T-cell Receptors for Clinical Therapy: <i>In Vitro</i> Assessment of Toxicity Risk

Kunert, Andre; Obenaus, Matthias; Lamers, Cor H.J.; Blankenstein, Thomas; Debets, Reno

doi:10.1158/1078-0432.ccr-17-1012

Cited by 38 publications

(38 citation statements)

References 44 publications

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“…15,[20][21][22][23] Suggested mechanisms for these toxicities include TCR reactivity against healthy tissues that is either on-target or off-target. 34 The peptide-specific nature of these responses makes in vivo toxicology studies unsuitable since the differences between human and murine proteomes preclude reliable detection of off-target cross-reactivity. Consequently, a robust, biologically relevant preclinical testing strategy for selecting target antigens and TCRs for clinical development is required.…”

Section: Discussionmentioning

confidence: 99%

“…Consequently, a robust, biologically relevant preclinical testing strategy for selecting target antigens and TCRs for clinical development is required. 34,35 We developed an extensive in vitro preclinical testing protocol to evaluate the safety and efficacy of SPEAR T-cell therapies ( Figure 5). These approaches were developed and expanded from techniques that retrospectively predicted clinical off-target toxicity induced by an affinity-enhanced MAGE-A3 TCR.…”

Section: Discussionmentioning

confidence: 99%

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Preclinical evaluation of an affinity-enhanced MAGE-A4-specific T-cell receptor for adoptive T-cell therapy

et al. 2019

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2020) Preclinical evaluation of an affinity-enhanced MAGE-A4-specific T-cell receptor for adoptive T-cell therapy, OncoImmunology, 9:1, 1682381, ABSTRACT A substantial obstacle to the success of adoptive T cell-based cancer immunotherapy is the sub-optimal affinity of T-cell receptors (TCRs) for most tumor antigens. Genetically engineered TCRs that have enhanced affinity for specific tumor peptide-MHC complexes may overcome this barrier. However, this enhancement risks increasing weak TCR cross-reactivity to other antigens expressed by normal tissues, potentially leading to clinical toxicities. To reduce the risk of such adverse clinical outcomes, we have developed an extensive preclinical testing strategy, involving potency testing using 2D and 3D human cell cultures and primary tumor material, and safety testing using human primary cell and cell-line crossreactivity screening and molecular analysis to predict peptides recognized by the affinity-enhanced TCR. Here, we describe this strategy using a developmental T-cell therapy, ADP-A2M4, which recognizes the HLA-A2-restricted MAGE-A4 peptide GVYDGREHTV. ADP-A2M4 demonstrated potent anti-tumor activity in the absence of major off-target cross-reactivity against a range of human primary cells and cell lines. Identification and characterization of peptides recognized by the affinity-enhanced TCR also revealed no cross-reactivity. These studies demonstrated that this TCR is highly potent and without major safety concerns, and as a result, this TCR is now being investigated in two clinical trials (NCT03132922, NCT04044768). ARTICLE HISTORY

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Preclinical evaluation of an affinity-enhanced MAGE-A4-specific T-cell receptor for adoptive T-cell therapy

et al. 2019

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“…For example, the EC 50 of cognate peptide for activating the MAGE-A10 specific TCR-Ts was 0.3-0.5 nM (23), and the EC 50 of NY-ESO1 157 peptide for activating cognate TCR-Ts was around 0.2 nM (28). In contrast, the EC 50 of cognate peptide for activating the MAGE-C2 specific TCR6-T cells was 3.3 nM (18). Next, based on the EC 50 , the EC 90 of AFP 158 peptide was calculated as 10 ng/ml ( Figures 1C,D).…”

Section: The Ec 50 and Ec 90 Of Target Afp 158 And The Peptide Concenmentioning

confidence: 99%

“…All three aspects of the TCR-T's toxicity must be properly evaluated prior to conducting clinical trials. As investigations of TCR-Ts in animal models offers little value in evaluating their toxicity in human, an in vitro preclinical toxicity study strategy was proposed to assess the TCR-T's risk (18). Ideally, tumor-specific or relatively tumor-specific antigens should be selected as the TCR-T's target to reduce on-target/offtumor reactivity.…”

Section: Introductionmentioning

confidence: 99%

Preclinical Studies of the Off-Target Reactivity of AFP158-Specific TCR Engineered T Cells

et al. 2020

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Autologous T cells engineered with T receptor genes (TCR) are being studied to treat cancers. We have recently identified a panel of mouse TCRs specific for the HLA-A0201/alpha fetoprotein epitope (AFP 158) complex and have shown that human T cells engineered with these TCR genes (TCR-Ts) can eradicate hepatocellular carcinoma (HCC) xenografts in NSG mice. However, due to TCR's promiscuity, their off-target cross-reactivity must be studied prior to conducting clinical trials. In this study, we conducted in vitro X-scan assay and in silico analysis to determine the off-target crossreactivity of 3 AFP 158-specific TCR-Ts. We found that the 3 AFP 158-specific TCR-Ts could be cross-activated by ENPP1 436 peptide and that the TCR3-Ts could also be activated by another off-target peptide, RCL1 215. However, compared to AFP 158 , it requires 250 times more ENPP1 436 and 10,000 times more RCL1 215 peptides to achieve the same level of activation. The EC 50 of ENPP1 436 peptide for activating TCR-Ts is approximately 17-33 times higher than AFP 158. Importantly, the ENPP1+ tumor cells did not activate TCR1-Ts and TCR2-Ts, and only weakly activated TCR3-Ts. The IFNγ produced by TCR3-Ts after ENPP1+ cell stimulation was >22x lower than that after HepG2 cells. And, all TCR-Ts did not kill ENPP1 + tumor cells. Furthermore, ectopic over-expression of ENPP1 protein in HLA-A2+ tumor cells did not activate TCR-Ts. In silico analysis showed that the ENPP1 436 peptide affinity for HLA-A0201 was ranked 40 times lower than AFP 158 and the chance of ENPP1 436 peptide being processed and presented by HLA-A0201 was 100 times less likely than AFP 158. In contrast, the two off-targets (Titin and MAGE-A3) that did cause severe toxicity in previous trials have the same or higher MHC-binding affinity and the same or higher chance of being processed and presented. In conclusion, our data shows that TCR-Ts can be activated by off-target ENPP1 436 peptide. But, compared to target AFP 158 , it requires at least 250 times more ENPP1 436 to achieve the same level of activation. Importantly, ENPP1 436 peptide in human cells is not processed and presented to a sufficient level to activate the AFP 158-specific TCR-Ts. Thus, these TCR-Ts, especially the TCR1-Ts and TCR2-Ts, will unlikely cause significant off-target toxicity.

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“…In order to limit on-target toxicity for oncoviral and germ-line antigens, their absence from panels of healthy tissue is tested in silico, using online databases (Human Protein Atlas, CGA database) and in vitro using PCR cDNA libraries and IHC in tissue panels [70,49]). TCRs are tested against random epitopes and allogeneic MHC molecules using for example lymphoblastoid B cell lines with various MHC allotypes [71,72].…”

Section: Toxicitiesmentioning

confidence: 99%