γδT cells play an important role in cancer immunosurveillance and are able to distinguish malignant cells from their healthy counterparts via their γδTCR. This characteristic makes γδT cells an attractive candidate for therapeutic application in cancer immunotherapy. Previously, we have identified a novel CD8α‐dependent tumor‐specific allo‐HLA‐A*24:02‐restricted Vγ5Vδ1TCR with potential therapeutic value when used to engineer αβT cells from HLA‐A*24:02 harboring individuals. αβT cells engineered to express this defined Vγ5Vδ1TCR (TEG011) have been suggested to recognize spatial changes in HLA‐A*24:02 present selectively on tumor cells but not their healthy counterparts. However, in vivo efficacy and toxicity studies of TEG011 are still limited. Therefore, we extend the efficacy and toxicity studies as well as the dynamics of TEG011 in vivo in a humanized HLA‐A*24:02 transgenic NSG (NSG‐A24:02) mouse model to allow the preparation of a first‐in‐men clinical safety package for adoptive transfer of TEG011. Mice treated with TEG011 did not exhibit any graft‐versus‐host disease‐like symptoms and extensive analysis of pathologic changes in NSG‐A24:02 mice did not show any off‐target toxicity of TEG011. However, loss of persistence of TEG011 in tumor‐bearing mice was associated with the outgrowth of extramedullary tumor masses as also observed for mock‐treated mice. In conclusion, TEG011 is well tolerated without harming HLA‐A*24:02+ expressing healthy tissues, and TEG011 persistence seems to be crucial for long‐term tumor control in vivo.

The population study of threatened species requires marking techniques that do not affect the survival of individuals. In this study, we evaluated the effectiveness of visible implant elastomer (VIE) in the identification and survival of individuals of the salamander Parvimolge townsendi. We compared three salamander groups under different treatments: intervened, simulated intervention and control. No significant mortality differences were observed between groups (with two, none, and one individual, respectively), but implant migration was observe in four of 10 intervened individuals. Although VIE does not have a significant effect on survival, implant migration should be considered before use in population studies.

T cell engineering strategies offer cures to patients and have entered clinical practice with chimeric antibody-based receptors; abT cell receptor (abTCR)-based strategies are, however, lagging behind. To allow a more rapid and successful translation to successful concepts also using abTCRs for engineering, incorporating a method for the purification of genetically modified T cells, as well as engineered T cell deletion after transfer into patients, could be beneficial. This would allow increased efficacy, reduced potential side effects, and improved safety of newly to-be-tested lead structures. By characterizing the antigen-binding interface of a good manufacturing process (GMP)-grade anti-abTCR antibody, usually used for depletion of abT cells from stem cell transplantation products, we developed a strategy that allows for the purification of untouched abTCR-engineered immune cells by changing 2 amino acids only in the TCRb chain constant domain of introduced TCR chains. Alternatively, we engineered an antibody that targets an extended mutated interface of 9 amino acids in the TCRb chain constant domain and provides the opportunity to further develop depletion strategies of engineered immune cells.

γδT cell receptors (γδTCRs) recognize a broad range of malignantly transformed cells in mainly a major histocompatibility complex (MHC)-independent manner, making them valuable additions to the engineered immune effector cell therapy that currently focuses primarily on αβTCRs and chimeric antigen receptors (CARs). As an exception to the rule, we have previously identified a γδTCR, which exerts antitumor reactivity against HLA-A*24:02-expressing malignant cells, however without the need for defined HLA-restricted peptides, and without exhibiting any sign of off-target toxicity in humanized HLA-A*24:02 transgenic NSG (NSG-A24:02) mouse models. This particular tumor-HLA-A*24:02-specific Vγ5Vδ1TCR required CD8αα co-receptor for its tumor reactive capacity when introduced into αβT cells engineered to express a defined γδTCR (TEG), referred to as TEG011; thus, it was only active in CD8+ TEG011. We subsequently explored the concept of additional redirection of CD4+ T cells through co-expression of the human CD8α gene into CD4+ and CD8+ TEG011 cells, later referred as TEG011_CD8α. Adoptive transfer of TEG011_CD8α cells in humanized HLA-A*24:02 transgenic NSG (NSG-A24:02) mice injected with tumor HLA-A*24:02+ cells showed superior tumor control in comparison to TEG011, and to mock control groups. The total percentage of mice with persisting TEG011_CD8α cells, as well as the total number of TEG011_CD8α cells per mice, was significantly improved over time, mainly due to a dominance of CD4+CD8+ double-positive TEG011_CD8α, which resulted in higher total counts of functional T cells in spleen and bone marrow. We observed that tumor clearance in the bone marrow of TEG011_CD8α-treated mice associated with better human T cell infiltration, which was not observed in the TEG011-treated group. Overall, introduction of transgenic human CD8α receptor on TEG011 improves antitumor reactivity against HLA-A*24:02+ tumor cells and further enhances in vivo tumor control.

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IntroductionWe have recently developed a novel T cell engager concept by utilizing γ9δ2TCR as tumor targeting domain, named gamma delta TCR anti-CD3 bispecific molecule (GAB), targeting the phosphoantigen-dependent orchestration of BTN2A1 and BTN3A1 at the surface of cancer cells. GABs are made by the fusion of the ectodomains of a γδTCR to an anti-CD3 single chain variable fragment (scFv) (γδECTO-αCD3), here we explore alternative designs with the aim to enhance GAB effectivity.MethodsThe first alternative design was made by linking the variable domains of the γ and δ chain to an anti-CD3 scFv (γδVAR-αCD3). The second alternative design was multimerizing γδVAR-αCD3 proteins to increase the tumor binding valency. Both designs were expressed and purified and the potency to target tumor cells by T cells of the alternative designs was compared to γδECTO-αCD3, in T cell activation and cytotoxicity assays.Results and discussionThe γδVAR-αCD3 proteins were poorly expressed, and while the addition of stabilizing mutations based on finding for αβ single chain formats increased expression, generation of meaningful amounts of γδVAR-αCD3 protein was not possible. As an alternative strategy, we explored the natural properties of the original GAB design (γδECTO-αCD3), and observed the spontaneous formation of γδECTO-αCD3-monomers and -dimers during expression. We successfully enhanced the fraction of γδECTO-αCD3-dimers by shortening the linker length between the heavy and light chain in the anti-CD3 scFv, though this also decreased protein yield by 50%. Finally, we formally demonstrated with purified γδECTO-αCD3-dimers and -monomers, that γδECTO-αCD3-dimers are superior in function when compared to similar concentrations of monomers, and do not induce T cell activation without simultaneous tumor engagement. In conclusion, a γδECTO-αCD3-dimer based GAB design has great potential, though protein production needs to be further optimized before preclinical and clinical testing.

Dry jungles harbor a large number of species with restricted distribution or endemic, and little is known about the ecological processes involving these species. Such as activity periods, diet, or predation. In October 2016, we registered a predation event on the Mexican endemic rodent Xenomys nelsoni by the colubrid snake Senticolis triapsis in the Chamela-Cuixmala Biosphere Reserve. This rodent species is listed as endangered by the Mexican law. This is the first report of predation on individuals of this threatened rodent genus.