The GD2 ganglioside, which is abundant on the surface of neuroblastoma cells, is targeted by an FDA-approved therapeutic monoclonal antibody and is an attractive tumor-associated antigen for cellular immunotherapy. Chimeric antigen receptor (CAR)–modified T cells can have potent antitumor activity in B-cell malignancies, and trials to harness this cytolytic activity toward GD2 in neuroblastoma are under way. In an effort to enhance the antitumor activity of CAR T cells that target GD2, we generated variant CAR constructs predicted to improve the stability and the affinity of the GD2-binding, 14G2a-based, single-chain variable fragment (scFv) of the CAR and compared their properties in vivo. We included the E101K mutation of GD2 scFv (GD2-E101K) that has enhanced antitumor activity against a GD2+ human neuroblastoma xenograft in vivo. However, this enhanced antitumor efficacy in vivo was concomitantly associated with lethal central nervous system (CNS) toxicity comprised of extensive CAR T-cell infiltration and proliferation within the brain and neuronal destruction. The encephalitis was localized to the cerebellum and basal regions of the brain that display low amounts of GD2. Our results highlight the challenges associated with target antigens that exhibit shared expression on critical normal tissues. Despite the success of GD2-specific antibody therapies in the treatment of neuroblastoma, the fatal neurotoxicity of GD2-specific CAR T-cell therapy observed in our studies suggests that GD2 may be a difficult target antigen for CAR T-cell therapy without additional strategies that can control CAR T-cell function within the CNS.
The variable (V) domains of antibodies and T cell receptors (TCRs) share sequence homology and striking structural similarity. Single-chain antibody V domain constructs (scFv) are routinely expressed in a variety of heterologous systems, both for production of soluble protein as well as for in vitro engineering. In contrast, single-chain T cell receptor V domain constructs (scTCR) are prone to aggregation and misfolding and are refractory to display on phage or yeast in their wild-type form. However, through random mutagenesis and yeast display engineering, it has been possible to isolate scTCR mutants that are properly folded and displayed on the yeast surface. These displayed mutants can serve not only as a scaffold for further engineering but also as scTCR variants that exhibit favorable biophysical properties in E. coli expression. Thus, a more comprehensive understanding of the V domain mutations that allowed display would be beneficial. Our goal here was to identify generalizable patterns of important mutations that can be applied to different TCRs. We compared five different scTCRs, four from mice and one from a human, for yeast surface display. Analysis of a collection of mutants revealed four distinct regions of TCR V domains that were most important for enabling surface expression: the Vα-Vβ interface, the HV4 of Vβ, and the region of the Vα and Vβ domains normally apposed against the constant (C) domains. Consistent with the role of the V-C interface in surface display, reconstitution of this interface, by including the constant domains of each chain, allowed V domain display and αβ chain association on the yeast surface, thus providing an alternative TCR scaffold. However, the surface levels of TCR achieved with engineered scTCR mutants were superior to that of the VαCα/VβCβ constructs. Therefore, we describe further optimization of the current strategy for surface display of the single-chain format in order to facilitate yeast display engineering of a broader range of scTCRs.
Blood chimerism has been reported sporadically among visceral transplant recipients, mostly in association with graft-vs-host disease (GVHD). We hypothesized that a higher degree of mixed chimerism would be observed in multivisceral (MVTx) than in isolated intestinal (iITx) and isolated liver transplant (iLTx) recipients, regardless of GVHD. We performed a longitudinal prospective study investigating multilineage blood chimerism with flow cytometry in 5 iITx and 4 MVTx recipients up to one year post-transplant. Although only one iITx patient experienced GVHD, T-cell mixed chimerism was detected in 8 out of 9 iITx/MVTx recipients. Chimerism was significantly lower in the four subjects who displayed early moderate to severe rejection. Pre-formed high titer donor-specific antibodies, bound in vivo to the circulating donor cells, were associated with an accelerated decline in chimerism. Blood chimerism was also studied in 10 iLTx controls. Among non-sensitized patients, MVTx recipients exhibited greater T and B-cell chimerism than either iITx and iLTx recipients. Myeloid lineage chimerism was present exclusively among iLTx and MVTx (6/13) recipients, suggesting that its presence required the hepatic allograft. Our study demonstrates, for the first time, frequent T cell chimerism without GVHD following visceral transplantation and a possible relationship with reduced rejection rate in MVTx recipients.
Transduction of exogenous T cell receptor (TCR) genes into patients’ activated peripheral blood T cells is a potent strategy to generate large numbers of specific T cells for adoptive therapy of cancer and viral diseases. However, the remarkable clinical promise of this powerful approach is still being overshadowed by a serious potential consequence: mispairing of the exogenous TCR chains with endogenous TCR chains. These “mixed” heterodimers can generate new specificities that result in graft-versus-host reactions. Engineering TCR constant regions of the exogenous chains with a cysteine promotes proper pairing and reduces the mispairing, but, as we show here, does not eliminate the formation of mixed heterodimers. By contrast, deletion of the constant regions, through use of a stabilized Vα/Vβ single-chain TCR (scTv), avoided mispairing completely. By linking a high-affinity scTv to intracellular signaling domains, such as Lck and CD28, the scTv was capable of activating functional T cell responses in the absence of either the CD3 subunits or the co-receptors, and circumvented mispairing with endogenous TCRs. Such transduced T cells can respond to the targeted antigen independent of CD3 subunits via the introduced scTv, without the transduced T cells acquiring any new undefined and potentially dangerous specificities.
Effective chimeric antigen receptor (CAR)-T cell therapy is dependent on optimal cell culture methods conducive to the activation and expansion of T cells ex vivo , as well as infection with CAR. Media formulations used in CAR-T cell manufacturing have not been optimized for gene delivery, cell expansion, and overall potency. Bioactive components and derivatives that support the generation of functionally-competent T cell progeny with long-lasting persistence are largely undefined. Current media formulations rely on fetal bovine serum (FBS) or human serum (HS), which suffer from a lack of consistency or supply issues. We recognize that components of blood cellular fractions that are absent in serum may have therapeutic value. Here we investigate whether a concentrated growth factor extract, purified from human transfusion grade whole blood fractions, and marketed as PhysiologixTM xeno-free (XF) hGFC (Phx), supports CAR-T cell expansion and function. We show that Phx supports T cell proliferation in clinical and research-grade media. We also show that Phx treatment enhances lentiviral-mediated gene expression across a wide range of multiplicity of infections (MOIs). We compared the ability of anti-GD-2 CAR-T cells expanded ex vivo in medium conditioned with either Phx or HS to clear tumor burden in a human xenograft model of neuroblastoma. We show that T cells expanded in Phx have superior engraftment and potency in vivo , as well as CAR-induced cytolytic activity in vitro . Metabolomic profiling revealed several factors unique to Phx that may have relevance for CAR-T cell preclinical discovery, process development, and manufacturing. In particular, we show that carnosine, a biogenic amine modestly enriched in Phx relative to HS, enhances lentiviral gene delivery in activated T cells. By limiting extracellular acidification, carnosine enhances the metabolic fitness of T cells, shifting their metabolic profile from an acidic, stressed state toward an oxidative, energetic state. These findings are very informative regarding potential derivatives to include in medium customized for gene delivery and overall potency for T cell adoptive immunotherapies.
Key Points High-affinity tumor/self antigen-specific TCRs that surpass the threshold for normal thymic selection can be safe for TCR gene therapy. T cells that express endogenous TCRs that are self-reactive can survive in the periphery with diminished TCR expression levels.
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