Chimeric Antigen Receptor T cells targeting malignancies expressing CD19 (CAR19) have been widely... more Chimeric Antigen Receptor T cells targeting malignancies expressing CD19 (CAR19) have been widely successful, with products approved to treat B cell lymphomas (NHL) and B cell leukemia. A major limitation of CAR19 therapy is the steep relapse rate within 6 months of treatment, often due to the loss or diminution of tumor cell CD19 expression. ALETA-001 is a CAR-T Engager protein that contains the CD19 extracellular domain (ECD), an anti-CD20 VHH, and an anti-albumin VHH for half-life extension. When combined with CAR19 T cells, ALETA-001 triggers cytotoxicity through CD19 bound to CD20, thus increasing total target antigen density and preventing relapse due to loss of CD19 expression. ALETA-001 will enter Cancer Research UK-sponsored Phase 1/2 clinical trials in CAR19-treated NHL patients next year. We made novel variants of ALETA-001 including CAR-T Engagers with alternative anti-CD20 modules and CAR-T Engagers that target both CD20 and a second B cell cancer antigen. Further, we extended our technology to target Acute Myeloid Leukemia (AML), using novel antigen binding domains and immunomodulatory functional domains. Site-directed mutagenesis was used to make ALETA-001 variants. We mutated the complementary determining regions, CDR2 and/or CDR3, within the anti-CD20 VHH. The novel CAR-T Engager Proteins were evaluated for binding to CD19-negative/CD20-positive lymphoma cells and for cytotoxicity against those cells in the presence of CAR19 T cells. Further, CAR-T Engagers were made that contained novel CD20 binding domains and binding domains to other B cell antigens. In addition, we created a novel CAR-T engager that contains the CD19 ECD and binding domains to two AML antigens, plus an immunomodulatory domain. This novel CAR-T engager is designed to treat CD19-positive Mixed Phenotype Leukemia (MPL) and other forms of AML. We evaluated the series of CDR mutations in the ALETA-001 anti-CD20 VHH. A spectrum of activities was observed, with mutations that retained full CD20 binding and cytotoxic activity to those with partial or no activity. A series of constructs with distinct anti-CD20 binding domains were also evaluated, with some found to be equipotent with ALETA-001. Constructs binding both CD20 and a second B cell tumor antigen were prepared and assayed and found to have potent activity against either or both antigens. Finally, CAR-T Engagers directed to AML antigens were able to bind each antigen individually, and mediated potent toxicity with the addition of CAR19 T cells. We conclude that the CAR-T Engager platform offers robust and modular anti-tumor functionality, featuring potent multi-antigen targeting for diverse indications. Finally, we can incorporate other useful immunomodulatory activities into Engager proteins. Citation Format: Paul D. Rennert, Lihe Su, Lan Wu, Roy R. Lobb, Christine Ambrose. CAR-T engager proteins for the treatment of B cell cancers and acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2759.
Introduction: CAR T-cells targeting CD19 (CAR19s) can eradicate B cell leukemias and lymphomas. T... more Introduction: CAR T-cells targeting CD19 (CAR19s) can eradicate B cell leukemias and lymphomas. The effectiveness of CAR19s is linked to their robust expansion properties but also their long-term persistence. Persistence is maintained by normal CD19+ B cells: a non-tumor dependent, self-renewing source of antigen. In this manner, CAR19s are quite unique. We have re-engineered CAR19s to secrete a wide variety of retargeting fusion proteins (FPs) by encoding expression cassettes downstream of the CAR sequence in lentiviral vectors. By hijacking CAR19s, we utilize their inherent persistence properties. By designing multispecific FP, we directly counter the clinically critical issues of tumor heterogeneity and antigen loss. Experimental Procedures: A lentiviral vector with an MCSV promoter was used to express the CAR19 construct and FPs. FPs and multispecific-FPs were designed to encode the extracellular domain of the CD19 protein, followed by one or two scFv sequences, separated from the CAR sequence by a P2A cleavage site, a design termed IMPACTtm (Integrated Modular Proteins for Adoptive Cell Therapy). The FPs therefore consist of the CD19 extracellular domain linked to one or more scFvs. The FPs redirect CAR19 T-cell cytotoxic activity to any tumor antigen of interest by coating that antigen with CD19 via the scFv. Further, multiple antigens can be coated with CD19 by encoding multiple scFv in the FP. Hijacked CAR19s therefore serve as a platform for targeting diverse antigens. Results: Here we describe one example in detail, focusing on Her2+ solid tumors. The CD19/anti-Her2 FP was highly potent in cytotoxicity assays targeting Her2+/CD19- solid tumor cell lines. The concentration of fusion protein required to reduce tumor cell number by 50% was 10 pM (0.7 ng/ml). Primary donor T-cells transduced with the CAR19 - CD19/anti-Her2 FP lentiviral vector secreted > 20 ng/ml of FP in cell culture. Cytotoxic activity of this redirected CAR19 against Her2+ SKOV3 tumor cells was demonstrated in vitro and in vivo. CD19-mediated persistence was demonstrated in serial restimulation assays. A bispecific FP containing CD19 linked to anti-Her2 and anti-EGFR scFv had specific activity against both antigens with a potency of 0.75 pM. For each antigen, the potent cytotoxicity was specifically mediated by the secreted fusion protein. Additional program examples of multispecific targeting for diverse hematologic and solid tumor types will be shown. Conclusions: The IMPACT platform addresses critical issues in cell therapy including CAR persistence, antigen escape and antigen heterogeneity, and provides important solutions for treating both hematologic and solid tumors. The potency of redirected cytotoxicity supports clinical development of CAR19/IMPACT programs, four of which are now ready for IND enabling studies. Citation Format: Paul Rennert, Fay Dufort, Lihe Su, Lan Wu, Alyssa Birt, Christine Ambrose, Roy Lobb. Hijacking CAR19 T-cells for use in targeting diverse hematopoietic and solid tumors [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A040.
The V3- and C4-coding regions in the envelope gene of the infectious, pathogenic SIVmac239 clone ... more The V3- and C4-coding regions in the envelope gene of the infectious, pathogenic SIVmac239 clone were replaced by the corresponding HIV-1 sequences. Viral particles were obtained after transfection of COS-1 cells. Chimeric SIVmac constructs were not replication competent in the human T cell lines CEMx174, AA2, H9, and MT-4 or in primary cultures of rhesus monkey peripheral blood mononuclear cells. The lack of infectivity of the hybrid constructs was associated with inefficient proteolytic processing of the gp160env precursor. Unlike the modular nature of some proteins, gp120 appears to be a highly ordered molecule whose function is dependent on the integration of many discontinuous, interactive regions.
Both lymphotoxin-alpha (LTalpha)-deficient mice and alymphoplasia (aly) mice, a natural mutant st... more Both lymphotoxin-alpha (LTalpha)-deficient mice and alymphoplasia (aly) mice, a natural mutant strain, manifest a quite similar phenotype: lack of lymph nodes (LN) and Peyer's patches (PP), with disturbed spleen architecture. The mechanisms underlying the defective lymphoid organogenesis in these mice were investigated by generating aggregation chimeras; ex vivo fused morulae were implanted into pseudo-pregnant host females and allowed to develop to term. Chimeric mice between LTalpha-deficient mice and wild-type mice restored LN and PP almost completely, suggesting that LTalpha expressed by circulating bone marrow-derived cells is essential for lymphoid organogenesis as well as for organization of spleen architecture. By contrast, chimeric mice between aly mice and wild-type mice showed only limited restoration of LN and PP. This suggests that the putative aly gene product does not act as a circulating ligand for lymphoid organogenesis, like LTalpha. Rather, abnormal development of lymphoid organs in aly mice seems most likely due to the defective development of the incipient stromal cells of the LN and PP. Supporting this hypothesis, up-regulation of VCAM-1 on aly mouse embryonic fibroblasts by signals through LTbetaR, which is exclusively expressed by nonlymphoid cells, was disturbed. These studies demonstrate that LTalpha and the putative aly gene product together control lymphoid organogenesis with a close mechanistic relationship in their biochemical pathways through governing the distinct cellular compartments, the former acting as a circulating ligand and the latter as a LTbetaR-signaling molecule expressed by the stroma of the lymphoid organs.
Introduction. CAR-CD19 T cell therapeutics are approved to treat B cell leukemias and lymphomas. ... more Introduction. CAR-CD19 T cell therapeutics are approved to treat B cell leukemias and lymphomas. Previously we showed that providing CAR19 T cells with a retargeting fusion protein (FP), consisting of soluble CD19 protein linked to an scFv, redirects CAR19 T cell cytotoxic activity to other tumor antigens. Further, exposure of CAR19 T cells to normal or malignant B cells expressing CD19 improves effector function and phenotype, due to the provision of costimulatory signals. Such signals are missing when CAR-T cells engage antigens on solid tumor cells. In addition, lack of sufficient antigen for expansion and persistence is a critical issue for CAR T therapeutics targeting solid tumors in the clinical setting. We have redirected CAR19 T cells to other tumor antigens via novel FP and bispecific FP (biFP) expression cassettes. CAR19 T cells that express FP or biFP solve critical issues in the cell therapy field by 1) promoting CAR19 T cell expansion, efficacy and persistence by engaging CD19 on B cells; 2) addressing antigen heterogeneity/escape in hematologic malignancies and solid tumors. FP- and biFP-mediated cytotoxicity is extremely potent at pM concentrations. Here we provide examples of in vitro and in vivo modeling to characterize the activity of the CAR19 T cells that are retargeted to kill other tumor types. Experimental Procedures. A CAR-CD19 construct was created as the recipient for FP and biFP expression cassettes, in a lentiviral vector with an MCSV promoter. The CAR consists of the FMC63 scFv, a stalk, TM and CD28 and/or 4-1BB cytoplasmic signaling domains, and the CD3ε cytoplasmic signaling domain. FP and biFP cassettes were designed to encode the extracellular domain of the CD19 protein, followed by an scFv or VHH to one (FP) or two (biFP) antigens, separated from the CAR sequence by a P2A cleavage site. Results. Flow cytometry and binding analyses demonstrated FP and bi-FP-mediated bridging between target tumor cells and CAR19 T cells. In vitro cytotoxicity studies showed robust redirected killing of antigen+ (CD19-) tumor cells by CAR19 T cells. This cytotoxicity is specifically mediated by the secreted FP or biFP and correlated with antigen affinity. The efficiency of cytotoxicity was enhanced by the presence of CD19+ B cells. Tumor xenograft models using Her2+ and CD38+ tumor lines demonstrated effective tumor killing in vivo, with or without the presence of CD19+ cells. Conclusions. Redirected CAR19 T cells potently kill diverse tumor cells in vitro and in vivo. The strategy of redirecting CAR19 T cells to other tumor cell types by encoding novel FP and biFP expression cassettes solves critical issues in cell therapy by supporting CAR19 T cell activation and expansion and by providing a simple and modular multi-antigen targeting system. Further technology refinements include the creation of inducible promoters for FP expression, adding anti-PD-L1 activity, and providing cytokine support. Citation Format: Paul Rennert, Fay Dufort, Lihe Su, Lan Wu, Alyssa Birt, Roy Lobb, Christine Ambrose. CAR19 T cells secreting antigen-retargeting fusion proteins have remarkable potency against diverse tumor types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2569.
Previous studies have shown that steroid hormones reduce concentrations of nerve growth factor (N... more Previous studies have shown that steroid hormones reduce concentrations of nerve growth factor (NGF) in medium conditioned by L-929 fibroblasts (L cells). In this study, we extend those observations and have measured in L cells the effects of hormone treatment on mRNA encoding NGF. L Cells were grown for 3 days in the presence or absence of hormones. NGF in conditioned medium was measured by NGF RIA; NGF mRNA was measured in cell extracts by Northern blot analysis. Cortisone reduced NGF levels in conditioned medium below the limit of detection of the RIA (less than 10% of control values) with an ED50 of 5 X 10(-9) M; NGF mRNA was reduced to 12% of control levels with an ED50 of 1 X 10(-8) M. Reductions in mRNA were maximal within 3 h and were completely reversed 12 h after removal of the hormone. Levels of NGF in conditioned medium were also undetectable in cultures treated with testosterone, and mRNA levels were reduced by 80%; the ED50 for both effects was 4 X 10(-9) M. Aldosterone (1 X 10(-6) M) reduced NGF to below detectable levels and NGF mRNA by 70%. Progesterone and thyroid hormone had no effect on NGF or NGF mRNA. 17 beta-Estradiol reduced levels of NGF in medium by 50%, but had no detectable effect on levels of NGF mRNA. These results suggest that cortisone, testosterone, and aldosterone decrease NGF levels in L cell-conditioned medium by reducing the cellular content of NGF mRNA.
Chimeric Antigen Receptor T cells targeting malignancies expressing CD19 (CAR19) have been widely... more Chimeric Antigen Receptor T cells targeting malignancies expressing CD19 (CAR19) have been widely successful, with products approved to treat B cell lymphomas (NHL) and B cell leukemia. A major limitation of CAR19 therapy is the steep relapse rate within 6 months of treatment, often due to the loss or diminution of tumor cell CD19 expression. ALETA-001 is a CAR-T Engager protein that contains the CD19 extracellular domain (ECD), an anti-CD20 VHH, and an anti-albumin VHH for half-life extension. When combined with CAR19 T cells, ALETA-001 triggers cytotoxicity through CD19 bound to CD20, thus increasing total target antigen density and preventing relapse due to loss of CD19 expression. ALETA-001 will enter Cancer Research UK-sponsored Phase 1/2 clinical trials in CAR19-treated NHL patients next year. We made novel variants of ALETA-001 including CAR-T Engagers with alternative anti-CD20 modules and CAR-T Engagers that target both CD20 and a second B cell cancer antigen. Further, we extended our technology to target Acute Myeloid Leukemia (AML), using novel antigen binding domains and immunomodulatory functional domains. Site-directed mutagenesis was used to make ALETA-001 variants. We mutated the complementary determining regions, CDR2 and/or CDR3, within the anti-CD20 VHH. The novel CAR-T Engager Proteins were evaluated for binding to CD19-negative/CD20-positive lymphoma cells and for cytotoxicity against those cells in the presence of CAR19 T cells. Further, CAR-T Engagers were made that contained novel CD20 binding domains and binding domains to other B cell antigens. In addition, we created a novel CAR-T engager that contains the CD19 ECD and binding domains to two AML antigens, plus an immunomodulatory domain. This novel CAR-T engager is designed to treat CD19-positive Mixed Phenotype Leukemia (MPL) and other forms of AML. We evaluated the series of CDR mutations in the ALETA-001 anti-CD20 VHH. A spectrum of activities was observed, with mutations that retained full CD20 binding and cytotoxic activity to those with partial or no activity. A series of constructs with distinct anti-CD20 binding domains were also evaluated, with some found to be equipotent with ALETA-001. Constructs binding both CD20 and a second B cell tumor antigen were prepared and assayed and found to have potent activity against either or both antigens. Finally, CAR-T Engagers directed to AML antigens were able to bind each antigen individually, and mediated potent toxicity with the addition of CAR19 T cells. We conclude that the CAR-T Engager platform offers robust and modular anti-tumor functionality, featuring potent multi-antigen targeting for diverse indications. Finally, we can incorporate other useful immunomodulatory activities into Engager proteins. Citation Format: Paul D. Rennert, Lihe Su, Lan Wu, Roy R. Lobb, Christine Ambrose. CAR-T engager proteins for the treatment of B cell cancers and acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2759.
Introduction: CAR T-cells targeting CD19 (CAR19s) can eradicate B cell leukemias and lymphomas. T... more Introduction: CAR T-cells targeting CD19 (CAR19s) can eradicate B cell leukemias and lymphomas. The effectiveness of CAR19s is linked to their robust expansion properties but also their long-term persistence. Persistence is maintained by normal CD19+ B cells: a non-tumor dependent, self-renewing source of antigen. In this manner, CAR19s are quite unique. We have re-engineered CAR19s to secrete a wide variety of retargeting fusion proteins (FPs) by encoding expression cassettes downstream of the CAR sequence in lentiviral vectors. By hijacking CAR19s, we utilize their inherent persistence properties. By designing multispecific FP, we directly counter the clinically critical issues of tumor heterogeneity and antigen loss. Experimental Procedures: A lentiviral vector with an MCSV promoter was used to express the CAR19 construct and FPs. FPs and multispecific-FPs were designed to encode the extracellular domain of the CD19 protein, followed by one or two scFv sequences, separated from the CAR sequence by a P2A cleavage site, a design termed IMPACTtm (Integrated Modular Proteins for Adoptive Cell Therapy). The FPs therefore consist of the CD19 extracellular domain linked to one or more scFvs. The FPs redirect CAR19 T-cell cytotoxic activity to any tumor antigen of interest by coating that antigen with CD19 via the scFv. Further, multiple antigens can be coated with CD19 by encoding multiple scFv in the FP. Hijacked CAR19s therefore serve as a platform for targeting diverse antigens. Results: Here we describe one example in detail, focusing on Her2+ solid tumors. The CD19/anti-Her2 FP was highly potent in cytotoxicity assays targeting Her2+/CD19- solid tumor cell lines. The concentration of fusion protein required to reduce tumor cell number by 50% was 10 pM (0.7 ng/ml). Primary donor T-cells transduced with the CAR19 - CD19/anti-Her2 FP lentiviral vector secreted > 20 ng/ml of FP in cell culture. Cytotoxic activity of this redirected CAR19 against Her2+ SKOV3 tumor cells was demonstrated in vitro and in vivo. CD19-mediated persistence was demonstrated in serial restimulation assays. A bispecific FP containing CD19 linked to anti-Her2 and anti-EGFR scFv had specific activity against both antigens with a potency of 0.75 pM. For each antigen, the potent cytotoxicity was specifically mediated by the secreted fusion protein. Additional program examples of multispecific targeting for diverse hematologic and solid tumor types will be shown. Conclusions: The IMPACT platform addresses critical issues in cell therapy including CAR persistence, antigen escape and antigen heterogeneity, and provides important solutions for treating both hematologic and solid tumors. The potency of redirected cytotoxicity supports clinical development of CAR19/IMPACT programs, four of which are now ready for IND enabling studies. Citation Format: Paul Rennert, Fay Dufort, Lihe Su, Lan Wu, Alyssa Birt, Christine Ambrose, Roy Lobb. Hijacking CAR19 T-cells for use in targeting diverse hematopoietic and solid tumors [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A040.
The V3- and C4-coding regions in the envelope gene of the infectious, pathogenic SIVmac239 clone ... more The V3- and C4-coding regions in the envelope gene of the infectious, pathogenic SIVmac239 clone were replaced by the corresponding HIV-1 sequences. Viral particles were obtained after transfection of COS-1 cells. Chimeric SIVmac constructs were not replication competent in the human T cell lines CEMx174, AA2, H9, and MT-4 or in primary cultures of rhesus monkey peripheral blood mononuclear cells. The lack of infectivity of the hybrid constructs was associated with inefficient proteolytic processing of the gp160env precursor. Unlike the modular nature of some proteins, gp120 appears to be a highly ordered molecule whose function is dependent on the integration of many discontinuous, interactive regions.
Both lymphotoxin-alpha (LTalpha)-deficient mice and alymphoplasia (aly) mice, a natural mutant st... more Both lymphotoxin-alpha (LTalpha)-deficient mice and alymphoplasia (aly) mice, a natural mutant strain, manifest a quite similar phenotype: lack of lymph nodes (LN) and Peyer's patches (PP), with disturbed spleen architecture. The mechanisms underlying the defective lymphoid organogenesis in these mice were investigated by generating aggregation chimeras; ex vivo fused morulae were implanted into pseudo-pregnant host females and allowed to develop to term. Chimeric mice between LTalpha-deficient mice and wild-type mice restored LN and PP almost completely, suggesting that LTalpha expressed by circulating bone marrow-derived cells is essential for lymphoid organogenesis as well as for organization of spleen architecture. By contrast, chimeric mice between aly mice and wild-type mice showed only limited restoration of LN and PP. This suggests that the putative aly gene product does not act as a circulating ligand for lymphoid organogenesis, like LTalpha. Rather, abnormal development of lymphoid organs in aly mice seems most likely due to the defective development of the incipient stromal cells of the LN and PP. Supporting this hypothesis, up-regulation of VCAM-1 on aly mouse embryonic fibroblasts by signals through LTbetaR, which is exclusively expressed by nonlymphoid cells, was disturbed. These studies demonstrate that LTalpha and the putative aly gene product together control lymphoid organogenesis with a close mechanistic relationship in their biochemical pathways through governing the distinct cellular compartments, the former acting as a circulating ligand and the latter as a LTbetaR-signaling molecule expressed by the stroma of the lymphoid organs.
Introduction. CAR-CD19 T cell therapeutics are approved to treat B cell leukemias and lymphomas. ... more Introduction. CAR-CD19 T cell therapeutics are approved to treat B cell leukemias and lymphomas. Previously we showed that providing CAR19 T cells with a retargeting fusion protein (FP), consisting of soluble CD19 protein linked to an scFv, redirects CAR19 T cell cytotoxic activity to other tumor antigens. Further, exposure of CAR19 T cells to normal or malignant B cells expressing CD19 improves effector function and phenotype, due to the provision of costimulatory signals. Such signals are missing when CAR-T cells engage antigens on solid tumor cells. In addition, lack of sufficient antigen for expansion and persistence is a critical issue for CAR T therapeutics targeting solid tumors in the clinical setting. We have redirected CAR19 T cells to other tumor antigens via novel FP and bispecific FP (biFP) expression cassettes. CAR19 T cells that express FP or biFP solve critical issues in the cell therapy field by 1) promoting CAR19 T cell expansion, efficacy and persistence by engaging CD19 on B cells; 2) addressing antigen heterogeneity/escape in hematologic malignancies and solid tumors. FP- and biFP-mediated cytotoxicity is extremely potent at pM concentrations. Here we provide examples of in vitro and in vivo modeling to characterize the activity of the CAR19 T cells that are retargeted to kill other tumor types. Experimental Procedures. A CAR-CD19 construct was created as the recipient for FP and biFP expression cassettes, in a lentiviral vector with an MCSV promoter. The CAR consists of the FMC63 scFv, a stalk, TM and CD28 and/or 4-1BB cytoplasmic signaling domains, and the CD3ε cytoplasmic signaling domain. FP and biFP cassettes were designed to encode the extracellular domain of the CD19 protein, followed by an scFv or VHH to one (FP) or two (biFP) antigens, separated from the CAR sequence by a P2A cleavage site. Results. Flow cytometry and binding analyses demonstrated FP and bi-FP-mediated bridging between target tumor cells and CAR19 T cells. In vitro cytotoxicity studies showed robust redirected killing of antigen+ (CD19-) tumor cells by CAR19 T cells. This cytotoxicity is specifically mediated by the secreted FP or biFP and correlated with antigen affinity. The efficiency of cytotoxicity was enhanced by the presence of CD19+ B cells. Tumor xenograft models using Her2+ and CD38+ tumor lines demonstrated effective tumor killing in vivo, with or without the presence of CD19+ cells. Conclusions. Redirected CAR19 T cells potently kill diverse tumor cells in vitro and in vivo. The strategy of redirecting CAR19 T cells to other tumor cell types by encoding novel FP and biFP expression cassettes solves critical issues in cell therapy by supporting CAR19 T cell activation and expansion and by providing a simple and modular multi-antigen targeting system. Further technology refinements include the creation of inducible promoters for FP expression, adding anti-PD-L1 activity, and providing cytokine support. Citation Format: Paul Rennert, Fay Dufort, Lihe Su, Lan Wu, Alyssa Birt, Roy Lobb, Christine Ambrose. CAR19 T cells secreting antigen-retargeting fusion proteins have remarkable potency against diverse tumor types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2569.
Previous studies have shown that steroid hormones reduce concentrations of nerve growth factor (N... more Previous studies have shown that steroid hormones reduce concentrations of nerve growth factor (NGF) in medium conditioned by L-929 fibroblasts (L cells). In this study, we extend those observations and have measured in L cells the effects of hormone treatment on mRNA encoding NGF. L Cells were grown for 3 days in the presence or absence of hormones. NGF in conditioned medium was measured by NGF RIA; NGF mRNA was measured in cell extracts by Northern blot analysis. Cortisone reduced NGF levels in conditioned medium below the limit of detection of the RIA (less than 10% of control values) with an ED50 of 5 X 10(-9) M; NGF mRNA was reduced to 12% of control levels with an ED50 of 1 X 10(-8) M. Reductions in mRNA were maximal within 3 h and were completely reversed 12 h after removal of the hormone. Levels of NGF in conditioned medium were also undetectable in cultures treated with testosterone, and mRNA levels were reduced by 80%; the ED50 for both effects was 4 X 10(-9) M. Aldosterone (1 X 10(-6) M) reduced NGF to below detectable levels and NGF mRNA by 70%. Progesterone and thyroid hormone had no effect on NGF or NGF mRNA. 17 beta-Estradiol reduced levels of NGF in medium by 50%, but had no detectable effect on levels of NGF mRNA. These results suggest that cortisone, testosterone, and aldosterone decrease NGF levels in L cell-conditioned medium by reducing the cellular content of NGF mRNA.
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