γ9 and δ2CDR3 domains regulate functional avidity of T cells harboring γ9δ2TCRs

Abstract

Immunotherapy with innate immune cells has recently evoked broad interest as a novel treatment option for cancer patients. ␥9␦2T cells in particular are emerging as an innate cell population with high frequency and strong antitumor reactivity, which makes them and their receptors promising candidates for immune interventions. However, clinical trials have so far reported only limited tumor control by adoptively transferred ␥9␦2T cells. As a potential explanation for this lack of efficacy, we found unexpectedly high variability in tumor recognition within the physiologic human ␥9␦2T-cell repertoire, which is substantially regulated by the CDR3 domains of individual ␥9␦2TCRs. In the present study, we demonstrate that the reported molecular requirements of CDR3 domains to interact with target cells shape the physiologic ␥9␦2T-cell repertoire and, most likely, limit the protective and thera- IntroductionImmunotherapy with innate immune cells has become widely used because this approach obviates the need to match a cellular product to a defined HLA haplotype, allowing adoptive immunotherapies to be used in virtually any cancer patient without extensive in vitro selection or manipulation of the cellular product. 1-4 ␥9␦2T cells are promising as an innate cell population for this purpose because they are usually observed at high frequencies in the human peripheral blood and provide a strong antitumor reactivity against various solid and hematologic cancers. 5 However, within ␥9␦2T-cell populations, individual clones display great diversity in the repertoire because of the activating or inhibitory receptors expressed. 6 Selecting innate cell products for certain cell types, such as those with a low level of inhibitory receptors, therefore seems plausible, especially considering the limited efficacy of adoptively transferred innate immune cells in clinical trials. 7,8 An alternative proposal is to engineer cells to express defined activating innate receptors that mediate strong antitumor reactivity, such as a defined ␥9␦2TCR, 9 which could pave the way for readily available and more effective cellular products. However, the molecular details of how a ␥9␦2TCR interacts with its target are not fully understood, making it challenging to select defined ␥9␦2T cells or to engineer T cells with defined ␥9␦2TCRs.In "classic" immunoreceptors such as ␣␤TCRs or Igs, the complementary determining regions (CDRs) determine affinity and specificity for a specific (peptide) epitope. V(D)J recombination allows the creation of a highly variable CDR repertoire ensuring recognition of an immense collection of antigens. ␥9␦2T cells also possess a rearranged TCR that mediates recognition. The phosphoantigen isopentenyl pyrophosphate (IPP) has been suggested to be a key player in ␥9␦2TCR-mediated activation, 5,10,11 but no direct interaction between a ␥9␦2TCR and IPP or any other phosphoantigen has ever been demonstrated. It was previously suggested that positively charged residues within the ␥9␦2TCR are crucial for the response to negatively...

Untouched GMP-Ready Purified Engineered Immune Cells to Treat Cancer

“…The highly tumor reactive g9d2TCR chain genes were obtained via combinatorial-gdTCR chain exchange (28). The TCRg chain was derived from gdT cell clone G115 (33) and TCRd chain from clone 5 (28), codon optimized (Geneart Life Technologies) and cloned into the retroviral vector pBullet as single TCR chain vectors containing either g-chain-IRES-neomycine or d-chain-IRES-puromycine.…”

“…51 Chromium-release assay for cell-mediated cytotoxicity was previously described (28). In brief, target cells were labeled overnight with 100 mCu 51 Cr and incubated for 4 to 5 h with transduced T cells in five effector-to-target ratios (E:T) between 30:1 and 0.3:1.…”

“…gdT cells are considered as an innate-like population of immune cells recognizing molecular stress signals on infected or malignant cells. A subset of gdT cells express a TCR composed of Vg9 and Vd2 chains that sense accumulated nonpeptidic pyrophosphate molecules (phosphoantigens), intermediates of a deregulated mevalonate pathway of isoprenoid synthesis, via BTN3A1 (CD277) and reported by us (27)(28)(29) and others (32) to display potent cytotoxicity against a broad range of tumor species. Other intriguing properties of gdTCRs are the absence of forming potentially self-reactive mixed TCR dimers, their HLA-independency, absence of immunogenicity, which can either result in reduced therapeutic efficacy or substantial toxicity and avoidance of education and long-term tolerance when expressing innate immune receptors out of the context of an innate immune cell (30,31).…”

“…By utilizing a novel tumor-specific immune receptor naturally interfering with endogenous abTCR chains (27)(28)(29)(30), an optimized expression system, and clinical grade anti-abTCR beads, we developed a highly pure engineered cellular product with improved antitumor activity. The production process can be readily translated to meet GMP-grade standards and the resulting cellular medicine can be applied as part of an immune intervention strategy in a broad population of cancer patients in autologous, allogeneic, and third-party situations.…”

“…Alternative simple and cost-effective solutions are clearly still needed. Therefore, classical methods such as the interference with endogenous abTCR chains by introducing physiological strong abTCR competitors for the components of the CD3 complex (24)(25)(26) such as gdTCR chains (27)(28)(29), remain valid alternatives in order to substantially reduce endogenous abTCR expression. Besides the reduction of endogenous abTCR expression, gdTCRs are promising immune receptors to retarget abT cells against cancer as reviewed extensively in refs.…”