighting the potential of CRISPR/Cas9 gene editing to advance immunotherapies.[xv]
 
The need for innovative approaches to apply CAR T-cell treatment to solid tumors
As nonviral gene modification technologies advance and immunology understanding grows, work is underway to address the challenge of ensuring efficacious CAR T-cell activity without off-target toxicity and to extend its application to solid tumors. While CAR T cells have been highly effective in treating blood cancers, CD19 has proven to be an unusually appropriate target thanks to its high level of expression on tumor cells compared with healthy cells.
In contrast with its use against hematopoietic malignancies, applying CAR T-cell treatments to fight solid tumors is more complex. Firstly, the potential for autoimmune side-effects is much greater, as there is often a lack of specifically targetable antigens.[xvi] In solid tumors, antigens are generally not tumor-selective but are simply overexpressed. This is increasing the focus on those antigens that are preferentially expressed in certain types of cancer. Other strategies under investigation are aimed at increasing targeting specificity, including dual antigen recognition techniques.
 
Secondly, there are significant issues in overcoming the hostile microenvironment in solid tumors. Potential strategies to combat these challenges include “armored CAR” T cells, which are engineered to be resistant to immunosuppression, as well as combination therapy approaches such as those based on checkpoint inhibitors. Ongoing efforts to tackle these major challenges are beginning to yield novel and innovative CAR T-cell therapy treatments for solid tumors. One recent review[xvii] has highlighted encouraging results in pediatric neuroblastoma, HER2-positive sarcoma and disseminated glioblastoma, and also noted that more than one-third of CAR T cell trials registered at the U.S. National Library of Medicine are investigating solid tumor indications.
 
Conclusion
CAR T cells are currently the focus of considerable research and development efforts in the field of immuno-oncology, with some notable therapeutic successes in the treatment of blood cancers. Work to improve gene transfer technologies is helping to ensure robust and sustained CAR T-cell activity and increase the safety and efficacy of CAR T-cell treatments for B-cell malignancies.
 
Despite this success, however, widening the scope of CAR T-cell therapy to solid tumors presents a variety of more complex challenges that include identifying unique TAAs, avoiding autoimmune side-effects and overcoming the immunosuppressive tumor microenvironment. Consequently, researchers are no longer simply searching for the next novel biomarker, but are looking to improve the underlying issues around treatment specificity and efficacy. The encouraging progress that has been made using innovative solutions suggests CAR T cells have the potential to usher in a new generation of effective cancer treatments.

Andrea Toell is a Senior Product Manager at Lonza Biosciences
 
References
 
[i]               . Cancer Research UK, 2014. Worldwide cancer incidence statistics. Available at: https://www.cancer.gov/news-events/cancer-currents-blog/2018/tisagenlecleucel-fda-lymphoma
[ii]           . Dai H, Wang Y, Lu X, Han W. Chimeric Antigen Receptors Modified T Cells for Cancer Therapy. J Natl Cancer Inst. 2016; 108(7). DOI: 10.1093/jnci/djv439
[iii]             . Zou W. Immunosuppressive networks in the tumour environment and their therapeutic relevance. Nature Reviews Cancer. 2005; 5(4), 263–274.
[iv]             . Rabinovich GA, Gabrilovich D. Sotomayor EM. Immunosuppressive Strategies that are Mediated by Tumor Cells. Annual Review of Immunology. 2007; 25(1), 267–296.
[v]              . Wu AA, Drake V. Huang HS, Chiu S, Zheng L. Reprogramming the tumor microenvironment: tumor-induced immunosuppressive factors paralyze T cells. Oncoimmunology. 2015; 4(7), e1016700.
[vi]             . Hay KA, Turtle CJ. Chimeric Antigen Receptor (CAR) T Cells: Lessons Learned from Targeting of CD19 in B-Cell Malignancies. Drugs. 2017; 77(3), 237–245.
[vii]            . National Cancer Institute. FDA Approves Second CAR T Cell Therapy for Lymphoma. Cancer Currents Blog. Published May 2018 updated 1 Dec 2018. Available at: https://www.cancer.gov/news-events/cancer-currents-blog/2018/tisagenlecleucel-fda-lymphoma
[viii]           . Melero I, Gaudernack G, Gerritsen W, Huber C, Parmiani G, Scholl S, Thatcher N, Wagstaff J, Zielinski C, Faulkner I, Mellstedt H. Therapeutic vaccines for cancer: an overview of clinical trials. Nat Rev Clin Onc. 2014; 11(9), 509–524.
[ix]           . Dotti G, Gottschalk S, Savoldo B, Brenner MK. Design and Development of Therapies using Chimeric Antigen Receptor-Expressing T cells. Immunol Rev. 2014; 257(1), DOI: 10.1111/imr.12131
[x]              . Saito S, Nakazawa Y, Sueki A, Matsuda K, Tanaka M, Yanagisawa R, Maeda Y, Sato Y, Okabe S, Inukai T, Sugita K, Wilson MH, Rooney CM, Koike K. Anti-leukemic potency of piggyBac-mediated CD19-specific T cells against refractory Philadelphia chromosome–positive acute lymphoblastic leukemia. Cytotherapy. 2014; 16(9), 1257–1269.
[xi]             . Ivics Z, Hackett PB, Plasterk RH, Izsvák Z. Molecular Reconstruction of Sleeping Beauty, a Tc1-like Transposon from Fish, and Its Transposition in Human Cells. Cell. 1997; 91(4), 501–510.
[xii]            . Singh H, Moyes JS, Huls MH, Cooper LJ. Manufacture of T cells using the Sleeping Beauty system to enforce expression of a CD19-specific chimeric antigen receptor. Cancer Gene Therapy. 2015; 22(2), 95–100.
[xiii]           . Monjezi, R. et al., 2017. Enhanced CAR T-cell engineering using non-viral Sleeping Beauty transposition from minicircle vectors. Leukemia, 31(1), pp.186–194. http://www.nature.com/leu/journal/v31/n1/full/leu2016180a.html
[xiv]            . Caruso HG, Torikai H, Zhang L, Maiti S, Dai J, Do KA, Singh H, Huls H, Lee DA, Champlin RE, Heimberger AB, Cooper LJ. Redirecting T Cell Specificity to EGFR Using mRNA to Self-limit Expression of Chimeric Antigen Receptor. Journal of Immunotherapy. 2016; 39(5), 205–217.
[xv]          . Su S, Hu B, Shao J, Shen B, Du J, Du Y, Zhou J, Yu L, Zhang L, Chen F, Sha H, Cheng L, Meng F, Zou Z, Huang X, Liu, B.Su, CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients. Nature Scientific Reports. 2016; 6, 20070.
[xvi]          . D’Aloia MM, Zizzari IG, Sacchetti B, Pierelli L, Alimandi M. CAR-T cells: the long and winding road to solid tumors. Cell Death & Disease. 2018; 9, 282.
[xvii]         . Schmidts A, Maus MV. Making CAR T Cells a Solid Option for Solid Tumors. Frontiers in Immunology. 2018; DOI: 10.3389/fimmu.2018.02593
 
 
 
 
 
 
 

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