Abstract:The ability to use a systemically injected agent to image tumor is influenced by tumor characteristics such as permeability and vascularity, and the size, shape, and affinity of the imaging agent. In this study, six different imaging biomolecules, with or without specificity to tumor, were examined for tumor uptake and internalization at the whole body, ex-vivo tissue, and cellular levels: antibodies, antibody fragments (Fab), serum albumin, and streptavidin. The time of peak tumor uptake was dependent solely … Show more
“…Cross-reactivity with its murine homologue enables examination of on-target, off-tumor toxicity of I domain CAR T cells concurrently with on-target, on-tumor efficacy in preclinical mouse models with human tumor xenografts. In comparison, the R6.5 scFv (derived from the mouse hybridoma clone, R6.5 33 ) has a K D of 10 nM for human ICAM-1 (Table 1 ) but does not cross-react with murine ICAM-1. Figure 1 Construction of ICAM-1 specific CARs with step-wise, 10 6 -fold variations in affinity.…”
Adoptive transfer of high-affinity chimeric antigen receptor (CAR) T cells targeting hematological cancers has yielded impressive clinical results. However, safety concerns regarding target expression on healthy tissue and poor efficacy have hampered application to solid tumors. Here, a panel of affinity-variant CARs were constructed targeting overexpressed ICAM-1, a broad tumor biomarker, using its physiological ligand, LFA-1. Anti-tumor T cell potency in vitro was directly proportional to CAR affinity and ICAM-1 density. In a solid tumor mouse model allowing simultaneous monitoring of anti-tumor potency and systemic off-tumor toxicity, micromolar affinity CAR T cells demonstrated superior anti-tumor efficacy and safety compared to their nanomolar counterparts. Longitudinal T cell tracking by PET/CT and concurrent cytokine measurement revealed superior expansion and contraction kinetics of micromolar affinity CAR T cells. Therefore, we developed an ICAM-1 specific CAR with broad anti-tumor applicability that utilized a reduced affinity targeting strategy to significantly boost efficacy and safety.
“…Cross-reactivity with its murine homologue enables examination of on-target, off-tumor toxicity of I domain CAR T cells concurrently with on-target, on-tumor efficacy in preclinical mouse models with human tumor xenografts. In comparison, the R6.5 scFv (derived from the mouse hybridoma clone, R6.5 33 ) has a K D of 10 nM for human ICAM-1 (Table 1 ) but does not cross-react with murine ICAM-1. Figure 1 Construction of ICAM-1 specific CARs with step-wise, 10 6 -fold variations in affinity.…”
Adoptive transfer of high-affinity chimeric antigen receptor (CAR) T cells targeting hematological cancers has yielded impressive clinical results. However, safety concerns regarding target expression on healthy tissue and poor efficacy have hampered application to solid tumors. Here, a panel of affinity-variant CARs were constructed targeting overexpressed ICAM-1, a broad tumor biomarker, using its physiological ligand, LFA-1. Anti-tumor T cell potency in vitro was directly proportional to CAR affinity and ICAM-1 density. In a solid tumor mouse model allowing simultaneous monitoring of anti-tumor potency and systemic off-tumor toxicity, micromolar affinity CAR T cells demonstrated superior anti-tumor efficacy and safety compared to their nanomolar counterparts. Longitudinal T cell tracking by PET/CT and concurrent cytokine measurement revealed superior expansion and contraction kinetics of micromolar affinity CAR T cells. Therefore, we developed an ICAM-1 specific CAR with broad anti-tumor applicability that utilized a reduced affinity targeting strategy to significantly boost efficacy and safety.
“…Its use for the treatment of stroke cases was associated with side effects due to a complement reaction attributable to the Fc portion of the mouse IgG2a (41). The F(ab) of R6.5 has an estimated affinity of 10 nM (42), and the R6.5 scFv in the present ICAM-1 CAR T construct endowed T cells with ICAM-1 specific cytotoxicity that recognizes ICAM-1-overexpressing target tumor cells while sparing normal tissue with basal ICAM-1 expression.…”
Purpose
Poorly-differentiated thyroid cancer and anaplastic thyroid cancer (ATC) are rare yet lethal malignancies with limited treatment options. Many malignant tumors including papillary thyroid cancer (PTC) and ATC are associated with increased expression of ICAM-1, providing a rationale for utilizing ICAM-1-targeting agents for the treatment of aggressive cancer. We developed a third-generation CAR targeting ICAM-1 to leverage adoptive T cell therapy as a new treatment modality.
Experimental Design
ICAM-1 CAR T cells were applied on multiple malignant and non-malignant target cells to investigate specific target cell death and ‘off-tumor’ toxicity in vitro. In vivo therapeutic efficacy of ICAM-1 CAR T cells was examined in ATC mouse models established from a cell line and patient-derived tumors that rapidly develop systemic metastases.
Results
ICAM-1 CAR T cells demonstrated robust and specific killing of PTC and ATC cell lines in vitro. Interestingly, although certain ATC cell lines showed heterogeneous levels of ICAM-1 expression, addition of cytotoxic CAR T cells induced increased ICAM-1 expression such that all cell lines became targetable. In mice with systemic ATC, a single administration of ICAM-1 CAR-T cells mediated profound tumor killing that resulted in long term remission and significantly improved survival. Patient-derived ATC cells overexpressed ICAM-1 and were largely eliminated by autologous ICAM-1 CAR T cells in vitro and in animal models.
Conclusions
Our findings are the first demonstration of CAR T therapy for metastatic, thyroid cancer cell line and advanced ATC patient-derived tumors that exhibit dramatic therapeutic efficacy and survival benefit in animal studies.
“…Construction of ICAM-1 CAR and SSTR2 reporter genes. The CAR gene specific to ICAM-1 was derived from the scFv sequence of a murine monoclonal anti-human R6.5 antibody -itself derived from hybridoma (ATCC) (56,57). The R6.5-specific scFv was then fused to the transmembrane and cytoplasmic domains of CD28, CD137, and CD3ζ of an independent third-generation pLenti plasmid (a gift from Carl June at the University of Pennsylvania, Philadelphia, Pennsylvania, USA) (35).…”
Section: Discussionmentioning
confidence: 99%
“…Flow cytometry gates were first determined by live-cell gating (PI negative) and subsequently by staining of respective antibodies. ICAM-1 expression on tumor cell lines was determined using a murine anti-human R6.5 monoclonal antibody (10 μg/ml) derived from hybridoma (ATCC) (57). R6.5-CAR expression on T cells was detected using FITC-conjugated goat anti-mouse F(ab′)2 secondary antibody (Thermo Fisher, 31543).…”
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