Commentary Open Access
Volume 4 | Issue 2 | DOI: https://doi.org/10.33696/cancerimmunol.4.066

Clutch Control: Changing the Speed and Direction of CAR-T Cell Therapy

  • 1Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
  • 2Department of Medicine, Harvard Medical School, Boston, MA 02115, USA
  • 3Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
  • 4Current Address: Dynamic Cell Therapies, Inc., 127 Western Ave., Allston, MA 02134, USA
  • 5Department of Surgery, Brigham and Women’s Hospital, Boston, MA 02115, USA
  • 6Department of Surgery, Harvard Medical School, Boston, MA 02115, USA
  • #These authors contributed equally
+ Affiliations - Affiliations

*Corresponding Author

Carl D. Novina, carl_novina@dfci.harvard.edu

Received Date: September 07, 2022

Accepted Date: October 21, 2022


The adoptive transfer of T cells expressing chimeric antigen receptors (CAR-T cells) has revolutionized the treatment of hematological malignancies, providing unmatched clinical responses in adults and children with relapsed or refractory B cell malignancies. To date, this has led the US Food and Drug Administration (FDA) to approve six separate CAR-T cell therapies [1-6]. The success of CAR-T cell therapy for treating solid tumors, however, has been limited [7-11]. There are several possible reasons for this including intratumoral heterogeneity, lack of tumor-specific proteins universally expressed on the surface of tumor cells, and adverse consequences due to offtumor and uncontrolled immune activity.

Indeed, less than half of the patients treated with CAR-T cells achieved long-term durable remission [4,12,13]. As of 2022, approximately 15,000 patients with hematological malignancies have been treated with CAR-T cells with an average of approximately 400 CAR-T cell infusions per month in the US alone, and response rates of 60-to-90% worldwide [14-16]. Despite widespread use, relapse has been shown to occur for many patients who initially respond; a phenomenon thought to be due, in part, to downregulation or loss of CAR-targeted protein expression on the surface of tumor cells [17,18]. Other obstacles to CAR-T cell therapy for solid tumors include poor persistence of CAR-T cells after infusion, insufficient CAR-T cell tumor infiltration, and T cell exhaustion [19], together resulting in inefficient or slow kinetics of tumor cell lysis, further increasing the chances of tumor evolution and escape from CAR-T cell therapy.

To overcome these limitations, we recently described a novel binary-activated CAR-T cell capable of simultaneously targeting multiple tumor antigens in a precise and controllable fashion [20]. By engineering the CAR-T cell to recognize a small molecule and then conjugating the same small molecule to distinct tumor-targeting antibodies, a single small-moleculespecific CAR-T cell can be directed against multiple separate targets to increase the kinetics of tumor-cell lysis. To enable safe targeting of cell surface tumor antigens shared with nonneoplastic cells, we incorporated an ultraviolet (UV) lightsensitive cage coupled to small molecules that inhibits CAR-T cells from interacting with the small molecules until exposure to UV light. This caging strategy can be used to increase the therapeutic window of CAR-T cells, allowing for temporal and spatial control of CAR-T cell activity. We believe that such binary-activated CAR-T cells provide a dynamic and adaptable approach that will accelerate the kinetics of tumor cell lysis while increasing the therapeutic window, allowing for safe and effective treatment of solid tumors. In this Commentary, we review this and other novel therapeutic approaches and discuss their potential utility for treating solid tumors.


Identifying safe and effective CAR-T cell target antigens

The majority of driver oncogenes are intracellular and are not targetable by conventional CAR-T cells [21]. Accordingly, all FDA-approved CAR-T cell therapies target abundantly expressed cell-surface proteins (e.g. CD19 and B-cell maturation antigen (BCMA)). Most of these transmembrane proteins are not related to oncogenesis, but instead are related to tumor cell lineage, with tumor cells originating from B cells or plasma cells, in the case of CD19- and BCMA-targeted CAR-T cells, respectively. Although non-neoplastic B cells are depleted by CD19-targeted CAR-T cell therapy, the depletion of B cells, and the resulting agammaglobulinemia is clinically manageable as elimination of B cells is compatible with life.

For treating solid tumors, however, target antigens are often also expressed on the surface of healthy cells, and even lowlevel expression of a target protein on the surface of healthy tissue can produce on-target but off-tumor toxicities. In some cases, CAR-T cell therapies have led to severe, and even fatal, toxicities due to the on-target but off-tumor activity directed against healthy tissues. For example, CAR-T cells directed against human epidermal growth factor 2 (HER2), a transmembrane tyrosine kinase in the ERBB family, has been used for the treatment of breast, gastric, and colorectal cancers as well as medulloblastoma [22-25]. Unfortunately, even lowlevel expression of HER2 on normal pulmonary epithelial cells led to pulmonary toxicity as well as cytokine release syndrome (CRS) [26-28]. CAR-T cells targeting carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) used for the treatment of breast, gastric, colorectal, lung, ovarian, and pancreatic cancers induced acute respiratory toxicity, likely due to CEACAM5 expression on non-neoplastic lung epithelium [29]. Similarly, metastatic renal cell carcinoma patients treated with CAR-T cells targeting carboxy-anhydrase- IX (CAIX) developed cholangitis due to the on-target but offtumor toxicity such as the destruction of CAIX-expressing bile duct epithelial cells [30]. Indeed, most solid tumors do not express a unique tumor-specific surface protein that is absent from healthy tissues. There is a need for new technologies that allow for safely targeting tumor-associated, cell-surface proteins without associated on-target, off-tumor toxicities.

Others have shown that CAR-T cells directed against tumorassociated cell surface targets can be used safely, without dose-limiting toxicities, though efficacy has been limited. This has included CAR-T cells directed against epidermal growth factor receptor (EGFR) [31,32], interleukin 13 receptor subunit alpha 2 (IL13Ra2) [33], mesothelin [34-36], carcinoembryonic antigen [CEA] [37,38], prostate-specific membrane antigen (PSMA) [39], and disaloganglioside GD2 [40,41]. While these CAR-T cells have demonstrated reasonable safety profiles, clinical benefit has been limited. The safety of these targets for CAR-T cell therapy may need to be reevaluated when effective anti-tumor responses are achieved.

Tumor heterogeneity

A major challenge to successfully implementing CAR-T cell therapy for patients with solid tumors is inter-tumoral and intra-tumoral heterogeneity. Targetable antigens can differ among patients with the same type and stage of cancer, limiting the utility of a single-antigen targeted CAR-T cell therapies. Furthermore, there is heterogeneity within individual patient’s tumors, with sub-populations of tumor cells expressing different levels of the same antigen, different isoforms of the same antigen or different antigens entirely. This is important as CAR-T cell-induced tumor cell lysis is directly proportional to the level of the target antigen expressed on the surface of tumor cells [42-46]. Tumor cells with reduced or absent expression of CAR-T cell-targeted antigens below the threshold required for CAR-T cell activation result in inefficient tumor cell lysis and are a potential source of treatment failure or recurrence, highlighting a limitation of single antigentargeted CAR-T cell therapy.

Although some patients achieve long-term durable responses after treatment with single-antigen targeted CAR-T cell therapies [4,12,13], recurrent disease associated with downregulation or loss of target antigen expression on tumor cells is a significant challenge [17,18]. In patients with CD19-positive lymphomas, durable, complete remission rates of 30-50% have been achieved with lymphodepleting chemotherapy followed by adoptive transfer of CD19-targeted CAR-T cells [47,48]. However, disease relapse is associated with the emergence of antigen-negative tumor cells, resulting in treatment failure [49-51].

To combat intratumoral evolution and antigen escape known to occur with the relatively slower kinetics of singleantigen targeted CAR-T cell therapy, we and others have evaluated strategies using multi-antigen targeted T cells in both hematological [52,53] and solid tumors [43,54-57]. For example, Ciltacabtagene autoleucel is a CAR-T cell therapy expressing two BCMA-targeting single-domain antibodies that has been approved for treating patients with multiple myeloma [58], demonstrating the safety of a multi-epitope targeting approach. Others have alternatively infused separate populations of CAR-T cells, each of which target different antigens. This has included clinical evaluation of CAR-T cells directed against CD19 and CD22 for B-lymphoblastic leukemia [59], BCMA and CD19 for multiple myeloma [60], and EGFR and CD133 for cholangiocarcinoma [57]. Some groups have used bivalent CAR-T cells targeting two distinct antigens. These include CAR-T cells simultaneously targeting CD19 and CD20 [61,62], CD19 and CD22 [63], CD19 and CD123 [53], BCMA and SLAM family member 7 (SLAMF7) [64], BCMA and CD38 [65], and glypican 2 (GPC2) and CD276 (B7-H3) [66,67]. Trivalent CAR-T cells simultaneously targeting CD19, CD20 and CD22 [68] and HER2, ephrin type-A receptor 2 (EphA2) and IL13Ra2 [69] have also been explored. These bivalent and trivalent CAR-T cells achieved better clinical and preclinical responses in comparison to monovalent CAR-T cells, highlighting the utility of such a multi-antigen targeted CAR-T cell approach.

Together, these data suggest that targeting multiple antigens simultaneously is safe and leads to more effective responses when compared to targeting single antigens alone. Moreover, targeting multiple epitopes simultaneously likely leads to more rapid clearance of tumors, decreasing the likelihood of intratumoral evolution and antigen escape, thereby producing more durable responses. While these strategies demonstrate the utility and safety of combinatorial epitope targeting with CAR-T cell therapy, they are limited in that they require separate viral transduction of multiple CAR-T cell constructs or are limited in scope to the initial targets of the infused CAR-T cell population [43,52-69]. In contrast, our strategy utilizing small-molecule targeted CAR-T cells is adaptable, being capable of simultaneously targeting multiple tumor antigens following the infusion of a single CAR-T cell construct [20].


Switch-controls increase the therapeutic window for CAR-T cell therapies

One method to redirect CAR-T cells against multiple antigens is to uncouple antigen binding from CAR-T cell engagement with tumor cells. This can be accomplished by using “switchcontrolled” CAR-T cells which consist of two-parts. The first part is a CAR-T cell directed against a peptide tag, such as the 5B9 tag-CAR (UniCAR) [70] or GCN4 peptide-CAR [71,72], or against a small molecule such as biotin [73] or fluorescein (FL) [20,71,74,75]. The second part utilizes a tumor-targeting antibody conjugated to a protein, peptide, or small molecule. Such two-part systems enable CAR-T cell-mediated killing only when the antibody-conjugate is administered. Unlike conventional CAR-T cells with fixed antigen specificity, switch CAR-T cells can be redirected to multiple targets on the surface of tumor cells by using antibodies with distinct specificities, each coupled to the same protein, peptide, or small molecule. This allows for the timing and strength of CAR-T cell cytotoxic activity to be controlled in a small molecule-dose-dependent manner [20].

Conventional CAR-T cells with fixed antigen specificity have a narrow therapeutic window when used for the treatment of solid tumors where targeted antigens are commonly also found on non-neoplastic tissue. This results in on-target but off-tumor dose-limiting toxicities, as induced lysis occurs when CAR-T cells encounter cells expressing the target antigen, regardless of whether the cell is a cancer cell or nonneoplastic cell [76]. In contrast, the switch CAR-T approach allows for the adoptive transfer of a single population of T cells that can be redirected against separate antigens through the administration of separate small molecule-conjugated monoclonal antibodies. By choosing a combination of monoclonal antibodies with distinct antigen specificities and different on-target, off-tumor activities, the dose of each antibody can be optimized to minimize on-target, off-tumor activity while producing synergistic anti-neoplastic activity. Together, this results in increased anti-tumor activity with potentially decreased on-target, off-tumor activity, widening the therapeutic window and increasing the safety and efficacy of CAR-T cells directed against solid tumors [20].

We recently described switch CAR-T cells directed against different small molecules, including fluorescein and 4-[(6-methylpyrazin-2-yl)oxy]benzoic acid (MPOB) [20]. We anticipate that simultaneously targeting multiple antigens will accelerate the kinetics of tumor cell lysis and reduce tumor evolution and antigen escape. Furthermore, if CAR T cell therapy results in target-antigen negative recurrence, persisting switch CAR-T cells can be re-directed against recurrent tumor cells by administering additional smallmolecule conjugated monoclonal antibodies specific for additional targets on recurrent tumor cells [70,77-80].

When compared to other switch-controlled CAR-T cells, our small-molecule strategy adds an additional level of control over CAR-T cell activity by allowing for titration of the density of small molecules conjugated to monoclonal antibodies. Consistent with the observation that CAR-T cell activation is correlated with the density of surface antigens on target cells [81,82], we observed increasing CAR-T cell activity with increasing density of small molecules conjugated to monoclonal antibodies. Thus, titrating the density of small molecules conjugated to antibodies along with the dose of the antibody-small molecule conjugate administered allows for further widening the therapeutic window of CAR-T cells for treating patients with solid tumors.

Additionally, we directed our switch CAR-T cells against small molecules, such as fluorescein, rather than peptides or larger proteins as others have done because small molecules are poorly immunogenic and have previously been used safely in the clinic. Indeed, fluorescein has been used in angiography without severe side effects [83] or as a tracer prior to tumor resection [84]. In contrast, switch CAR-T cells directed against peptides and proteins may be limited in efficacy by host immunity directed against the peptide or protein molecule. Previously developed switch CAR-T cells directed against the Fc portion of FDA-approved antibodies have also led to fatal side effects due to non-specific T cell activation, suggesting that careful selection of adaptor protein or peptide tags is important to precisely control switch CAR-T cell activity [85]. These data suggest that careful consideration must be given to the adaptor and linker molecules for precise control over CAR-T cell activity [85].

We have shown that anti-fluorescein CAR-T cells (FL-CAR-T cells) can target multiple antigens including EGFR, HER2, and CD38 expressed on the surface of the breast cancer, ovarian cancer, Burkitt lymphoma, and multiple myeloma cells. FLCAR- T cells induced dose-dependent cytotoxicity of tumor cells labeled with antibody-fluorescein conjugates; this tumor cell killing was proportional to both the concentration of antibody-fluorescein complexes and the density of fluorescein conjugated to a tumor-targeting antibody. These data suggest that our small molecule switch CAR-T cells may be a potent, safe, and effective CAR-T cell with “customizable kinetics” of target cell lysis that may be useful for treating patients with multiple types of cancer [20].

Furthermore, to model CAR-T cell-induced lysis of heterogenous populations of tumor cells, we mixed EGFRexpressing tumor cells with CD38-expressing tumor cells. Addition of fluorescein-conjugated anti-EGFR monoclonal antibody resulted in cell lysis of EGFR-expressing tumor cells while addition of fluorescein-conjugated anti-CD38 monoclonal antibody resulted in cell lysis of CD38-expressing tumor cells. The combination of both monoclonal antibodies, however, resulted in the killing of both EGFR and CD38 expressing tumor cells, demonstrating that single-specificity CAR-T cells can be used to simultaneously target multiple tumor antigens and kill heterogeneous populations of cells [20]. Such an approach could accelerate the kinetics of tumor cell lysis and prevent antigen escape while maintaining a high level of safety.

Local control over access to CAR-T cell antigens, and future directions

While our switch CAR-T cell system allows for antigen multiplexing and titratable control over CAR-T cell activity, finding the right combination of antibodies with both meaningful on-tumor and minimal off-tumor activities can be challenging, especially in the context of treating patients with solid tumors. To overcome this limitation and further improve the safety of our switch CAR-T cell system, we implemented ultraviolet (UV) light-sensitive cage technology to our small molecule switch CAR-T cell system. Two near UV light-sensitive 5-carboxymethoxy-2-nitrobenzyl (CMNB) caging groups covalently coupled to the CAR-T cell-targeted small molecule prevented recognition of small molecules by CAR-T cells. This CMNB-cage is stable under the light of an operating room or in physiologic environments but is destabilized by exposure to the UV-light at 365nm, leading to the release of the CMNBcage [20]. Once uncaged, the anti-small molecule CAR-T cell can recognize the small molecule and engage in tumor cell lysis. This strategy is particularly relevant in the context of solid tumors where small molecule-coupled antibodies bound to the surface of the tumor cells can be selectively exposed to UV light. Exposing the small molecule to UV light results in uncaging, CAR-T cell recognition, and subsequent tumor cell lysis, while small molecule-coupled antibodies bound to targets on distant, non-neoplastic cells remain caged and thus protected from CAR-T cell attack. This caging strategy enables temporal and spatial control over tumor cell killing while reducing the risk of on-target, off-tumor toxicities. The caging strategy can be alternated with dosing uncaged small molecule-coupled antibodies or mixed with small moleculecoupled antibodies directed against antigens with a more tumor-specific expression pattern, thereby generating a wider therapeutic window overall [20].

While this caging strategy may be useful for targeting tumor antigens broadly expressed on healthy tissue, it may also be implemented to safely enhance CAR-T cell tumor penetration. Stromal cells in the tumor microenvironment secrete extracellular matrix and create a physical barrier that prevents the infiltration of CAR-T cells and other immune cells into tumors [86]. CAR-T cells have been developed to attack fibroblasts in the tumor microenvironment, resulting in increased stromal permeability and improved immune cell infiltration into solid tumors [86]. However, most proteins expressed by tumor stroma are also widely expressed in non-neoplastic tissue, the targeting of which can lead to life-threatening on-target, off-tumor toxicities, especially secondary to widespread destruction of the bone marrow [86]. An intermittent CAR-T cell activity, like the one provided by the use of the caged small molecule and switch CAR-T cell platform, however, may allow for precise targeting of intratumoral stromal cells without associated wide-spread tissue destruction [87].

One limitation of translating this UV-caging strategy to clinical practice is insufficient tissue penetration by UV-light to achieve significant uncaging of small molecules within deeper tissues [88]. However, several photocaging groups are destabilized within the optical window for photomedicine applications, between 650 to 1300 nm [89], such as BODIPY or cyanine, and can be uncaged at longer (near infrared) wavelengths [90,91]. As such, our caged small molecule-targeted CAR-T cell system can be integrated with novel photocaging technologies and clinical applications as they are developed.

Other caging strategies that circumvent the need for light take advantage of secondary signals that are specific to the tumor microenvironment, such as tumor-specific proteases or the release of reactive oxygen or nitrogen species (ROS and RNS) [92-94]. The Probody approach developed by CytomX [95] uses an antibody coupled to a peptide that masks the antigen recognition site. The mask is connected via a proteasecleavable linker that prevents the binding of the antibody to the antigen. The Probody can be unmasked by proteases commonly found in the tumor microenvironment. Combining antibody masking and small molecule caging strategies could increase tumor-specificity and expand the number of targetable antigens. These modular approaches will increase the specificity and efficacy of CAR-T cell therapies and reduce the risk of toxicity and relapse for heterogenous solid tumors.

Our caged small molecule CAR-T cell system may be complemented by bio-orthogonal technologies. For example, fluorescein- or metal-labelled antibodies could be used to track initial tumor heterogeneity and tumor evolution in response to therapy [96-98]. Image segmentation could be used to (i) predict the density and distribution of target proteins in tumors (innate and acquired tumor heterogeneity), (ii) mark the boundaries of the targeted region, and (iii) provide visual confirmation of which target(s) were effective in the therapy of heterogeneous tumors [99-102].

While CAR-T cell therapy is approved for relapsed, refractory disease, recent clinical trials have suggested that CD19- directed CAR-T cell therapy may be more effective than the standard of care second-line therapy for patients with non- Hodgkin lymphoma [103-105]. These results support the use of CAR-T cell therapy for earlier stage disease. As this occurs, it will be important to adopt novel CAR-T cell strategies that enhance the safety profile by widening the therapeutic window while allowing for increased diversity of targets.

Conflict of Interests

A.N., A.K., and C.D.N. filed patents on the small molecule CAR-T cell and caging technologies described in this manuscript. A.N. and C.D.N. and have equity in Dynamic Cell Therapies, Inc. C.D.N. is a member of the Scientific Advisory Board and Board of Directors of Dynamic Cell Therapies, Inc.


Author Contributions

A.N., A.K., P.C.G., and C.D.N. wrote the manuscript. A.N., A.K., P.C.G. contributed equally to this work. C.D.N supervised the project. The manuscript was revised and approved by all authors.


This work was supported by grants from the National Institute of Health (DP1 DK105602) and Curing Kids Cancer Foundation to C.D.N and generous gifts from the Paula and Rodger Riney Foundation and the Ellie Kavalieros DIPG Research Fund.


1. Porter DL, Hwang WT, Frey NV, Lacey SF, Shaw PA, Loren AW, et al. Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia. Sci Transl Med. 2015;7(303):303ra139.

2. Bouchkouj N, Kasamon YL, de Claro RA, George B, Lin X, Lee S, et al. FDA Approval Summary: Axicabtagene Ciloleucel for Relapsed or Refractory Large B-cell Lymphoma. Clin Cancer Res. 2019;25(6):1702-8.

3. Locke FL, Neelapu SS, Bartlett NL, Siddiqi T, Chavez JC, Hosing CM, et al. Phase 1 Results of ZUMA-1: A Multicenter Study of KTE-C19 Anti-CD19 CAR T Cell Therapy in Refractory Aggressive Lymphoma. Mol Ther. 2017;25(1):285-95.

4. Neelapu SS, Locke FL, Bartlett NL, Lekakis LJ, Miklos DB, Jacobson CA, et al. Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B-Cell Lymphoma. N Engl J Med. 2017;377(26):2531-44.

5. Munshi NC, Anderson LD, Jr., Shah N, Madduri D, Berdeja J, Lonial S, et al. Idecabtagene Vicleucel in Relapsed and Refractory Multiple Myeloma. N Engl J Med. 2021;384(8):705-16.

6. Anagnostou T, Riaz IB, Hashmi SK, Murad MH, Kenderian SS. Anti-CD19 chimeric antigen receptor T-cell therapy in acute lymphocytic leukaemia: a systematic review and meta-analysis. Lancet Haematol. 2020;7(11):e816-e26.

7. Wagner J, Wickman E, DeRenzo C, Gottschalk S. CAR T Cell Therapy for Solid Tumors: Bright Future or Dark Reality? Mol Ther. 2020;28(11):2320-39.

8. Fuca G, Reppel L, Landoni E, Savoldo B, Dotti G. Enhancing Chimeric Antigen Receptor T-Cell Efficacy in Solid Tumors. Clin Cancer Res. 2020;26(11):2444-51.

9. Sakemura R, Cox MJ, Hefazi M, Siegler EL, Kenderian SS. Resistance to CART cell therapy: lessons learned from the treatment of hematological malignancies. Leuk Lymphoma. 2021;62(9):2052-63.

10. Kankeu Fonkoua LA, Sirpilla O, Sakemura R, Siegler EL, Kenderian SS. CAR T cell therapy and the tumor microenvironment: Current challenges and opportunities. Mol Ther Oncolytics. 2022;25:69-77.

11. Hou AJ, Chen LC, Chen YY. Navigating CAR-T cells through the solid-tumour microenvironment. Nat Rev Drug Discov. 2021.

12. Schuster SJ, Bishop MR, Tam CS, Waller EK, Borchmann P, McGuirk JP, et al. Tisagenlecleucel in Adult Relapsed or Refractory Diffuse Large B-Cell Lymphoma. N Engl J Med. 2019;380(1):45-56.

13. Wang M, Munoz J, Goy A, Locke FL, Jacobson CA, Hill BT, et al. KTE-X19 CAR T-Cell Therapy in Relapsed or Refractory Mantle-Cell Lymphoma. N Engl J Med. 2020;382(14):1331-42.

14. June CH, Sadelain M. Chimeric Antigen Receptor Therapy. N Engl J Med. 2018;379(1):64-73.

15. News PM. Penn Medicine and Children's Hospital of Philadelphia Announce Partnership with Costa Rica for CAR T Cell Therapy 2022 [Available from: https://www.pennmedicine.org/news/news-releases/2022/may/penn-medicine-and-chop-announce-partnership-with-costa-rica-for-car-t-cell-therapy.

16. Saez-Ibañez AR, Upadhaya S, Partridge T, Shah M, Correa D, Campbell J. Landscape of cancer cell therapies: trends and real-world data. Nat Rev Drug Discov. 2022.

17. Samur MK, Fulciniti M, Aktas Samur A, Bazarbachi AH, Tai YT, Prabhala R, et al. Biallelic loss of BCMA as a resistance mechanism to CAR T cell therapy in a patient with multiple myeloma. Nat Commun. 2021;12(1):868.

18. Majzner RG, Mackall CL. Tumor Antigen Escape from CAR T-cell Therapy. Cancer Discov. 2018;8(10):1219-26.

19. Hou AJ, Chen LC, Chen YY. Navigating CAR-T cells through the solid-tumour microenvironment. Nat Rev Drug Discov. 2021;20:531-50.

20. Kobayashi A, Nobili A, Neier SC, Sadiki A, Distel R, Zhou ZS, et al. Light-Controllable Binary Switch Activation of CAR T Cells. ChemMedChem. 2022;17(12):e202100722.

21. Futreal PA, Coin L, Marshall M, Down T, Hubbard T, Wooster R, et al. A census of human cancer genes. Nat Rev Cancer. 2004;4(3):177-83.

22. Yan M, Schwaederle M, Arguello D, Millis SZ, Gatalica Z, Kurzrock R. HER2 expression status in diverse cancers: review of results from 37,992 patients. Cancer and Metastasis Rev. 2015;34(1):157-64.

23. Han Y, Liu C, Li G, Li J, Lv X, Shi H, et al. Antitumor effects and persistence of a novel HER2 CAR T cells directed to gastric cancer in preclinical models. Am J Cancer Res. 2018;8(1):106-19.

24. Priceman SJ, Tilakawardane D, Jeang B, Aguilar B, Murad JP, Park AK, et al. Regional Delivery of Chimeric Antigen Receptor–Engineered T Cells Effectively Targets HER2+ Breast Cancer Metastasis to the Brain. Clin Cancer Res. 2018;24(1):95-105.

25. Nellan A, Rota C, Majzner R, Lester-McCully CM, Griesinger AM, Mulcahy Levy JM, et al. Durable regression of Medulloblastoma after regional and intravenous delivery of anti-HER2 chimeric antigen receptor T cells. J Immunother Cancer. 2018;6(1):30.

26. Morgan RA, Yang JC, Kitano M, Dudley ME, Laurencot CM, Rosenberg SA. Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther. 2010;18(4):843-51.

27. Ahmed N, Brawley VS, Hegde M, Robertson C, Ghazi A, Gerken C, et al. Human Epidermal Growth Factor Receptor 2 (HER2) -Specific Chimeric Antigen Receptor-Modified T Cells for the Immunotherapy of HER2-Positive Sarcoma. J Clin Oncol. 2015;33(15):1688-96.

28. Feng K, Liu Y, Guo Y, Qiu J, Wu Z, Dai H, et al. Phase I study of chimeric antigen receptor modified T cells in treating HER2-positive advanced biliary tract cancers and pancreatic cancers. Protein Cell. 2018;9(10):838-47.

29. Thistlethwaite FC, Gilham DE, Guest RD, Rothwell DG, Pillai M, Burt DJ, et al. The clinical efficacy of first-generation carcinoembryonic antigen (CEACAM5)-specific CAR T cells is limited by poor persistence and transient pre-conditioning-dependent respiratory toxicity. Cancer Immunol Immunother. 2017;66(11):1425-36.

30. Lamers CHJ, Sleijfer S, Vulto AG, Kruit WHJ, Kliffen M, Debets R, et al. Treatment of Metastatic Renal Cell Carcinoma With Autologous T-Lymphocytes Genetically Retargeted Against Carbonic Anhydrase IX: First Clinical Experience. J Clin Oncol. 2006;24(13):e20-e2.

31. Guo Y, Feng K, Liu Y, Wu Z, Dai H, Yang Q, et al. Phase I Study of Chimeric Antigen Receptor–Modified T Cells in Patients with EGFR-Positive Advanced Biliary Tract Cancers. Clin Cancer Res. 2018;24(6):1277-86.

32. Liu Y, Guo Y, Wu Z, Feng K, Tong C, Wang Y, et al. Anti-EGFR chimeric antigen receptor-modified T cells in metastatic pancreatic carcinoma: A phase I clinical trial. Cytotherapy. 2020;22(10):573-80.

33. Brown CE, Alizadeh D, Starr R, Weng L, Wagner JR, Naranjo A, et al. Regression of Glioblastoma after Chimeric Antigen Receptor T-Cell Therapy. N Engl J Med. 2016;375(26):2561-9.

34. Maus MV, Haas AR, Beatty GL, Albelda SM, Levine BL, Liu X, et al. T cells expressing chimeric antigen receptors can cause anaphylaxis in humans. Cancer Immunol Res. 2013;1(1):26-31.

35. Beatty GL, Haas AR, Maus MV, Torigian DA, Soulen MC, Plesa G, et al. Mesothelin-specific chimeric antigen receptor mRNA-engineered T cells induce anti-tumor activity in solid malignancies. Cancer Immunol Res. 2014;2(2):112-20.

36. Beatty GL, O'Hara MH, Lacey SF, Torigian DA, Nazimuddin F, Chen F, et al. Activity of Mesothelin-Specific Chimeric Antigen Receptor T Cells Against Pancreatic Carcinoma Metastases in a Phase 1 Trial. Gastroenterology. 2018;155(1):29-32.

37. Katz SC, Burga RA, McCormack E, Wang LJ, Mooring W, Point GR, et al. Phase I Hepatic Immunotherapy for Metastases Study of Intra-Arterial Chimeric Antigen Receptor-Modified T-cell Therapy for CEA+ Liver Metastases. Clin Cancer Res. 2015;21(14):3149-59.

38. Zhang C, Wang Z, Yang Z, Wang M, Li S, Li Y, et al. Phase I Escalating-Dose Trial of CAR-T Therapy Targeting CEA(+) Metastatic Colorectal Cancers. Mol Ther. 2017;25(5):1248-58.

39. Junghans RP, Ma Q, Rathore R, Gomes EM, Bais AJ, Lo AS, et al. Phase I Trial of Anti-PSMA Designer CAR-T Cells in Prostate Cancer: Possible Role for Interacting Interleukin 2-T Cell Pharmacodynamics as a Determinant of Clinical Response. Prostate. 2016;76(14):1257-70.

40. Majzner RG, Ramakrishna S, Yeom KW, Patel S, Chinnasamy H, Schultz LM, et al. GD2-CAR T cell therapy for H3K27M-mutated diffuse midline gliomas. Nature. 2022;603(7903):934-41.

41. Mount CW, Majzner RG, Sundaresh S, Arnold EP, Kadapakkam M, Haile S, et al. Potent antitumor efficacy of anti-GD2 CAR T cells in H3-K27M+ diffuse midline gliomas. Nat Med. 2018;24(5):572-9.

42. Song DG, Ye Q, Poussin M, Chacon JA, Figini M, Powell DJ, Jr. Effective adoptive immunotherapy of triple-negative breast cancer by folate receptor-alpha redirected CAR T cells is influenced by surface antigen expression level. J Hematol Oncol. 2016;9(1):56.

43. Anurathapan U, Chan RC, Hindi HF, Mucharla R, Bajgain P, Hayes BC, et al. Kinetics of tumor destruction by chimeric antigen receptor-modified T cells. Mol Ther. 2014;22(3):623-33.

44. Walker AJ, Majzner RG, Zhang L, Wanhainen K, Long AH, Nguyen SM, et al. Tumor Antigen and Receptor Densities Regulate Efficacy of a Chimeric Antigen Receptor Targeting Anaplastic Lymphoma Kinase. Molecular Therapy. 2017;25(9):2189-201.

45. Majzner RG, Rietberg SP, Sotillo E, Dong R, Vachharajani VT, Labanieh L, et al. Tuning the Antigen Density Requirement for CAR T-cell Activity. Cancer Discov. 2020;10(5):702-23.

46. Hudecek M, Lupo-Stanghellini M-T, Kosasih PL, Sommermeyer D, Jensen MC, Rader C, et al. Receptor Affinity and Extracellular Domain Modifications Affect Tumor Recognition by ROR1-Specific Chimeric Antigen Receptor T Cells. Clin Cancer Res. 2013;19(12):3153-64.

47. Leick MB, Maus MV, Frigault MJ. Clinical Perspective: Treatment of Aggressive B Cell Lymphomas with FDA-Approved CAR-T Cell Therapies. Mol Ther. 2021;29(2):433-41.

48. Mian A, Hill BT. Brexucabtagene autoleucel for the treatment of relapsed/refractory mantle cell lymphoma. Expert Opin Biol Ther. 2021;21(4):435-41.

49. Shah NN, Fry TJ. Mechanisms of resistance to CAR T cell therapy. Nat Rev Clin Oncol. 2019;16(6):372-85.

50. Singh N, Orlando E, Xu J, Xu J, Binder Z, Collins MA, et al. Mechanisms of resistance to CAR T cell therapies. Semin Cancer Biol. 2020;65:91-8.

51. Majzner RG, Mackall CL. Clinical lessons learned from the first leg of the CAR T cell journey. Nat Med. 2019;25(9):1341-55.

52. Zah E, Lin MY, Silva-Benedict A, Jensen MC, Chen YY. T Cells Expressing CD19/CD20 Bispecific Chimeric Antigen Receptors Prevent Antigen Escape by Malignant B Cells. Cancer Immunol Res. 2016;4(6):498-508.

53. Ruella M, Barrett DM, Kenderian SS, Shestova O, Hofmann TJ, Perazzelli J, et al. Dual CD19 and CD123 targeting prevents antigen-loss relapses after CD19-directed immunotherapies. J Clin Invest. 2016;126(10):3814-26.

54. Hegde M, Mukherjee M, Grada Z, Pignata A, Landi D, Navai SA, et al. Tandem CAR T cells targeting HER2 and IL13Ralpha2 mitigate tumor antigen escape. J Clin Invest. 2016;126(8):3036-52.

55. Genssler S, Burger MC, Zhang C, Oelsner S, Mildenberger I, Wagner M, et al. Dual targeting of glioblastoma with chimeric antigen receptor-engineered natural killer cells overcomes heterogeneity of target antigen expression and enhances antitumor activity and survival. Oncoimmunology. 2016;5(4):e1119354.

56. Wei X, Lai Y, Li J, Qin L, Xu Y, Zhao R, et al. PSCA and MUC1 in non-small-cell lung cancer as targets of chimeric antigen receptor T cells. Oncoimmunology. 2017;6(3):e1284722.

57. Feng KC, Guo YL, Liu Y, Dai HR, Wang Y, Lv HY, et al. Cocktail treatment with EGFR-specific and CD133-specific chimeric antigen receptor-modified T cells in a patient with advanced cholangiocarcinoma. J Hematol Oncol. 2017;10(1):4.

58. Berdeja JG, Madduri D, Usmani SZ, Jakubowiak A, Agha M, Cohen AD, et al. Ciltacabtagene autoleucel, a B-cell maturation antigen-directed chimeric antigen receptor T-cell therapy in patients with relapsed or refractory multiple myeloma (CARTITUDE-1): a phase 1b/2 open-label study. Lancet. 2021;398(10297):314-24.

59. Hua J, Qian W, Wu X, Zhou L, Yu L, Chen S, et al. Sequential Infusion of Anti-CD22 and Anti-CD19 Chimeric Antigen Receptor T Cells for a Pediatric Ph-Like B-ALL Patient That Relapsed After CART-Cell and Haplo-HSCT Therapy: A Case Report and Review of Literature. Onco Targets Ther. 2020;13:2311-7.

60. Yan L, Qu S, Shang J, Shi X, Kang L, Xu N, et al. Sequential CD19 and BCMA-specific CAR T-cell treatment elicits sustained remission of relapsed and/or refractory myeloma. Cancer Med. 2021;10(2):563-74.

61. Shah NN, Johnson BD, Schneider D, Zhu F, Szabo A, Keever-Taylor CA, et al. Bispecific anti-CD20, anti-CD19 CAR T cells for relapsed B cell malignancies: a phase 1 dose escalation and expansion trial. Nat Med. 2020;26(10):1569-75.

62. Tong C, Zhang Y, Liu Y, Ji X, Zhang W, Guo Y, et al. Optimized tandem CD19/CD20 CAR-engineered T cells in refractory/relapsed B-cell lymphoma. Blood. 2020;136(14):1632-44.

63. Qin H, Ramakrishna S, Nguyen S, Fountaine TJ, Ponduri A, Stetler-Stevenson M, et al. Preclinical Development of Bivalent Chimeric Antigen Receptors Targeting Both CD19 and CD22. Mol Ther Oncolytics. 2018;11:127-37.

64. Zah E, Nam E, Bhuvan V, Tran U, Ji BY, Gosliner SB, et al. Systematically optimized BCMA/CS1 bispecific CAR-T cells robustly control heterogeneous multiple myeloma. Nat Commun. 2020;11(1):2283.

65. Feng Y, Liu X, Li X, Zhou Y, Song Z, Zhang J, et al. Novel BCMA-OR-CD38 tandem-dual chimeric antigen receptor T cells robustly control multiple myeloma. Oncoimmunology. 2021;10(1):1959102.

66. Pant A, Jackson CM. Supercharged chimeric antigen receptor T cells in solid tumors. J Clin Invest. 2022;132(16).

67. Tian M, Cheuk AT, Wei JS, Abdelmaksoud A, Chou H-C, Milewski D, et al. An optimized bicistronic chimeric antigen receptor against GPC2 or CD276 overcomes heterogeneous expression in neuroblastoma. J Clin Invest. 2022;132(16).

68. Schneider D, Xiong Y, Wu D, Hu P, Alabanza L, Steimle B, et al. Trispecific CD19-CD20-CD22–targeting duoCAR-T cells eliminate antigen-heterogeneous B cell tumors in preclinical models. Sci Transl Med. 2021;13(586):eabc6401.

69. Bielamowicz K, Fousek K, Byrd TT, Samaha H, Mukherjee M, Aware N, et al. Trivalent CAR T cells overcome interpatient antigenic variability in glioblastoma. Neuro Oncol. 2018;20(4):506-18.

70. Cartellieri M, Feldmann A, Koristka S, Arndt C, Loff S, Ehninger A, et al. Switching CAR T cells on and off: a novel modular platform for retargeting of T cells to AML blasts. Blood Cancer J. 2016;6(8):e458.

71. Cao Y, Rodgers DT, Du J, Ahmad I, Hampton EN, Ma JS, et al. Design of Switchable Chimeric Antigen Receptor T Cells Targeting Breast Cancer. Angew Chem Int Ed Engl. 2016;55(26):7520-4.

72. Qi J, Tsuji K, Hymel D, Burke Jr. TR, Hudecek M, Rader C, et al. Chemically Programmable and Switchable CAR-T Therapy. Angew Chem Int Ed Engl. 2020;59(29):12178-85.

73. Urbanska K, Lanitis E, Poussin M, Lynn RC, Gavin BP, Kelderman S, et al. A universal strategy for adoptive immunotherapy of cancer through use of a novel T-cell antigen receptor. Cancer Res. 2012;72(7):1844-52.

74. Tamada K, Geng D, Sakoda Y, Bansal N, Srivastava R, Li Z, et al. Redirecting gene-modified T cells toward various cancer types using tagged antibodies. Clin Cancer Res. 2012;18(23):6436-45.

75. Kim MS, Ma JS, Yun H, Cao Y, Kim JY, Chi V, et al. Redirection of genetically engineered CAR-T cells using bifunctional small molecules. J Am Chem Soc. 2015;137(8):2832-5.

76. Melenhorst JJ, Chen GM, Wang M, Porter DL, Chen C, Collins MA, et al. Decade-long leukaemia remissions with persistence of CD4+ CAR T cells. Nature. 2022;602(7897):503-9.

77. Wermke M, Kraus S, Ehninger A, Bargou RC, Goebeler M-E, Middeke JM, et al. Proof of concept for a rapidly switchable universal CAR-T platform with UniCAR-T-CD123 in relapsed/refractory AML. Blood. 2021;137(22):3145-8.

78. Feldmann A, Arndt C, Bergmann R, Loff S, Cartellieri M, Bachmann D, et al. Retargeting of T lymphocytes to PSCA- or PSMA positive prostate cancer cells using the novel modular chimeric antigen receptor platform technology “UniCAR”. Oncotarget. 2017;8(19).

79. Kittel-Boselli E, Soto KEG, Loureiro LR, Hoffmann A, Bergmann R, Arndt C, et al. Targeting Acute Myeloid Leukemia Using the RevCAR Platform: A Programmable, Switchable and Combinatorial Strategy. Cancers. 2021;13(19):4785.

80. Lee YG, Marks I, Srinivasarao M, Kanduluru AK, Mahalingam SM, Liu X, et al. Use of a Single CAR T Cell and Several Bispecific Adapters Facilitates Eradication of Multiple Antigenically Different Solid Tumors. Cancer Res. 2019;79(2):387-96.

81. Walker AJ, Majzner RG, Zhang L, Wanhainen K, Long AH, Nguyen SM, et al. Tumor Antigen and Receptor Densities Regulate Efficacy of a Chimeric Antigen Receptor Targeting Anaplastic Lymphoma Kinase. Mol Ther. 2017;25(9):2189-201.

82. Harris DT, Hager MV, Smith SN, Cai Q, Stone JD, Kruger P, et al. Comparison of T Cell Activities Mediated by Human TCRs and CARs That Use the Same Recognition Domains. J Immunol. 2018;200(3):1088-100.

83. Alford R, Simpson HM, Duberman J, Hill GC, Ogawa M, Regino C, et al. Toxicity of Organic Fluorophores Used in Molecular Imaging: Literature Review. Mol Imaging. 2009;8(6).

84. Shinoda J, Yano H, Yoshimura S-I, Okumura A, Kaku Y, Iwama T, et al. Fluorescence-guided resection of glioblastoma multiforme by using high-dose fluorescein sodium: Technical note. J Neurosurg. 2003;99(3):597-603.

85. Unum-Therapeutics. FDA puts Unum T cell therapy trial on hold after adverse events 2019 [Available from: https://www.fiercebiotech.com/biotech/fda-puts-unum-t-cell-therapy-trial-hold-after-adverse-events.

86. Bughda R, Dimou P, D'Souza RR, Klampatsa A. Fibroblast Activation Protein (FAP)-Targeted CAR-T Cells: Launching an Attack on Tumor Stroma. Immunotargets Ther. 2021;10:313-23.

87. Rurik JG, Tombácz I, Yadegari A, Méndez Fernández PO, Shewale SV, Li L, et al. CAR T cells produced in vivo to treat cardiac injury. Science. 2022;375(6576):91-6.

88. Ash C, Dubec M, Donne K, Bashford T. Effect of wavelength and beam width on penetration in light-tissue interaction using computational methods. Lasers Med Sci. 2017;32(8):1909-18.

89. Castano AP, Demidova TN, Hamblin MR. Mechanisms in photodynamic therapy: part one-photosensitizers, photochemistry and cellular localization. Photodiagnosis Photodyn Ther. 2004;1(4):279-93.

90. Nani RR, Gorka AP, Nagaya T, Kobayashi H, Schnermann MJ. Near-IR Light-Mediated Cleavage of Antibody–Drug Conjugates Using Cyanine Photocages. Angew Chem Int Ed Engl. 2015;54(46):13635-8.

91. Peterson JA, Wijesooriya C, Gehrmann EJ, Mahoney KM, Goswami PP, Albright TR, et al. Family of BODIPY Photocages Cleaved by Single Photons of Visible/Near-Infrared Light. J Am Chem Soc. 2018;140(23):7343-6.

92. Kirkpatrick JD, Warren AD, Soleimany AP, Westcott PMK, Voog JC, Martin-Alonso C, et al. Urinary detection of lung cancer in mice via noninvasive pulmonary protease profiling. Sci Transl Med. 2020;12(537):eaaw0262.

93. Bauer G. Targeting extracellular ROS signaling of tumor cells. Anticancer Res. 2014;34(4):1467-82.

94. Chang MCY, Pralle A, Isacoff EY, Chang CJ. A Selective, Cell-Permeable Optical Probe for Hydrogen Peroxide in Living Cells. J Am Chem Soc. 2004;126(47):15392-3.

95. Desnoyers LR, Vasiljeva O, Richardson JH, Yang A, Menendez EE, Liang TW, et al. Tumor-specific activation of an EGFR-targeting probody enhances therapeutic index. Sci Transl Med. 2013;5(207):207ra144.

96. Sakemura R, Can I, Siegler EL, Kenderian SS. In vivo CART cell imaging: Paving the way for success in CART cell therapy. Mol Ther Oncolytics. 2021;20:625-33.

97. Arnouk S, De Groof TWM, Van Ginderachter JA. Imaging and therapeutic targeting of the tumor immune microenvironment with biologics. Adv Drug Deliv Rev. 2022;184:114239.

98. Jarrett AM, Hormuth DA, Adhikarla V, Sahoo P, Abler D, Tumyan L, et al. Towards integration of 64Cu-DOTA-trastuzumab PET-CT and MRI with mathematical modeling to predict response to neoadjuvant therapy in HER2?+?breast cancer. Sci Rep. 2020;10(1):20518.

99. Dakua SP, Sahambi JS. Automatic left ventricular contour extraction from cardiac magnetic resonance images using cantilever beam and random walk approach. Cardiovasc Eng. 2010;10(1):30-43.

100. Dakua SP, Sahambi JS. Modified active contour model and Random Walk approach for left ventricular cardiac MR image segmentation. Int J Numer Methods Biomed Eng. 2011;27(9):1350-61.

101. Dakua SP, Abinahed J, Al-Ansari AA. Pathological liver segmentation using stochastic resonance and cellular automata. J Vis Commun Image Represent. 2016;34:89-102.

102. Dakua SP. LV Segmentation Using Stochastic Resonance and Evolutionary Cellular Automata. Intern J Pattern Recognit Artif Intell. 2015;29(03):1557002.

103. Bishop MR, Dickinson M, Purtill D, Barba P, Santoro A, Hamad N, et al. Second-Line Tisagenlecleucel or Standard Care in Aggressive B-Cell Lymphoma. N Engl J Med. 2022;386(7):629-39.

104. Kamdar M, Solomon SR, Arnason J, Johnston PB, Glass B, Bachanova V, et al. Lisocabtagene maraleucel versus standard of care with salvage chemotherapy followed by autologous stem cell transplantation as second-line treatment in patients with relapsed or refractory large B-cell lymphoma (TRANSFORM): results from an interim analysis of an open-label, randomised, phase 3 trial. Lancet. 2022;399(10343):2294-308.

105. Locke FL, Miklos DB, Jacobson CA, Perales MA, Kersten MJ, Oluwole OO, et al. Axicabtagene Ciloleucel as Second-Line Therapy for Large B-Cell Lymphoma. N Engl J Med. 2022;386(7):640-54.

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