Pub Date : 2019-02-01DOI: 10.1158/2326-6074.CRICIMTEATIAACR18-A045
Dongrui Wang, Vanessa D. Jonsson, Sarah L Wright, W. Chang, Xin Yang, R. Starr, Alfonso Brito, B. Aguilar, A. Sarkissian, L. Weng, S. Forman, M. Barish, Christine E. Brown
Glioblastoma (GBM) is the most common type of primary brain tumor, with the standard therapy only modestly improving the prognosis, highlighting the necessity to develop advanced treatments. We and others have established the platform to potentiate immune response against GBMs using chimeric antigen receptor (CAR) engineered T-cells. Specifically, we have shown that intracranial administration of CAR T-cells can be well tolerated in patients with recurrent GBMs, together with some early clinical evidence of antitumor response. However, CAR T-cell therapy against GBMs is complicated by the inter- and intratumoral heterogeneity, while the single-targeting therapies only respond to a subset of tumor cells. The development of new CAR therapy would thus aim for targeting a wider range of tumor cells and bypassing antigen escape. Here, we took an approach different from conventional strategies of tumor antigen discovery, exploiting the tumor-binding potential of a natural peptide to develop CAR T-cells that broadly target GBMs. Chlorotoxin (CLTX) is a 36-amino acid peptide with demonstrated GBM-binding capability. Inspired by the utilization of CLTX in GBM tumor imaging, we used a fluorescence-conjugated CLTX to screen the freshly-dispersed primary GBM cells and patient-derived GBM neurospheres, and found that CLTX binding was more homogeneous than the expression of other GBM-associated antigens including EGFR, HER2 and IL13Rα2. Although CLTX has limited inhibitory effect on GBM growth, its broad binding to GBM cells illustrates the potential to be conjugated with a cytotoxic agent. Therefore, we generated CAR T-cells bearing CLTX as the antigen targeting domain. CLTX-CAR T-cells were able to get activated after stimulating with GBM cells, as indicated by their degranulation, cytokine production and immuno-synapse formation. Modification of CAR constructs revealed that CLTX-CAR T-cells with CD28 costimulatory signal exhibited potent effector activity, while the 4-1BB costimulation resulted in inadequate CAR activation. In both in vitro and in vivo models, CLTX-CAR T-cells effectively eliminated GBM cells and tumors, including the ones with no/low expression of EGFR, HER2 and IL13Rα2. Importantly, CLTX peptide exhibited negligible binding to a panel of normal cells from neural and other tissues, and CLTX-CAR T-cells showed no off-target effect against normal organs in tumor-bearing mouse models. Screening on patient-derived GBM neurospheres, we discovered that the expression of metalloproteinase (MMP)-2 on targeT-cells was correlated with the effector function of CLTX-CAR T-cells. Further, the antitumor function of CLTX-CAR T-cells was severely diminished against GBMs with MMP-2 knockdown. Consistent with the cytotoxicity of CLTX-CAR T-cells, MMP-2 expression was also present in a subgroup of GBM cells with undetectable levels of EGFR, HER2 and IL13Rα2 expression. Our results demonstrate for the first time that a peptide toxin can be successfully used a
胶质母细胞瘤(Glioblastoma, GBM)是最常见的原发性脑肿瘤类型，标准治疗仅能适度改善预后，突出了开发先进治疗方法的必要性。我们和其他人已经建立了一个平台，利用嵌合抗原受体(CAR)工程t细胞增强对GBMs的免疫反应。具体来说，我们已经证明CAR - t细胞颅内给药对复发性GBMs患者具有良好的耐受性，并有一些早期临床证据显示抗肿瘤反应。然而，针对GBMs的CAR -t细胞治疗由于肿瘤间和肿瘤内的异质性而变得复杂，而单靶向治疗仅对一部分肿瘤细胞有反应。因此，新的CAR疗法的发展将瞄准更大范围的肿瘤细胞，并绕过抗原逃逸。在这里，我们采用了一种不同于传统肿瘤抗原发现策略的方法，利用天然肽的肿瘤结合潜力来开发广泛靶向GBMs的CAR - t细胞。氯毒素(CLTX)是一种含有36个氨基酸的肽，具有与gbm结合的能力。受CLTX在GBM肿瘤成像中的应用启发，我们使用荧光偶联的CLTX筛选新分散的原代GBM细胞和患者来源的GBM神经球，发现CLTX结合比其他GBM相关抗原(包括EGFR、HER2和IL13Rα2)的表达更为均匀。尽管CLTX对GBM生长的抑制作用有限，但其与GBM细胞的广泛结合表明其与细胞毒性药物结合的潜力。因此，我们产生了以CLTX为抗原靶向结构域的CAR - t细胞。CLTX-CAR - t细胞在GBM细胞刺激后能够被激活，这表明它们的脱颗粒、细胞因子的产生和免疫突触的形成。CAR结构的修饰表明，CD28共刺激信号的CLTX-CAR t细胞表现出强大的效应活性，而4-1BB共刺激导致CAR激活不足。在体外和体内模型中，CLTX-CAR - t细胞均能有效清除GBM细胞和肿瘤，包括EGFR、HER2和IL13Rα2无表达或低表达的肿瘤。重要的是，在荷瘤小鼠模型中，CLTX肽与来自神经和其他组织的正常细胞的结合可以忽略不计，CLTX- car -t细胞对正常器官没有脱靶作用。筛选患者源性GBM神经球，我们发现靶细胞上金属蛋白酶(MMP)-2的表达与CLTX-CAR - t细胞的效应功能相关。此外，CLTX-CAR - t细胞对MMP-2敲低的GBMs的抗肿瘤功能严重减弱。与CLTX-CAR - t细胞的细胞毒性一致，在EGFR、HER2和IL13Rα2表达水平未检测到的GBM细胞亚组中也存在MMP-2表达。我们的研究结果首次证明，肽毒素可以成功地用作CAR的肿瘤靶向结构域，以高效率和选择性消除GBMs。CLTX-CAR有可能限制GBM的异质性，并弥补目前针对实体瘤的CAR - t细胞疗法。引文格式:王东瑞，Vanessa Jonsson, Sarah Wright，张文忠，杨欣，Renate Starr, Alfonso Brito, Brenda Aguilar, Aniee Sarkissian, wlihong, Stephen J Forman, Michael E Barish, Christine E. Brown氯毒素重定向t细胞特异性和有效靶向胶质母细胞瘤[摘要]。第四届CRI-CIMT-EATI-AACR国际癌症免疫治疗会议:将科学转化为生存;2018年9月30日至10月3日;纽约，纽约。费城(PA): AACR;癌症免疫学杂志2019;7(2增刊):摘要nr A045。
{"title":"Abstract A045: Chlorotoxin redirects T-cells for specific and effective targeting against glioblastomas","authors":"Dongrui Wang, Vanessa D. Jonsson, Sarah L Wright, W. Chang, Xin Yang, R. Starr, Alfonso Brito, B. Aguilar, A. Sarkissian, L. Weng, S. Forman, M. Barish, Christine E. Brown","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A045","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A045","url":null,"abstract":"Glioblastoma (GBM) is the most common type of primary brain tumor, with the standard therapy only modestly improving the prognosis, highlighting the necessity to develop advanced treatments. We and others have established the platform to potentiate immune response against GBMs using chimeric antigen receptor (CAR) engineered T-cells. Specifically, we have shown that intracranial administration of CAR T-cells can be well tolerated in patients with recurrent GBMs, together with some early clinical evidence of antitumor response. However, CAR T-cell therapy against GBMs is complicated by the inter- and intratumoral heterogeneity, while the single-targeting therapies only respond to a subset of tumor cells. The development of new CAR therapy would thus aim for targeting a wider range of tumor cells and bypassing antigen escape. Here, we took an approach different from conventional strategies of tumor antigen discovery, exploiting the tumor-binding potential of a natural peptide to develop CAR T-cells that broadly target GBMs. Chlorotoxin (CLTX) is a 36-amino acid peptide with demonstrated GBM-binding capability. Inspired by the utilization of CLTX in GBM tumor imaging, we used a fluorescence-conjugated CLTX to screen the freshly-dispersed primary GBM cells and patient-derived GBM neurospheres, and found that CLTX binding was more homogeneous than the expression of other GBM-associated antigens including EGFR, HER2 and IL13Rα2. Although CLTX has limited inhibitory effect on GBM growth, its broad binding to GBM cells illustrates the potential to be conjugated with a cytotoxic agent. Therefore, we generated CAR T-cells bearing CLTX as the antigen targeting domain. CLTX-CAR T-cells were able to get activated after stimulating with GBM cells, as indicated by their degranulation, cytokine production and immuno-synapse formation. Modification of CAR constructs revealed that CLTX-CAR T-cells with CD28 costimulatory signal exhibited potent effector activity, while the 4-1BB costimulation resulted in inadequate CAR activation. In both in vitro and in vivo models, CLTX-CAR T-cells effectively eliminated GBM cells and tumors, including the ones with no/low expression of EGFR, HER2 and IL13Rα2. Importantly, CLTX peptide exhibited negligible binding to a panel of normal cells from neural and other tissues, and CLTX-CAR T-cells showed no off-target effect against normal organs in tumor-bearing mouse models. Screening on patient-derived GBM neurospheres, we discovered that the expression of metalloproteinase (MMP)-2 on targeT-cells was correlated with the effector function of CLTX-CAR T-cells. Further, the antitumor function of CLTX-CAR T-cells was severely diminished against GBMs with MMP-2 knockdown. Consistent with the cytotoxicity of CLTX-CAR T-cells, MMP-2 expression was also present in a subgroup of GBM cells with undetectable levels of EGFR, HER2 and IL13Rα2 expression. Our results demonstrate for the first time that a peptide toxin can be successfully used a","PeriodicalId":254712,"journal":{"name":"Genetically Engineered T-cells","volume":"89 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"122656286","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-02-01DOI: 10.1158/2326-6074.CRICIMTEATIAACR18-A043
D. Stenger, Tanja A. Stief, Theresa Käuferle, Semjon Willier, F. Rataj, Kilian Schober, R. Lotfi, Beate Wagner, D. Busch, S. Kobold, F. Blaeschke, T. Feuchtinger
Overall survival of pediatric B-precursor ALL patients reached 90% in recent years. However, the outcome for refractory or relapsed children remains very poor. Anti-CD19 chimeric antigen receptor T-cells (CD19-CAR) showed significant antileukemic activity in relapsed and refractory B-precursor ALL. Especially in children, isolation of a suitable T-cell amount for autologous CAR T-cell manufacturing can be challenging due to low blood volume, low T-cell counts and clinical condition. In this case, the adoptive transfer of CAR T-cells from an unmatched healthy third-party donor provides a promising strategy. In order to prevent life-threatening graft-versus-host disease, a knockout (KO) of the endogenous T-cell receptor (TCR) has to be performed. Here, we generated CD19-CARs with a CRISPR/Cas9 mediated TCR KO, which remain highly functional and show strongly reduced alloreactivity compared to conventional CAR T-cells introduced into third-party T-cells. T-cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors and activated via anti-CD3/anti-CD28 stimulation. Retroviral transduction of a second generation anti-CD19 CAR (containing CD3zeta and 4-1BB stimulatory domains) was performed, followed by CRISPR/Cas9 mediated KO of the T-cell receptor beta chain via electroporation. After eleven days of expansion in the presence of IL-7 and IL-15, cells were purified for TCR KO-CD19-CAR T-cells via magnetic separation. Finally, the cell product was analyzed for cellular characteristics, functionality and alloreactivity by flow cytometry. A mean transduction rate of 37% for CD19-CARs and 40% for TCR KO-CD19-CARs was reached as well as a mean TCR KO rate of 78%. Both CD19-CARs as well as TCR KO-CD19-CARs showed suitable amounts of CD4- (45% vs. 33%) and CD8-T-cells (37% vs. 48%). The phenotype of CD19-CARs and TCR KO-CD19-CARs were comparable with mainly central memory (CM) (38% vs. 40%) and effector memory (EM) (57% vs. 51%) T-cells. The expansion of TCR KO-CD19-CARs was significantly reduced compared to conventional CD19-CARs (54-fold vs. 109-fold). This effect was not mediated by the loss of the TCR, but due to electroporation procedure. While CD19-CARs with or without TCR KO showed almost no background expression of the activation marker CD25 (2% vs 1%), contact with CD19-expressing targeT-cells resulted in a comparable upregulation of CD25 in both groups (95% vs. 94%). Co-culture with a CD19-expressing targeT-cell line led to an increased Interferon-γ secretion compared to unstimulated CARs, which was not significantly altered by the TCR KO (17% CD19-CAR vs. 14% TCR KO-CD19-CAR). CD19-dependent proliferative capacity of CAR T-cells was not influenced by loss of the TCR, as in both cases 97% of the T-cells proliferated after antigen recognition. Both CD19-CARs as well as TCR KO-CD19-CARs showed high, antigen-specific killing of 86% vs. 87% of the CD19-expressing targeT-cells at a 1:1 effector to target ratio. To evaluate the a
{"title":"Abstract A043: Anti-CD19 CAR T-cells with a CRISPR/Cas9-mediated T-cell receptor knockout show high functionality in the absence of alloreactivity in vitro","authors":"D. Stenger, Tanja A. Stief, Theresa Käuferle, Semjon Willier, F. Rataj, Kilian Schober, R. Lotfi, Beate Wagner, D. Busch, S. Kobold, F. Blaeschke, T. Feuchtinger","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A043","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A043","url":null,"abstract":"Overall survival of pediatric B-precursor ALL patients reached 90% in recent years. However, the outcome for refractory or relapsed children remains very poor. Anti-CD19 chimeric antigen receptor T-cells (CD19-CAR) showed significant antileukemic activity in relapsed and refractory B-precursor ALL. Especially in children, isolation of a suitable T-cell amount for autologous CAR T-cell manufacturing can be challenging due to low blood volume, low T-cell counts and clinical condition. In this case, the adoptive transfer of CAR T-cells from an unmatched healthy third-party donor provides a promising strategy. In order to prevent life-threatening graft-versus-host disease, a knockout (KO) of the endogenous T-cell receptor (TCR) has to be performed. Here, we generated CD19-CARs with a CRISPR/Cas9 mediated TCR KO, which remain highly functional and show strongly reduced alloreactivity compared to conventional CAR T-cells introduced into third-party T-cells. T-cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors and activated via anti-CD3/anti-CD28 stimulation. Retroviral transduction of a second generation anti-CD19 CAR (containing CD3zeta and 4-1BB stimulatory domains) was performed, followed by CRISPR/Cas9 mediated KO of the T-cell receptor beta chain via electroporation. After eleven days of expansion in the presence of IL-7 and IL-15, cells were purified for TCR KO-CD19-CAR T-cells via magnetic separation. Finally, the cell product was analyzed for cellular characteristics, functionality and alloreactivity by flow cytometry. A mean transduction rate of 37% for CD19-CARs and 40% for TCR KO-CD19-CARs was reached as well as a mean TCR KO rate of 78%. Both CD19-CARs as well as TCR KO-CD19-CARs showed suitable amounts of CD4- (45% vs. 33%) and CD8-T-cells (37% vs. 48%). The phenotype of CD19-CARs and TCR KO-CD19-CARs were comparable with mainly central memory (CM) (38% vs. 40%) and effector memory (EM) (57% vs. 51%) T-cells. The expansion of TCR KO-CD19-CARs was significantly reduced compared to conventional CD19-CARs (54-fold vs. 109-fold). This effect was not mediated by the loss of the TCR, but due to electroporation procedure. While CD19-CARs with or without TCR KO showed almost no background expression of the activation marker CD25 (2% vs 1%), contact with CD19-expressing targeT-cells resulted in a comparable upregulation of CD25 in both groups (95% vs. 94%). Co-culture with a CD19-expressing targeT-cell line led to an increased Interferon-γ secretion compared to unstimulated CARs, which was not significantly altered by the TCR KO (17% CD19-CAR vs. 14% TCR KO-CD19-CAR). CD19-dependent proliferative capacity of CAR T-cells was not influenced by loss of the TCR, as in both cases 97% of the T-cells proliferated after antigen recognition. Both CD19-CARs as well as TCR KO-CD19-CARs showed high, antigen-specific killing of 86% vs. 87% of the CD19-expressing targeT-cells at a 1:1 effector to target ratio. To evaluate the a","PeriodicalId":254712,"journal":{"name":"Genetically Engineered T-cells","volume":"2 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"125654961","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-02-01DOI: 10.1158/2326-6074.CRICIMTEATIAACR18-A034
M. Klinger, Peter Ebert, E. Osborne, R. Taniguchi, Joyce K. Hu, Tim Hayes, S. Benzeno, Adria Carbo, Melanie B Laur, Erica L. Eggers, H. Robins
The growing use of immunotherapy to treat advanced cancers has brought about a revolution in techniques to mobilize the immune system to antitumor effect. Chimeric antigen receptor (CAR) T-cells targeting CD19 constitute the first modified T-cell products to garner FDA approval for clinical use. However, CAR technologies can only targeT-cell surface antigens, representing approximately one quarter of potential targets. In contrast, T-cell receptor (TCR) therapies can target peptides presented by the major histocompatibility complex (MHC), including those derived from intracellular antigens. Hitherto, such receptors have generally been identified through low-throughput techniques using cancer patients’ blood, followed by affinity-maturation of TCRs, a step that can decrease safety. Here, we demonstrate a novel pipeline for the identification of potential therapeutic TCRs from the naive repertoire of healthy individuals. Using a technique called Multiplexed Identification of T-cell Receptor Antigen specificity (MIRA), we input hundreds of antigens of interest, including tumor-associated antigens, viral antigens and neoantigens, and identify thousands of TCRs to these antigens. These TCRs then undergo evaluation for affinity, avidity, cytokine release, cytotoxicity and safety. Cytotoxicity is demonstrated using both peptide-loaded and endogenously presented peptides. Safety is evaluated using alanine-glycine scans; evaluation of reactivity of TCRs againsT-cell lines and primary cells is planned. We have fully characterized several TCRs targeting clinically relevant targets, which demonstrated improved avidity and cytolysis relative to a benchmark TCR, and a promising preliminary safety profile. In the recent year, cancer neoantigens as targets for natural and therapeutic antitumor responses have also gained momentum given their attractive product profile. Hereto, we also show data for TCRs against shared neoantigens. Citation Format: Mark Klinger, Peter Ebert, Edward Osborne, Ruth Taniguchi, Joyce Hu, Tim Hayes, Sharon Benzeno, Adria Carbo, Melanie Laur, Erica Eggers, Harlan Robins. High-throughput identification of naturally occurring T-cell receptors with therapeutic potential against tumor-associated, viral and neoantigens [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 A034.
越来越多的使用免疫疗法治疗晚期癌症带来了一场技术革命，动员免疫系统的抗肿瘤作用。靶向CD19的嵌合抗原受体(CAR) t细胞是第一个获得FDA批准用于临床应用的修饰t细胞产品。然而，CAR技术只能靶向细胞表面抗原，约占潜在靶标的四分之一。相比之下，t细胞受体(TCR)疗法可以靶向主要组织相容性复合体(MHC)呈递的肽，包括来自细胞内抗原的肽。到目前为止，这种受体通常是通过使用癌症患者血液的低通量技术来识别的，然后是tcr的亲和成熟，这一步骤可能会降低安全性。在这里，我们展示了一种从健康个体的原始曲目中识别潜在治疗性tcr的新管道。使用一种称为多重识别t细胞受体抗原特异性(MIRA)的技术，我们输入数百种感兴趣的抗原，包括肿瘤相关抗原，病毒抗原和新抗原，并识别出数千种针对这些抗原的tcr。然后对这些tcr进行亲和力、亲和性、细胞因子释放、细胞毒性和安全性评估。细胞毒性是用多肽负载和内源性肽来证明的。安全性评估使用丙氨酸-甘氨酸扫描;计划评价tcr对抗细胞系和原代细胞的反应性。我们已经充分表征了几种靶向临床相关靶点的TCR，与基准TCR相比，这些TCR表现出更好的亲和力和细胞溶解性，并且具有良好的初步安全性。近年来，肿瘤新抗原作为天然和治疗性抗肿瘤反应的靶点也因其具有吸引力的产品概况而获得了发展势头。在这里，我们也展示了针对共享新抗原的tcr的数据。引文格式:Mark Klinger, Peter Ebert, Edward Osborne, Ruth Taniguchi, Joyce Hu, Tim Hayes, Sharon Benzeno, Adria Carbo, Melanie Laur, Erica Eggers, Harlan Robins。高通量鉴定具有抗肿瘤相关、病毒和新抗原治疗潜力的天然t细胞受体[摘要]。第四届CRI-CIMT-EATI-AACR国际癌症免疫治疗会议:将科学转化为生存;2018年9月30日至10月3日;纽约，纽约。费城(PA): AACR;癌症免疫学杂志2019;7(2增刊):摘要nr A034。
{"title":"Abstract A034: High-throughput identification of naturally occurring T-cell receptors with therapeutic potential against tumor-associated, viral and neoantigens","authors":"M. Klinger, Peter Ebert, E. Osborne, R. Taniguchi, Joyce K. Hu, Tim Hayes, S. Benzeno, Adria Carbo, Melanie B Laur, Erica L. Eggers, H. Robins","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A034","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A034","url":null,"abstract":"The growing use of immunotherapy to treat advanced cancers has brought about a revolution in techniques to mobilize the immune system to antitumor effect. Chimeric antigen receptor (CAR) T-cells targeting CD19 constitute the first modified T-cell products to garner FDA approval for clinical use. However, CAR technologies can only targeT-cell surface antigens, representing approximately one quarter of potential targets. In contrast, T-cell receptor (TCR) therapies can target peptides presented by the major histocompatibility complex (MHC), including those derived from intracellular antigens. Hitherto, such receptors have generally been identified through low-throughput techniques using cancer patients’ blood, followed by affinity-maturation of TCRs, a step that can decrease safety. Here, we demonstrate a novel pipeline for the identification of potential therapeutic TCRs from the naive repertoire of healthy individuals. Using a technique called Multiplexed Identification of T-cell Receptor Antigen specificity (MIRA), we input hundreds of antigens of interest, including tumor-associated antigens, viral antigens and neoantigens, and identify thousands of TCRs to these antigens. These TCRs then undergo evaluation for affinity, avidity, cytokine release, cytotoxicity and safety. Cytotoxicity is demonstrated using both peptide-loaded and endogenously presented peptides. Safety is evaluated using alanine-glycine scans; evaluation of reactivity of TCRs againsT-cell lines and primary cells is planned. We have fully characterized several TCRs targeting clinically relevant targets, which demonstrated improved avidity and cytolysis relative to a benchmark TCR, and a promising preliminary safety profile. In the recent year, cancer neoantigens as targets for natural and therapeutic antitumor responses have also gained momentum given their attractive product profile. Hereto, we also show data for TCRs against shared neoantigens. Citation Format: Mark Klinger, Peter Ebert, Edward Osborne, Ruth Taniguchi, Joyce Hu, Tim Hayes, Sharon Benzeno, Adria Carbo, Melanie Laur, Erica Eggers, Harlan Robins. High-throughput identification of naturally occurring T-cell receptors with therapeutic potential against tumor-associated, viral and neoantigens [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 A034.","PeriodicalId":254712,"journal":{"name":"Genetically Engineered T-cells","volume":"8 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"132804056","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-02-01DOI: 10.1158/2326-6074.CRICIMTEATIAACR18-A031
R. Hernandez-Lopez, W. Lim, Wei Yu
T-cells can be redirected to kill tumor cells via synthetic T-cell receptors known as chimeric antigen receptors (CARs); this approach is becoming a highly promising therapeutic strategy for cancer treatment. Current CAR T-cells, while effective at killing cells expressing the target antigen, fail to discriminate between cells expressing high and low levels of common antigens. Potential target antigens are often also found on normal cells at lower expression levels, and CAR T-cells get activated even by low amounts of target antigen, resulting in potentially deadly toxic effects. We are approaching this problem of antigen density discrimination by using common biochemical mechanisms for cooperative recognition to design new synthetic T-cell receptors. CAR T-cells are highly sensitive to even low densities of the target antigen. Current strategies for generating CAR T-cells consist of selecting antibodies with high affinity since previous studies have shown that the CAR T-cell activity is inversely correlated with the antibody’s affinity. However, these CARs are unable to discriminate between cancer and normal cells based on antigen density. Recent studies suggest that lowering the receptor affinity on a CAR T-cell could increase its selectivity against targeT-cells with different antigen densities. Nevertheless, lowering the affinity is unlikely to provide a sharp density threshold of activation. Instead for a sharp threshold transition from off to on, one would require cooperativity and nonlinear recognition. We have designed a two-step recognition-activation circuit that involves two receptors. In this circuit an initial recognition event alters the potency of a subsequent response. We combined a weak receptor that turns on the activity of a high-affinity receptor that fully activates the cell. For this circuits we have made used of a new class of modular receptors called synthetic notch receptors (SynNotch). SynNotch receptors use antibody-based domains to recognize a target antigen, but when activated, they control transcription via a cleaved transcriptional domain. We have designed and expressed several synNotch/CAR circuits and have tested in vitro their ability to achieve antigen density sensing. These circuits use a synNotch receptor to control the expression of a CAR—all recognizing the same Her2 ligand. The constructs are introduced into T-cells via standard lentiviral infection, and sorted for receptor expression using flow cytometry (anti-myc stain). We have screened candidate receptors for expression and activation response in the human Jurkat T-cell line and human primary CD4+ and CD8+ T-cells from several donors, using standard flow-based assays (e.g. CD69 Activation, T-cell proliferation) and cytokine assays (e.g., IL-2, INF-γ). We are now taking the best circuit candidate and proceeding in mouse models. We have first characterized the growth curves of tumors made by engineered targeT-cells to measure the baseline tumor growth and
{"title":"Abstract A031: Engineering antigen density sensors for T-cell immunotherapy","authors":"R. Hernandez-Lopez, W. Lim, Wei Yu","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A031","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A031","url":null,"abstract":"T-cells can be redirected to kill tumor cells via synthetic T-cell receptors known as chimeric antigen receptors (CARs); this approach is becoming a highly promising therapeutic strategy for cancer treatment. Current CAR T-cells, while effective at killing cells expressing the target antigen, fail to discriminate between cells expressing high and low levels of common antigens. Potential target antigens are often also found on normal cells at lower expression levels, and CAR T-cells get activated even by low amounts of target antigen, resulting in potentially deadly toxic effects. We are approaching this problem of antigen density discrimination by using common biochemical mechanisms for cooperative recognition to design new synthetic T-cell receptors. CAR T-cells are highly sensitive to even low densities of the target antigen. Current strategies for generating CAR T-cells consist of selecting antibodies with high affinity since previous studies have shown that the CAR T-cell activity is inversely correlated with the antibody’s affinity. However, these CARs are unable to discriminate between cancer and normal cells based on antigen density. Recent studies suggest that lowering the receptor affinity on a CAR T-cell could increase its selectivity against targeT-cells with different antigen densities. Nevertheless, lowering the affinity is unlikely to provide a sharp density threshold of activation. Instead for a sharp threshold transition from off to on, one would require cooperativity and nonlinear recognition. We have designed a two-step recognition-activation circuit that involves two receptors. In this circuit an initial recognition event alters the potency of a subsequent response. We combined a weak receptor that turns on the activity of a high-affinity receptor that fully activates the cell. For this circuits we have made used of a new class of modular receptors called synthetic notch receptors (SynNotch). SynNotch receptors use antibody-based domains to recognize a target antigen, but when activated, they control transcription via a cleaved transcriptional domain. We have designed and expressed several synNotch/CAR circuits and have tested in vitro their ability to achieve antigen density sensing. These circuits use a synNotch receptor to control the expression of a CAR—all recognizing the same Her2 ligand. The constructs are introduced into T-cells via standard lentiviral infection, and sorted for receptor expression using flow cytometry (anti-myc stain). We have screened candidate receptors for expression and activation response in the human Jurkat T-cell line and human primary CD4+ and CD8+ T-cells from several donors, using standard flow-based assays (e.g. CD69 Activation, T-cell proliferation) and cytokine assays (e.g., IL-2, INF-γ). We are now taking the best circuit candidate and proceeding in mouse models. We have first characterized the growth curves of tumors made by engineered targeT-cells to measure the baseline tumor growth and","PeriodicalId":254712,"journal":{"name":"Genetically Engineered T-cells","volume":"22 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"122251054","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-02-01DOI: 10.1158/2326-6074.CRICIMTEATIAACR18-A030
Yukai He, Wei Zhu, Yibing Peng, Lan Wang, Yuan Hong, Juan Wu, E. Celis
Hepatocellular carcinoma (HCC) is the major form of liver cancer for which there is no effective therapy. Genetic modification with T-cell receptors (TCR) specific for HCC-associated antigens, such as α-fetoprotein (AFP), can potentially redirect human T-cells to specifically recognize and kill HCC tumor cells to achieve antitumor effects. In this study, by using lentivector and peptide immunization, we identified a population of CD8-T-cells in HLA-A2 transgenic AAD mice that recognized AFP158 epitope on human HCC cells. Adoptive transfer of the AFP158-specific mouse CD8-T-cells eradicated HepG2 tumor xenografts as large as 2cm in diameter in immunocompromised NSG mice. We then established T-cell hybridoma clones from the AFP158-specific mouse CD8-T-cells and identified three sets of paired TCR genes out of 5 hybridomas. Expression of the murine TCR genes redirected primary human T-cells to bind HLA-A2/AFP158 tetramer. The TCR gene-engineered human T-cells (TCR-T) also specifically recognized HLA-A2+AFP+ HepG2 HCC tumor cells and produced effector cytokines. Importantly, the TCR-T-cells could specifically kill HLA-A2+AFP+ HepG2 tumor cells without significant toxicity to normal primary hepatocytes in vitro. Adoptive transfer of the AFP-specific human TCR-T-cells could eradicate HepG2 tumors in NSG mice. Conclusion: We have identified novel AFP-specific murine TCR genes that can redirect human T-cells to specifically recognize and kill HCC tumor cells, and those AFP158-specific TCRs have a great potential to engineer a patient’s autologous T-cells to treat HCC tumors. Citation Format: Yukai He, Wei Zhu, Yibing Peng, Lan Wang, Yuan Hong, Juan Wu, Esteban Celis. Identification of α-fetoprotein-specific T-cell receptors for hepatocellular carcinoma immunotherapy [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 A030.
{"title":"Abstract A030: Identification of α-fetoprotein-specific T-cell receptors for hepatocellular carcinoma immunotherapy","authors":"Yukai He, Wei Zhu, Yibing Peng, Lan Wang, Yuan Hong, Juan Wu, E. Celis","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A030","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A030","url":null,"abstract":"Hepatocellular carcinoma (HCC) is the major form of liver cancer for which there is no effective therapy. Genetic modification with T-cell receptors (TCR) specific for HCC-associated antigens, such as α-fetoprotein (AFP), can potentially redirect human T-cells to specifically recognize and kill HCC tumor cells to achieve antitumor effects. In this study, by using lentivector and peptide immunization, we identified a population of CD8-T-cells in HLA-A2 transgenic AAD mice that recognized AFP158 epitope on human HCC cells. Adoptive transfer of the AFP158-specific mouse CD8-T-cells eradicated HepG2 tumor xenografts as large as 2cm in diameter in immunocompromised NSG mice. We then established T-cell hybridoma clones from the AFP158-specific mouse CD8-T-cells and identified three sets of paired TCR genes out of 5 hybridomas. Expression of the murine TCR genes redirected primary human T-cells to bind HLA-A2/AFP158 tetramer. The TCR gene-engineered human T-cells (TCR-T) also specifically recognized HLA-A2+AFP+ HepG2 HCC tumor cells and produced effector cytokines. Importantly, the TCR-T-cells could specifically kill HLA-A2+AFP+ HepG2 tumor cells without significant toxicity to normal primary hepatocytes in vitro. Adoptive transfer of the AFP-specific human TCR-T-cells could eradicate HepG2 tumors in NSG mice. Conclusion: We have identified novel AFP-specific murine TCR genes that can redirect human T-cells to specifically recognize and kill HCC tumor cells, and those AFP158-specific TCRs have a great potential to engineer a patient’s autologous T-cells to treat HCC tumors. Citation Format: Yukai He, Wei Zhu, Yibing Peng, Lan Wang, Yuan Hong, Juan Wu, Esteban Celis. Identification of α-fetoprotein-specific T-cell receptors for hepatocellular carcinoma immunotherapy [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 A030.","PeriodicalId":254712,"journal":{"name":"Genetically Engineered T-cells","volume":"220 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"115653145","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-02-01DOI: 10.1158/2326-6074.CRICIMTEATIAACR18-A026
Jian Chen, Xiaofeng Xia
CAR-T-cells are currently manufactured for clinical use by infection of human T-cells with viral vectors containing the CAR gene. T lymphocytes have to be stimulated and expanded ex vivo because the viral vectors infect fresh natural lymphocytes very poorly. The viral vector approach is extremely expensive due to the high cost of virus production and the high cost of long-term cell expansion that could take 10-14 days in a GMP facility. The viral vector approach also has a huge biologic downside: ex vivo expanded T-cells become bulky and lose efficacy against tumor cells. The use of electroporation technology in CAR-T-cell manufacturing has attracted increasing interests for its low cost and the wide range of applications, including transposon based stable CAR expression, transient expression and genome editing. However, actual clinical use of electroporation technology in CAR-T has been difficult and several clinical trials have met significant problems due to the poor transfection efficiency and decreased T-cell survival with traditional electroporation methods. Through theoretical analysis of the other existing electroporation technologies, we found that they all have problems in electrophysical design. The problems include physical design of devices, electrical pulse selection, and buffer composition. With a redesigned electroporation system, we can now achieve very high transfection efficiency for T-cells while maintaining cell survival. For Sleeping Beauty transposon-based CAR expression, we found that over a period of two to three weeks the efficiency can get to 60% to 90% with fasT-cell proliferation. The protein expression time after electroporation is very short. For simple GFP plasmids we can observe GFP expression after only 30 minutes. Unlike viral vectors, electroporation works well on fresh natural T-cells, thereby eliminating the need for expensive cell expansion and virus production altogether and cutting the huge economic burden of CAR-T therapy. By reinfusion of more natural T-cells, the antitumor efficacy of CAR-T-cells could be improved while the side effects of cytokine release syndrome could be minimized. Citation Format: Jian Chen, Xiaofeng Xia. Novel electroporation method for quick CAR-T-cell manufacture [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 A026.
{"title":"Abstract A026: Novel electroporation method for quick CAR-T-cell manufacture","authors":"Jian Chen, Xiaofeng Xia","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A026","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A026","url":null,"abstract":"CAR-T-cells are currently manufactured for clinical use by infection of human T-cells with viral vectors containing the CAR gene. T lymphocytes have to be stimulated and expanded ex vivo because the viral vectors infect fresh natural lymphocytes very poorly. The viral vector approach is extremely expensive due to the high cost of virus production and the high cost of long-term cell expansion that could take 10-14 days in a GMP facility. The viral vector approach also has a huge biologic downside: ex vivo expanded T-cells become bulky and lose efficacy against tumor cells. The use of electroporation technology in CAR-T-cell manufacturing has attracted increasing interests for its low cost and the wide range of applications, including transposon based stable CAR expression, transient expression and genome editing. However, actual clinical use of electroporation technology in CAR-T has been difficult and several clinical trials have met significant problems due to the poor transfection efficiency and decreased T-cell survival with traditional electroporation methods. Through theoretical analysis of the other existing electroporation technologies, we found that they all have problems in electrophysical design. The problems include physical design of devices, electrical pulse selection, and buffer composition. With a redesigned electroporation system, we can now achieve very high transfection efficiency for T-cells while maintaining cell survival. For Sleeping Beauty transposon-based CAR expression, we found that over a period of two to three weeks the efficiency can get to 60% to 90% with fasT-cell proliferation. The protein expression time after electroporation is very short. For simple GFP plasmids we can observe GFP expression after only 30 minutes. Unlike viral vectors, electroporation works well on fresh natural T-cells, thereby eliminating the need for expensive cell expansion and virus production altogether and cutting the huge economic burden of CAR-T therapy. By reinfusion of more natural T-cells, the antitumor efficacy of CAR-T-cells could be improved while the side effects of cytokine release syndrome could be minimized. Citation Format: Jian Chen, Xiaofeng Xia. Novel electroporation method for quick CAR-T-cell manufacture [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 A026.","PeriodicalId":254712,"journal":{"name":"Genetically Engineered T-cells","volume":"56 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"127681243","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-02-01DOI: 10.1158/2326-6074.CRICIMTEATIAACR18-IA17
K. Anderson, D. Egan, S. Hingorani, S. Oda, K. Paulson, R. Perret, L. Schmidt, Thomas M. Schmitt, Ingunn M. Stromnes, A. Chapuis, P. Greenberg
We have been exploring in preclinical models and clinical trials methods to reproducibly provide therapeutic T cell responses by transfer of genetically engineered T cells. Our largest clinical experience is in treating human Acute Myelogenous Leukemia (AML). After identifying that WT1, a gene associated with promoting leukemic transformation, is over-expressed in human leukemic stem cells, and demonstrating in a clinical trial that in vitro expanded WT1-specific CD8 T cell clones can be safely transferred, exhibit anti-leukemic activity, and provide therapeutic benefit to AML patients, we extensively screened normal human repertoires and isolated a high affinity TCR specific for WT1 for genetically engineering CD8 T cells to reproducibly create cells with high avidity for leukemic cells. We have initially pursued this strategy in a 2 Arm trial for leukemia patients either at high risk of relapse (Arm 1) after hematopoietic cell transplant (HCT) or who have already relapsed after HCT (Arm 2). The prophylactic arm is now completed, with very encouraging results- all patients treated with engineered T cells remain alive and relapse free at a median of 38 months, compared to a relapse rate of ~50% in a concurrent matched cohort. Results in relapsed patients (Arm 2) have been less effective, and we have used high-dimensional analyses including single cell RNAseq both to elucidate the reasons for failure to eradicate the leukemia and to design strategies to overcome these obstacles. Our results have identified several mechanisms by which the leukemia escapes, and we have been testing approaches that employ further genetic modification of the T cells to enhance efficacy. The predominant reason for leukemia progression despite targeted therapy with T cells is the inability of the T cells to persist and maintain function in the context of encountering a rapidly proliferating myeloid leukemia. This reflects both engagement of pathways inhibitory to T cells by the leukemic cells, and apoptosis of the T cells from repetitive stimulation. We are addressing this issue by creating immuno-modulatory fusion proteins (IFPs) that have the ectodomain of an inhibitory or death receptor fused to a survival costimulatory domain. Results with two such IFPs will be discussed, a CD200R/CD28 fusion that binds the inhibitory ligand CD200 commonly expressed on leukemic cells but provides a CD28 costimulatory signal and a Fas/4-1BB fusion that binds FasL but rather than induce death promotes proliferation and survival. A more uncommon reason explaining progression is loss of expression of the WT1 epitope being targeted. This has occurred in 2 patients, but for distinct reasons. In one patient this reflected loss of a component of the immunoproteasome, and we have now isolated a TCR that recognizes an epitope not dependent on the immunoproteasome. In a second patient the level of WT1 expression declined- interestingly, this patient was treated with Vidaza, which can increase
我们一直在探索临床前模型和临床试验方法，通过基因工程T细胞的转移可重复地提供治疗性T细胞反应。我们最大的临床经验是治疗人类急性髓性白血病(AML)。在确定与促进白血病转化相关的基因WT1在人类白血病干细胞中过度表达，并在临床试验中证明体外扩增的WT1特异性CD8 T细胞克隆可以安全转移，表现出抗白血病活性，并为AML患者提供治疗益处后，我们广泛筛选了正常人的基因库，并为基因工程CD8 T细胞分离出一种高亲和力的WT1特异性TCR，以可重复地创造出对白血病细胞具有高亲和力的细胞。我们最初在一项针对造血细胞移植(HCT)后复发风险高(第1组)或HCT后已经复发(第2组)的白血病患者的2组试验中采用了这一策略。预防组现已完成，结果非常令人鼓舞-所有接受工程化T细胞治疗的患者在中位38个月时仍然存活且无复发，而同期匹配队列的复发率约为50%。复发患者(第2组)的结果不太有效，我们使用了高维分析，包括单细胞RNAseq来阐明根除白血病失败的原因，并设计了克服这些障碍的策略。我们的研究结果已经确定了白血病逃逸的几种机制，我们一直在测试进一步对T细胞进行遗传修饰以提高疗效的方法。尽管使用T细胞进行靶向治疗，但白血病进展的主要原因是T细胞在遇到快速增殖的髓性白血病时无法持续和维持功能。这既反映了白血病细胞对T细胞的抑制通路的参与，也反映了T细胞在重复刺激下的凋亡。我们正在通过创建免疫调节融合蛋白(IFPs)来解决这个问题，IFPs具有抑制或死亡受体的外域融合到生存共刺激域。我们将讨论两种IFPs的结果，一种是CD200R/CD28融合，它结合抑制配体CD200，通常在白血病细胞上表达，但提供CD28共刺激信号;另一种是Fas/4-1BB融合，它结合FasL，但不诱导死亡，促进增殖和存活。一个更不常见的解释进展的原因是WT1表位被靶向的表达缺失。2例患者出现过这种情况，但原因各不相同。在一个患者中，这反映了免疫蛋白酶体的一个组成部分的丢失，我们现在已经分离出一个识别不依赖于免疫蛋白酶体的表位的TCR。在第二例患者中，WT1表达水平下降-有趣的是，该患者接受了维达扎治疗，它可以增加WT1的表达，维达扎后，在患者骨髓中持续存在的转移的T细胞在体内识别并应答复发的白血病。我们在白血病中开发和测试的方法和技术也适用于实体肿瘤，并将讨论胰腺癌和卵巢癌的临床前研究。引文格式:Kristin G. Anderson, Dan Egan, Sunil R. Hingorani, Shannon K. Oda, Kelly Paulson, Rachel Perret, Leah Schmidt, Thomas Schmitt, Ingunn Stromnes, Aude Chapuis, Philip D. Greenberg。利用合成生物学和高维探测来解决治疗障碍，并赋予工程T细胞根除肿瘤的能力[摘要]。第四届CRI-CIMT-EATI-AACR国际癌症免疫治疗会议:将科学转化为生存;2018年9月30日至10月3日;纽约，纽约。费城(PA): AACR;癌症免疫学杂志，2019;7(2增刊):摘要1 - 17。
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