Biology subjects

Kuball, J.

Publications and source records attributed to Kuball, J..

4 recordsLinked to original sources

bioRxiv2021

Challenges of γ9δ2TCR affinity maturation when using phage display

BackgroundOver the past years we showed that the efficacy of {beta}T cells engineered to express a defined {gamma}{delta}TCR (TEG) depends on the functional avidity of the {gamma}9{delta}2TCR. We hypothesized that functional avidity mediated through {gamma}9{delta}2TCR in the TEG format could be further enhanced by increasing affinity of the {gamma}9{delta}2TCR. MethodsWe attempted to overcome limited affinity of natural occurring {gamma}9{delta}2TCRs through affinity maturation by phage display using a library containing mutations in CDR1 and CDR2 of both TCR chains. ConclusionAffinity maturation of {gamma}9{delta}2TCR by using phage display was not successful. The largest hurdle was the periplasmic expression of {gamma}9{delta}2TCR constructs in E.coli which is a prerequisite for successful phage display. The underlying reason for this lack of expression was the instability of the single chain (sc)TCR format. Expression of scTCR formats in HEK293F cells yielded only 15-20% correctly folded scTCR.

immunology↗

bioRxiv2021

Behavioral-transcriptomic landscape of engineered T cells targeting human cancer organoids

Cellular immunotherapies are rapidly gaining clinical importance, yet predictive platforms for modeling their mode of action are lacking. Here, we developed a dynamic immuno-organoid 3D imaging-transcriptomics platform; BEHAV3D, to unravel the behavioral and underlying molecular mechanisms of solid tumor targeting. Applied to an emerging cancer metabolome-sensing immunotherapy: TEGs, we first demonstrate targeting of multiple breast cancer subtypes. Live-tracking of over 120,000 TEGs revealed a diverse behavioral landscape and identified a super engager cluster with serial killing capability. Inference of single-cell behavior with transcriptomics identified the gene signature of super engager killer TEGs, which contained 27 genes with no previously described T cell function. Furthermore, guided by a dynamic type 1 interferon (IFN-I) signaling module induced by high TEG-sensitive organoids, we show that IFN-I can prime resistant organoids for TEG-mediated killing. Thus, BEHAV3D characterizes behavioral-phenotypic heterogeneity of cellular immunotherapies and holds promise for improving solid tumor-targeting in a patient-specific manner.

cancer biology↗

bioRxiv2021

Tumor-reactivity of d2 negative gdT cells and the role of the gdT cell receptor

{beta}T cells engineered to express a defined {gamma}{delta}TCR (TEG) to attack cancer cells have shown great promise when using a {gamma}9{delta}2TCR to redirect {beta}T cells (1-9). Reports by us (1-9) and recent reports by others (10, 11) support the key role of the {gamma}9{delta}2TCR in cancer recognition. We further emphasized the crucial role of the {delta}TCR chain and that differences in CDR3 sequences of the {delta}TCR chain modulates functional avidity of TEGs (2, 8). We and others demonstrated that also {delta}2 negative {gamma}{delta}TCRs are able to redirect {beta}T cells towards different tumor cell lines (12-15). However, some studies suggest that {delta}2 negative {gamma}{delta}TCRs play a minor role in the tumor recognition by {delta}2 negative {gamma}{delta}T cells (16, 17). In addition for both modes of action for tumor-recognition, {delta}2 negative {gamma}{delta}TCR-dependent and -independent, it has been suggested that CMV infection is not only a major driver of {delta}2 negative {gamma}{delta}T cell expansion (18) but also induces tumor-cross reactive {delta}2 negative {gamma}{delta}T cells (19-21). Therefore, we aimed to systematically explore frequencies of tumor reactive {delta}2 negative {gamma}{delta}T cells in naive repertoires (cord blood) and patients with or without CMV infection and examined the potential role the parental {delta}2 negative {gamma}{delta}TCR in anti-tumor reactivity of selected clones. We observed that approximately 30% of all tested clones were tumor-reactive, though no differences were observed between different sources. Surprisingly, none of the so far tested {gamma}{delta}TCR did mediate strong anti-tumor reactivity of the parental clones. Though numbers of tested TCR sequences are still low, our data imply that tumor-reactivity of {delta}2 negative {gamma}{delta}T cells is frequently not mediated by the {delta}2 negative {gamma}{delta}TCR alone.

immunology↗

bioRxiv2021

Enhancing cancer targeting of γ9δ2TCR through modified NKG2D co-stimulation

Despite the ability of {gamma}{delta}T cells to mediate tumor killing independently of MHC recognition, all the clinical trials that have been carried out using these cells showed low response rate in patients, in part due to its poor proliferation ability. Recently, a new generation of CAR-T cells called {beta}T cells engineered to express a defined {gamma}{delta}TCR (TEG) has been developed. TEGs are {beta}T cells engineered to express a defined {gamma}{delta}TCR. These cells are able to mediate effective antitumor reactivity without showing any reactivity towards healthy tissue, and combine the best qualities of both {beta}T and {gamma}{delta}T cells. In fact, the high affinity {gamma}9{delta}2TCR clone 5 has recently been selected within the TEG format as a clinical candidate (TEG001). Here we present a strategy to improve the antitumor activity of TEG001 by co-expressing an activating chimeric co-receptor together with {gamma}{delta}TCR-Cl5.Therefore, we developed three different co-receptors by fusing the extracellular domain of the activating cell surface receptor NKG2D, that is able to bind stress induced ligands typically expressed on tumor cells, to the cytoplasmic signaling domains of the T cell costimulatory proteins ICOS, CD28 and 4-1BB. We determined that introduction of the chimeric co-receptors NKG2D-CD28wt and NKG2D-4-1BBCD28TM improved the activity of TEG001 against tumors that were recognized by {gamma}{delta}TCR-Cl5 and expressed NKG2D ligands, but did not affect tumors that either were not recognized by {gamma}{delta}TCR-Cl5 or did not express NKG2D ligands. This chimeric co-receptors approach open a wide range of opportunities that lead to a next generation of TEGs.

immunology↗