Chimeric anti-GPC3 sFv-CD3ε receptor-modified T cells with IL7 co-expression for the treatment of solid tumors

Abstract

Chimeric antigen receptor (CAR) T cells targeting glypican-3 (GPC3) demonstrated early signs of therapeutic efficacy to hepatocellular carcinoma patients with a risk of cytokine release syndrome (CRS). Several adoptive cell therapies (ACTs) with T cells using the natural T cell receptor (TCR) signaling induced more efficient antitumor function and reduced cytokine production relative to CARs in solid tumors. To improve the efficacy and safety of GPC3-targeted ACTs, T cells were modified with anti-GPC3 single-chain fragment variable(sFv) linked to CD3ε, which could be incorporated into the entire TCR/CD3 complex to form chimeric sFv-CD3ε receptor (sFv-ε). sFv-ε T cells showed competitive antitumor activity and lower cytokine release compared to 28ζ or BBζ CAR T cells, which may be ascribed to moderately less activated Ca2+-calcineurin-NFAT signaling pathway. We further generated murine sFv-ε T cells with interleukin-7 co-expression (7sFv-ε) to promote T cell survival and to mobilize the endogenous immune system. In immunocompetent mouse models, 7sFv-ε T cells showed superior persistence, antitumor efficacy, and immunological memory while preserving the low production of cytokines associated with CRS compared to conventional sFv-ε T cells. These results indicate that GPC3-specific 7sFv-ε T cells could serve as a promising therapeutic strategy for solid tumors. Chimeric antigen receptor (CAR) T cells targeting glypican-3 (GPC3) demonstrated early signs of therapeutic efficacy to hepatocellular carcinoma patients with a risk of cytokine release syndrome (CRS). Several adoptive cell therapies (ACTs) with T cells using the natural T cell receptor (TCR) signaling induced more efficient antitumor function and reduced cytokine production relative to CARs in solid tumors. To improve the efficacy and safety of GPC3-targeted ACTs, T cells were modified with anti-GPC3 single-chain fragment variable(sFv) linked to CD3ε, which could be incorporated into the entire TCR/CD3 complex to form chimeric sFv-CD3ε receptor (sFv-ε). sFv-ε T cells showed competitive antitumor activity and lower cytokine release compared to 28ζ or BBζ CAR T cells, which may be ascribed to moderately less activated Ca2+-calcineurin-NFAT signaling pathway. We further generated murine sFv-ε T cells with interleukin-7 co-expression (7sFv-ε) to promote T cell survival and to mobilize the endogenous immune system. In immunocompetent mouse models, 7sFv-ε T cells showed superior persistence, antitumor efficacy, and immunological memory while preserving the low production of cytokines associated with CRS compared to conventional sFv-ε T cells. These results indicate that GPC3-specific 7sFv-ε T cells could serve as a promising therapeutic strategy for solid tumors. IntroductionImproved understanding of tumor immunity over the past decades has led to the accelerated development of adoptive cell therapies (ACTs), including autologous tumor-infiltrating lymphocytes (TILs), T cell receptor (TCR), and chimeric antigen receptor (CAR) engineered T cells. TCR and CAR T cells are generated by modifying peripheral T cells to express a precisely defined antigen-specific receptor. TCR T cells recognize antigenic peptides presented on major histocompatibility complex (MHC) molecules, the function of which may be impaired by reduced or absent MHC on tumor cells or disruption of antigen processing and presentation, and was constrained to patients with a particular MHC allele.1Morgan R.A. Dudley M.E. Wunderlich J.R. Hughes M.S. Yang J.C. Sherry R.M. Royal R.E. Topalian S.L. Kammula U.S. Restifo N.P. et al.Cancer regression in patients after transfer of genetically engineered lymphocytes.Science. 2006; 314: 126-129https://doi.org/10.1126/science.1129003Google Scholar,2Marincola F.M. Jaffee E.M. Hicklin D.J. Ferrone S. Escape of human solid tumors from T-cell recognition: molecular mechanisms and functional significance.Adv. Immunol. 2000; 74: 181-273https://doi.org/10.1016/s0065-2776(08)60911-6Google Scholar In contrast, CARs containing an extracellular antigen-binding domain, mostly antibody-derived single-chain fragment variable (sFv), could successfully target antigens on tumor cells in an MHC-independent fashion.3Eshhar Z. Waks T. Gross G. Schindler D.G. Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T-cell receptors.Proc. Natl. Acad. Sci. U S A. 1993; 90: 720-724https://doi.org/10.1073/pnas.90.2.720Google Scholar For second-generation CARs, the co-stimulatory signaling domain (e.g., CD28, 4-1BB) and the intracellular region of CD3ζ subunit were included to recapitulate signals of T cell activation.4Maher G. and by a single chimeric Chimeric with signaling CAR T cell therapies targeting or cell antigen for and with a of cytokine release syndrome A. et safety and activity of in a R.E. S. et and of patients with cell after on S. A. A. et of with in and S. J.R. et in or A. D.G. et for patients with or a S. et and of CAR T cell in cell A. A. et a chimeric antigen receptor T-cell in patients with or a Scholar For solid therapeutic in a of patients with CAR T cells in of cell solid and to of CAR T-cell for patients with solid M.S. et T cells in solid and Scholar or TCR T cells that on TCR signaling antitumor activity in and cell carcinoma with lower and of compared with CAR T cells, chimeric TCR natural TCR signaling may be a strategy to efficacy with reduced cytokine S. S. S. G. et of adoptive cell transfer of tumor-infiltrating lymphocytes after for R.A. Yang J.C. Sherry R.M. Dudley M.E. Wunderlich J.R. et regression in patients with cell and using genetically engineered lymphocytes with R.A. Yang J.C. Dudley M.E. Wunderlich J.R. Sherry R.M. et using lymphocytes genetically engineered with an T-cell and with Scholar In et that to could T cells to antigen in an the of sFv-ε endogenous TCR and and with an to the to J.C. of T antigen and of by Scholar a in solid in of hepatocellular in a promising target for Yang S. Z. of T cells to glypican-3 for the of hepatocellular a of tumor with functional of a Scholar early signs of antitumor of GPC3-specific 28ζ CAR T cells to of patients or CRS and CRS et antigen T-cell for hepatocellular results of Scholar To improve the safety and of GPC3-targeted T cell genetically modified T cells with anti-GPC3 linked to CD3ε, which be incorporated into the TCR/CD3 complex sFv-ε TCR in and TCR signaling of In the functional immune the tumor could the and function of adoptive T cells, T cells may memory tumor G. G. chimeric antigen receptor T-cell efficacy in solid A. T cells tumor immune adoptive T cell Scholar as a for cytotoxic T lymphocytes and memory T cells, which antitumor with S. T-cell after with of on memory T cell are by the of in Z. of and efficacy of cells in solid tumors T. and in cells immune cell and cell survival in the Scholar generated sFv-ε T cells interleukin-7 (7sFv-ε) and antitumor in mouse sFv-ε T cells signaling with compared to CAR T human sFv-ε was by the anti-GPC3 and the a was for of 28ζ and BBζ of sFv-ε or CAR were in To the sFv-CD3ε could be incorporated into the human cells sFv-ε or CAR were that of the endogenous TCR subunits CD3ε, and with sFv-ε with CAR that sFv-ε could form an TCR/CD3 complex from CARs, which an CD3ζ signaling sFv-ε could generated anti-GPC3 sFv-ε T or CAR T cells using human T cells. sFv-ε T and CAR T cells presented of T cells with T cells as the and of TCR signaling in sFv-ε T or CAR T cells activated with TCR signaling by of activation the domain of in in the of sFv-ε T and CAR T cells showed of endogenous CD3ζ while CAR T cells demonstrated of antigen sFv-ε CARs induced the of endogenous which that sFv-ε could antigen activation through the entire TCR/CD3 sFv-ε and CARs signaling including the of and For the TCR signaling 28ζ and BBζ CAR T cells showed and of sFv-ε T cells sFv-ε induced of and a of the target of to 28ζ and BBζ CARs after antigen for or 28ζ and BBζ CAR T cells and sFv-ε and T cells, and We further the signaling T cell and S. of A. to chimeric antigen receptor Scholar of memory T cells presented as of T cells, in 28ζ and BBζ CAR T cells, in 28ζ CAR T cells. the sFv-ε T cells a defined by of T cells and 28ζ and BBζ CAR T cells a of immune and compared to sFv-ε T cells and These results that sFv-ε signaling compared to sFv-ε T cells showed competitive antitumor efficacy with reduced cytokine sFv-ε T cells sFv-ε T or CAR T cells were with human cell or of the engineered T cells cells in an to cells CRS associated with CAR T cell by peripheral including tumor and were to be the major of in G. 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A. et and are for syndrome and to CAR T Scholar demonstrated that human sFv-ε T cells to less release of and CAR T cells in models, and was from cells. sFv-ε and 7sFv-ε T cell therapies less and to peripheral relative to BBζ CAR T cell In the of human sFv-ε and CAR T cells in mouse may more of cytokine production could the of T cells relative to CARs and antigen-specific human and murine conventional sFv-ε T cells or superior antitumor efficacy with reduced cytokine production compared with 28ζ and BBζ CAR T cells. GPC3-specific 7sFv-ε T cells further the antitumor immunity while preserving low production of cytokines associated with that could be an and therapeutic strategy for the of solid and of or human anti-GPC3 sFv-ε was of the targeting human and by a 28ζ CAR was S. Z. Z. of GPC3-specific chimeric antigen natural cells for the of hepatocellular Scholar BBζ CAR was of linked by the and region of the and intracellular and CD3ζ signaling domains in or BBζ CAR was into the for murine anti-GPC3 sFv-ε was of and murine by a 7sFv-ε was generated by the murine sFv-ε and a murine 28ζ or BBζ CAR and or domains from or CAR was into the for cells were from the of and with and in with and cells were from the cells were from the of and cells were by and were modified to express chimeric as S. Z. antitumor of and T cells in mouse of hepatocellular Scholar and cells were in with and of sFv-ε or CAR T cells from were by the T cells were activated with for and with and in with human and human T cells were from using the mouse T cell and activated with for T cells were with and in containing and human on T cells was using or with To in T cells, T cells were with and cells were with in with cells for T cells were by To tumor-infiltrating T cells, tumor were into by disruption and with were with and were as and and and cells were with for with and for For the cell was with and for was to the of was by and by with immunoglobulin or To TCR human T cells were and with was by and To the murine T cells for were in with and of was by and by cells were with tumor cells of and for of T cells was by the release in the using the T cells were with cells with or in with for human T cells were activated with cells with or for and murine T cells were with cells for was in with and the were after in or or tumor were using for and or for or to the cells were with for was using the were generated using for was on an and were as which were using and with in and BBζ CAR T cells. of the from and was by 28ζ sFv-ε BBζ of the were in with the of the or were were by of tumor cells in were into with the tumor of and with an of or T cells. For models, tumor cells were in the of were into with the tumor of and with T cells. For models, tumor cells were in in the the tumor were with T cells. In the after the tumors were by 7sFv-ε T cells, were with cells in the and were was by the and and were with and in and a were with and tumor were with or results were using the in was using the to the tumor were and and the endogenous activity was target and the were with targeting and was using the of and by with were using and the results were by of the were using by or were to or and the were using the are presented as IntroductionImproved understanding of tumor immunity over the past decades has led to the accelerated development of adoptive cell therapies (ACTs), including autologous tumor-infiltrating lymphocytes (TILs), T cell receptor (TCR), and chimeric antigen receptor (CAR) engineered T cells. TCR and CAR T cells are generated by modifying peripheral T cells to express a precisely defined antigen-specific receptor. TCR T cells recognize antigenic peptides presented on major histocompatibility complex (MHC) molecules, the function of which may be impaired by reduced or absent MHC on tumor cells or disruption of antigen processing and presentation, and was constrained to patients with a particular MHC allele.1Morgan R.A. Dudley M.E. Wunderlich J.R. Hughes M.S. Yang J.C. Sherry R.M. Royal R.E. Topalian S.L. Kammula U.S. Restifo N.P. et al.Cancer regression in patients after transfer of genetically engineered lymphocytes.Science. 2006; 314: 126-129https://doi.org/10.1126/science.1129003Google Scholar,2Marincola F.M. Jaffee E.M. Hicklin D.J. Ferrone S. Escape of human solid tumors from T-cell recognition: molecular mechanisms and functional significance.Adv. Immunol. 2000; 74: 181-273https://doi.org/10.1016/s0065-2776(08)60911-6Google Scholar In contrast, CARs containing an extracellular antigen-binding domain, mostly antibody-derived single-chain fragment variable (sFv), could successfully target antigens on tumor cells in an MHC-independent fashion.3Eshhar Z. Waks T. Gross G. Schindler D.G. Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T-cell receptors.Proc. Natl. Acad. Sci. U S A. 1993; 90: 720-724https://doi.org/10.1073/pnas.90.2.720Google Scholar For second-generation CARs, the co-stimulatory signaling domain (e.g., CD28, 4-1BB) and the intracellular region of CD3ζ subunit were included to recapitulate signals of T cell activation.4Maher G. and by a single chimeric Chimeric with signaling CAR T cell therapies targeting or cell antigen for and with a of cytokine release syndrome A. et safety and activity of in a R.E. S. et and of patients with cell after on S. A. A. et of with in and S. J.R. et in or A. D.G. et for patients with or a S. et and of CAR T cell in cell A. A. et a chimeric antigen receptor T-cell in patients with or a Scholar For solid therapeutic in a of patients with CAR T cells in of cell solid and to of CAR T-cell for patients with solid M.S. et T cells in solid and Scholar or TCR T cells that on TCR signaling antitumor activity in and cell carcinoma with lower and of compared with CAR T cells, chimeric TCR natural TCR signaling may be a strategy to efficacy with reduced cytokine S. S. S. G. et of adoptive cell transfer of tumor-infiltrating lymphocytes after for R.A. Yang J.C. Sherry R.M. Dudley M.E. Wunderlich J.R. et regression in patients with cell and using genetically engineered lymphocytes with R.A. Yang J.C. Dudley M.E. Wunderlich J.R. Sherry R.M. et using lymphocytes genetically engineered with an T-cell and with Scholar In et that to could T cells to antigen in an the of sFv-ε endogenous TCR and and with an to the to J.C. of T antigen and of by Scholar a in solid in of hepatocellular in a promising target for Yang S. Z. of T cells to glypican-3 for the of hepatocellular a of tumor with functional of a Scholar early signs of antitumor of GPC3-specific 28ζ CAR T cells to of patients or CRS and CRS et antigen T-cell for hepatocellular results of Scholar To improve the safety and of GPC3-targeted T cell genetically modified T cells with anti-GPC3 linked to CD3ε, which be incorporated into the TCR/CD3 complex sFv-ε TCR in and TCR signaling of In the functional immune the tumor could the and function of adoptive T cells, T cells may memory tumor G. G. chimeric antigen receptor T-cell efficacy in solid A. T cells tumor immune adoptive T cell Scholar as a for cytotoxic T lymphocytes and memory T cells, which antitumor with S. T-cell after with of on memory T cell are by the of in Z. of and efficacy of cells in solid tumors T. and in cells immune cell and cell survival in the Scholar generated sFv-ε T cells interleukin-7 (7sFv-ε) and antitumor in mouse

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