New Cell Contenders in the CAR Field (Part 2 of 2)

Why do we need new cell contenders in the CAR field? Well, different CAR immune cells have different advantages and sometimes unique challenges or limitations.

So, we began our discussion of alternative CAR-cell types with CAR-NK cells due to current their standing as the most popular alternative CAR-based therapy. However, the expansion of CARs to other cell types has already begun and shows promising results. The utility of CAR endowed macrophages (CAR-MF) has recently been demonstrated and their amazing potential is only beginning to be realized. The novelty of CAR-MFs means much of the experimental data remains proprietary. However, the available information on CAR-MFs in combination with what is known about macrophage biology allows us to develop solid insights into their potential.

Macrophages are prominent member of the innate immune system and have a number of diverse roles in the body. Scientists typically characterize macrophages based on various surface markers and secreted factors that are closely associated with inflammation pathogen defense, and tumor defense (M1), anti-inflammation and wound healing (M2a), Immunoregulation (M2b), Immunosuppression and tissue remodeling (M2c), and angiogenesis and tumor promotion (M2d) [1]. It is important to note that the remarkable fluidity of macrophage polarization means that these cells can exist as combinations of these subsets making this classification system a guideline instead of a steadfast rule [2].

cells

Overview of CAR macrophages and activation of the immune response.

While triggered by different receptors, the effector functions of T-cells and NK-cells are centered around activation of apoptotic pathways in target cells [3]. The functions macrophages can exert towards tumors are quite different and focus on tumor cell ingestion (phagocytosis and trogocytocis), antigen presentation, and cytokine and effector molecule secretion [4–6]. CARs for macrophages include cytoplasmic signaling domains from phagocytotic receptors  (CD64 and Megf10), Toll like receptors, as well as those more similar to CARs for CAR-T (CD3) [6–8]. The extracellular domain of these CARs instigate the identification of target molecules on cancer cells by macrophages, which can trigger 1) phagocytosis (full cancer cell engulfment) or 2)  trogocytosis (biting pieces off of a cancer cell), both of which can induce cancer cell death directly [6,7,9]. In addition, CAR-MF are able to incite other therapeutic effects, which are discussed in the following sections.

Clinically approved CAR-T therapies rely on a single target antigen to mount their anti-tumor response [10]. However, cancers often mutate leading to antigen escape [11]. Targets phagocytosed or trogocytosed by macrophages can be processed for antigen presentation to T-cells [9]. Previous studies have demonstrated the importance of macrophage antigen presentation for tumor immune responses [12,13]. For example, many clinical trials are currently studying the benefit of anti-CD47 for treating cancer. CD47 is a cell surface marker commonly over-expressed in many cancers, which inhibits phagocytosis by macrophages. By blocking this receptor, the rate of macrophage phagocytosis increases which allows macrophages to more efficiently present the antigens to CD8+ T-cells (primarily through an MHC I pathway) [13]. CAR-MF are able to present antigens from tumor cells to T-cells [6,7]. T-cell recognition of neoantigens could act as a second line of defense in cases where cancer cells adapt to evade CAR recognition

The makeup of the tumor microenvironment is such that it often mimics a chronic wound [14]. Because of this, the immune system responds in a manner appropriate to wound healing leading to immunosuppression, tumor metastasis, apoptosis resistance, and angiogenesis [14]. In addition to phagocytosis, target identification by CAR-MF promotes the release of inflammatory cytokines such as TNF-a, IL-6, IFN-g, and IL-12 [7,8,15]. These factors shift the tumor microenvironment and immune response towards tumor rejection through actions such as increasing Teff cell recruitment and function, tumor growth suppression, angiogenesis suppression, and increased sensitivity to apoptosis [15].  Because of the high prevalence of the target antigen in a tumor, CAR-MF are likely under constant stimulation allowing them help maintain this anti-tumor environment [7,8].

CAR-T and CAR-NK cells have struggled to effectively treat solid tumors due to the immunosuppressive nature of the tumor microenvironment, which actively inhibits their migration [16,17]. Research has demonstrated that macrophages are normally abundant in solid tumors and in some cases can make up 50% of their mass [18]. This high prevalence indicates that CAR-MF could have better targeting capabilities towards solid tumors than other cell types improving their efficacy [7].

One challenges that CAR-MF cells could face is inefficient gene incorporation [19]. The use of viral vectors CAR-T manufacturing produces a production bottleneck and is a major cost driver [20,21]. Unfortunately, macrophages are notoriously resistant to viral transduction due to 1) their ability to identify and destroy the viruses via phagocytosis and 2) their low rates of cell division [19]. These issues result in low average macrophage transfection efficiencies for g-retroviruses (25%) and lentiviruses (37.5%), which are the vectors currently used in clinically approved CAR-T therapies and some preclinical CAR-MF therapies [8,22,23]. Some reports suggest adenoviral transduction CAR-MF cells has promise with CAR-expression rates up to 70% [7,8]. However, adenoviruses can be highly immunogenic and induce an inflammatory phenotype in edited cells. This adds significant risk. An early clinical trial involving adenovirus-based transduction reported a patient death due to a systemic inflammatory reaction caused by the viral vector. Since this time, these vectors have been refined to minimize these hazards [19]. However, these risks should still be taken seriously in therapeutic environments.

Attempts to overcome these issues with non-viral methods have been challenging. The use of electroporation with macrophages is inefficient and induces wide spread cell death [19]. We believe the microfluidic vortex shedding (µVS) hardware developed at Indee labs could provide a solution to the challenges facing macrophage gene delivery. Unlike viruses and particle-based methods, µVS allows for efficient gene delivery through the creation of vortices-induced holes in the cell membrane, which should help it avoid phagocytosis. In addition, results with other cell types indicate that µVS can provide high transfection efficiency and recovery without the cytotoxicity seen from electroporation [24].

About the Author

imageMichael A. Evans, BSci MSci is a bioengineering Ph.D. candidate at Harvard University with 7 years of experience in immunology, cell therapies, drug delivery, and organic chemistry. His current research focuses on using macrophages as carriers for nanoparticles to improve their targeting towards inflamed tissues. He is an author of 4 peer reviewed publications with three more in press. He attended Furman University and the University of California, Santa Barbara (UCSB), where he received a B.S. and M.S. in chemistry, respectively. He received the 2014 John Sampey Award for the Chemical Sciences and was a 2017 UCSB New Venture Competition Finalist and the People’s Choice Award recipient. Michael will be finishing his PhD in late 2019 and is excited to begin his career in gene-modified cell therapy.

References

[1] L. xun Wang, S. xi Zhang, H. juan Wu, X. lu Rong, J. Guo, M2b macrophage polarization and its roles in diseases, J. Leukoc. Biol. (2018) 1–14. doi:10.1002/JLB.3RU1018-378RR.

[2] D.M. Mosser, J.P. Edwards, Exploring the full spectrum of macrophage activation, Nat. Rev. Immunol. 8 (2008) 958–969. doi:10.1038/nri2448.

[3] L. Martínez-Lostao, A. Anel, J. Pardo, How Do Cytotoxic Lymphocytes Kill Cancer Cells?, Clin. Cancer Res. 21 (2015) 5047–5056. doi:10.1158/1078-0432.CCR-15-0685.

[4] E.W. Curren Smith, Macrophage Polarization and Its Role in Cancer, J. Clin. Cell. Immunol. 06 (2015) 4–11. doi:10.4172/2155-9899.1000338.

[5] T. Chanmee, P. Ontong, K. Konno, N. Itano, Tumor-associated macrophages as major players in the tumor microenvironment, Cancers (Basel). 6 (2014) 1670–1690. doi:10.3390/cancers6031670.

[6] M.B. Headley, E.W. Roberts, N. Kern, A.M. Steinbach, A.P. Williamson, R.D. Vale, M.A. Morrissey, Chimeric antigen receptors that trigger phagocytosis, Elife. 7 (2018) e36688. doi:10.7554/elife.36688.

[7] M. Klichinsky, M. Ruella, O. Shestova, S.S. Kenderian, M.Y. Kim, R. O’Connor, J. Scholler, C. June, G. Saar, Abstract 4575: Chimeric antigen receptor macrophages (CARMA) for adoptive cellular immunotherapy of solid tumors, in: AACR Annu. Meet., 2017: p. Abstract nr 4575.

[8] E.J. Velazquez, J.E. Lattin, T.D. Brindley, Z.Z. Reinstein, R. Chu, L. Liu, E.G. Weagel, M.H. Townsend, K. V. Whitley, E.L. Lawrence, B.T. Garcia, S. Weber, R.A. Robison, K.L. O’Neill, Abstract 2563: Macrophage Toll-like receptor-chimeric antigen receptors (MOTO-CARs) as a novel adoptive cell therapy for the treatment of solid malignancies, in: Immunology, American Association for Cancer Research, 2018: pp. 2563–2563. doi:10.1158/1538-7445.AM2018-2563.

[9] R. Velmurugan, D.K. Challa, S. Ram, R.J. Ober, E.S. Ward, Macrophage-Mediated Trogocytosis Leads to Death of Antibody-Opsonized Tumor Cells, Mol. Cancer Ther. 15 (2016) 1879–1889. doi:10.1158/1535-7163.mct-15-0335.

[10] P.P. Zheng, J.M. Kros, J. Li, Approved CAR T cell therapies: ice bucket challenges on glaring safety risks and long-term impacts, Drug Discov. Today. 23 (2018) 1175–1182. doi:10.1016/j.drudis.2018.02.012.

[11] R.G. Majzner, C.L. Mackall, Tumor antigen escape from car t-cell therapy, Cancer Discov. 8 (2018) 1219–26. doi:10.1158/2159-8290.CD-18-0442.

[12] K. Asano, A. Nabeyama, Y. Miyake, C.H. Qiu, A. Kurita, M. Tomura, O. Kanagawa, S. ichiro Fujii, M. Tanaka, CD169-Positive Macrophages Dominate Antitumor Immunity by Crosspresenting Dead Cell-Associated Antigens, Immunity. 34 (2011) 85–95. doi:10.1016/j.immuni.2010.12.011.

[13] D. Tseng, J.-P. Volkmer, S.B. Willingham, H. Contreras-Trujillo, J.W. Fathman, N.B. Fernhoff, J. Seita, M.A. Inlay, K. Weiskopf, M. Miyanishi, I.L. Weissman, Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response, Proc. Natl. Acad. Sci. 110 (2013) 11103–11108. doi:10.1073/pnas.1305569110.

[14] H.F. Dvorak, Tumors: Wounds That Do Not Heal--Redux, Cancer Immunol. Res. 3 (2015) 1–11. doi:10.1158/2326-6066.cir-14-0209.

[15] M. Najafi, N. Hashemi Goradel, B. Farhood, E. Salehi, M.S. Nashtaei, N. Khanlarkhani, Z. Khezri, J. Majidpoor, M. Abouzaripour, M. Habibi, I.R. Kashani, K. Mortezaee, Macrophage polarity in cancer: A review, J. Cell. Biochem. 120 (2019) 2756–2765. doi:10.1002/jcb.27646.

[16] B. Valipour, K. Velaei, A. Abedelahi, M. Karimipour, M. Darabi, H.N. Charoudeh, NK cells: An attractive candidate for cancer therapy, J. Cell. Physiol. (2019) jcp.28657. doi:10.1002/jcp.28657.

[17] M.M. D’Aloia, I.G. Zizzari, B. Sacchetti, L. Pierelli, M. Alimandi, CAR-T cells: The long and winding road to solid tumors, Cell Death Dis. 9 (2018). doi:10.1038/s41419-018-0278-6.

[18] C. Murdoch, A. Giannoudis, C.E. Lewis, Mechanisms regulating the recruitment of macrophages into hypoxic areas of tumors and other ischemic tissues, Blood. 104 (2004) 2224–2234. doi:10.1182/blood-2004-03-1109.

[19] X. Zhang, J.P. Edwards, D.M. Mosser, The Expression of Exogenous Genes in Macrophages: Obstacles and Opportunities, in: Methods Mol. Biol., 2009: pp. 123–143. doi:10.1007/978-1-59745-396-7_9.

[20] V. Raper, Crafting a More Efficient CAR T-Cell Industry, Genet. Eng. Biotechnol. News. 39 (2019).

[21] R.K. Iyer, P.A. Bowles, H. Kim, A. Dulgar-Tulloch, Industrializing Autologous Adoptive Immunotherapies: Manufacturing Advances and Challenges, Front. Med. 5 (2018) 1–9. doi:10.3389/fmed.2018.00150.

[22] F.J. Leyva, J.J. Anzinger, J.P. McCoy, H.S. Kruth, Evaluation of transduction efficiency in macrophage colony-stimulating factor differentiated human macrophages using HIV-1 based lentiviral vectors, BMC Biotechnol. 11 (2011). doi:10.1186/1472-6750-11-13.

[23] L. Jarrosson-Wuilleme, C. Goujon, J. Bernaud, D. Rigal, J.-L. Darlix, A. Cimarelli, Transduction of Nondividing Human Macrophages with Gammaretrovirus-Derived Vectors, J. Virol. 80 (2006) 1152–1159. doi:10.1128/jvi.80.3.1152-1159.2006.

[24] J.A. Jarrell, A.A. Twite, K.H.W.J. Lau, M.N. Kashani, A.A. Lievano, J. Acevedo, C. Priest, J. Nieva, D. Gottlieb, R.S. Pawell, Intracellular delivery of mRNA to human primary T cells with microfluidic vortex shedding, Sci. Rep. 9 (2019) 1–11. doi:10.1038/s41598-019-40147-y.