Human cytomegalovirus (CMV) infections and relapse of disease remain major problems after allogeneic stem cell transplantation (allo-SCT), in particular in combination with CMV-negative donors or cordblood transplantations. Recent data suggest a paradoxical association between CMV reactivation after allo-SCT and reduced leukemic relapse. Given the potential of Vδ2-negative γδT cells to recognize CMV-infected cells and tumor cells, the molecular biology of distinct γδT-cell subsets expanding during CMV reactivation after allo-SCT was investigated. Vδ2(neg) γδT-cell expansions after CMV reactivation were observed not only with conventional but also cordblood donors. Expanded γδT cells were capable of recognizing both CMV-infected cells and primary leukemic blasts. CMV and leukemia reactivity were restricted to the same clonal population, whereas other Vδ2(neg) T cells interact with dendritic cells (DCs). Cloned Vδ1 T-cell receptors (TCRs) mediated leukemia reactivity and DC interactions, but surprisingly not CMV reactivity. Interestingly, CD8αα expression appeared to be a signature of γδT cells after CMV exposure. However, functionally, CD8αα was primarily important in combination with selected leukemia-reactive Vδ1 TCRs, demonstrating for the first time a co-stimulatory role of CD8αα for distinct γδTCRs. Based on these observations, we advocate the exploration of adoptive transfer of unmodified Vδ2(neg) γδT cells after allo-SCT to tackle CMV reactivation and residual leukemic blasts, as well as application of leukemia-reactive Vδ1 TCR-engineered T cells as alternative therapeutic tools.

Major limitations of currently investigated␣␤T cells redirected against cancer by transfer of tumor-specific ␣␤TCR arise from their low affinity, MHC restriction, and risk to mediate self-reactivity after pairing with endogenous ␣ or ␤TCR chains. Therefore, the ability of a defined ␥9␦2TCR to redirect ␣␤T cells selectively against tumor cells was tested and its molecular interaction with a variety of targets investigated. Functional analysis revealed that a ␥9␦2TCR efficiently reprograms both CD4 ؉ and CD8 ؉ IntroductionThe major challenge in the field of adoptive immunotherapy is the generation of tumor-reactive ␣␤T cells which can be applied to a broad variety of cancer patients. To facilitate the rapid generation of tumor-reactive ␣␤T cells, it has been proposed that ␣␤T cells can be reprogrammed with genes encoding for a tumor-specific ␣␤TCR or a chimeric receptor. 1 Several such receptors are already being used to redirect ␣␤T cells in phase 1 clinical trials. 1,2 However, reprogramming ␣␤T cells with defined ␣␤TCRs is substantially hampered by their restriction to HLA types, thus limiting the number of patients who can be treated with one ␣␤TCR. In addition, pairing of introduced with endogenous ␣␤TCR chains can induce life-threatening autoreactivity. 3,4 One attractive alternative to mediate a selective antitumor reactivity with a high-affinity TCR might arise from the ability of ␥␦T cells to mediate antitumor reactivity while ignoring a healthy environment. [5][6][7] Isolated ␥9␦2T cells efficiently kill tumor cells of hematologic malignancies and from solid tumors. 7 However, the function and proliferation capacity of ␥␦T cells is frequently heavily impaired in cancer patients 8 making autologous ␥␦T cells less attractive for immune interventions. On the other hand, as end-stage cancer patients can easily elicit ␣␤T-cell immune responses against, for example, viral Ags, 9,10 ␣␤T cells might serve as carriers for broadly tumor-reactive ␥␦TCRs.The recognition of mevalonate metabolites (phosphoantigens) 11 which are overexpressed in a broad range of tumor cells has been suggested as an important mechanism by which multiple ␥9␦2TCR can sense malignant transformation as the recognition involves TCR domains which are conserved in most ␥9␦2TCRs. [12][13][14] In addition, ␥9␦2TCR G115 has been also suggested to bind to a complex of Apolipoprotein AI (ApoAI) and F1-ATPase, 15 a complex mitochondrial enzyme found on the surface of many malignant cells. 16 This knowledge might allow a rational design of ␥␦T cell-based immunotherapies. Therefore, we investigated whether a defined ␥9␦2TCR can be efficiently expressed in ␣␤T cells, mediate tumor-specific proliferation of ␣␤T cells, and redirect both effector CD8 ϩ and helper CD4 ϩ ␣␤T-cell subsets against a broad panel of tumor cell lines while ignoring normal cells in vitro and in vivo. MethodsCell lines, Abs, the retroviral transduction and expansion of ␣␤T cells, functional T-cell assays 11,[17][18][19][20] as well as the animal model used are described in supp...

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...

Purpose: Engineering T cells with receptors to redirect the immune system against cancer has most recently been described as a scientific breakthrough. However, a main challenge remains the GMP-grade purification of immune cells selectively expressing the introduced receptor in order to reduce potential side effects due to poorly or nonengineered cells.Experimental Design: In order to test a novel purification strategy, we took advantage of a model gdT cell receptor (TCR), naturally interfering with endogenous TCR expression and designed the optimal retroviral expression cassette to achieve maximal interference with endogenous TCR chains. Following retroviral transduction, nonengineered and poorly engineered immune cells characterized by a high endogenous abTCR expression were efficiently depleted with GMP-grade anti-abTCR beads. Next, the engineered immune cells were validated for TCR expression, function against a panel of tumor cell lines and primary tumors and potential allo-reactivity. Engineered immune cells were further validated in two humanized mouse tumor models.Results: The untouched enrichment of engineered immune cells translated into highly purified receptor-engineered cells with strong antitumor reactivity both in vitro and in vivo. Importantly, this approach eliminated residual allo-reactivity of engineered immune cells. Our data demonstrate that even with long-term suboptimal interference with endogenous TCR chains such as in resting cells, allo-reactivity remained absent and tumor control preserved.Conclusions: We present a novel enrichment method for the production of untouched engineered immune cells, ready to be translated into a GMP-grade method and potentially applicable to all receptor-modified cells even if interference with endogenous TCR chains is far from complete.

γ9δ2T cells play a critical role in daily cancer immune surveillance by sensing cancer-mediated metabolic changes. However, a major limitation of the therapeutic application of γ9δ2T cells is their diversity and regulation through innate co-receptors. In order to overcome natural obstacles of γ9δ2T cells, we have developed the concept of T cells engineered to express a defined γδT cell receptor (TEGs). This next generation of chimeric antigen receptor engineered T (CAR-T) cells not only allows for targeting of hematological but also of solid tumors and, therefore, overcomes major limitations of many CAR-T and γδT cell strategies. Here, we report on the development of a robust manufacturing procedure of T cells engineered to express the high affinity Vγ9Vδ2T cell receptor (TCR) clone 5 (TEG001). We determined the best concentration of anti-CD3/CD28 activation and expansion beads, optimal virus titer, and cell density for retroviral transduction, and validated a Good Manufacturing Practice (GMP)-grade purification procedure by utilizing the CliniMACS system to deplete non- and poorly-engineered T cells. To the best of our knowledge, we have developed the very first GMP manufacturing procedure in which αβTCR depletion is used as a purification method, thereby delivering untouched clinical grade engineered immune cells. This enrichment method is applicable to any engineered T cell product with a reduced expression of endogenous αβTCRs. We report on release criteria and the stability of TEG001 drug substance and TEG001 drug product. The GMP-grade production procedure is now approved by Dutch authorities and allows TEG001 to be generated in cell numbers sufficient to treat patients within the approved clinical trial NTR6541. NTR6541 will investigate the safety and tolerability of TEG001 in patients with relapsed/refractory acute myeloid leukemia, high-risk myelodysplastic syndrome, and relapsed/refractory multiple myeloma.

Key Points We describe a novel allo-tumor–reactive and CD8α-dependent Vγ5Vδ1TCR. The molecular interface with proximity to the peptide-binding groove of HLA-A*24:02 is an essential determinant of recognition.

Backgroundγ9δ2T cells, which express Vγ9 and Vδ2 chains of the T cell receptor (TCR), mediate cancer immune surveillance by sensing early metabolic changes in malignant leukemic blast and not their healthy hematopoietic stem counterparts via the γ9δ2TCR targeting joined conformational and spatial changes of CD277 at the cell membrane (CD277J). This concept led to the development of next generation CAR-T cells, so-called TEGs: αβT cells Engineered to express a defined γδTCR. The high affinity γ9δ2TCR clone 5 has recently been selected within the TEG format as a clinical candidate (TEG001). However, exploring safety and efficacy against a target, which reflects an early metabolic change in tumor cells, remains challenging given the lack of appropriate tools. Therefore, we tested whether TEG001 is able to eliminate established leukemia in a primary disease model, without harming other parts of the healthy hematopoiesis in vivo.MethodsSeparate sets of NSG mice were respectively injected with primary human acute myeloid leukemia (AML) blasts and cord blood-derived human progenitor cells from healthy donors. These mice were then treated with TEG001 and mock cells. Tumor burden and human cells engraftment were measured in peripheral blood and followed up over time by quantifying for absolute cell number by flow cytometry. Statistical analysis was performed using non-parametric 2-tailed Mann-Whitney t-test.ResultsWe successfully engrafted primary AML blasts and healthy hematopoietic cells after 6–8 weeks. Here we report that metabolic cancer targeting through TEG001 eradicated established primary leukemic blasts in vivo, while healthy hematopoietic compartments derived from human cord-blood remained unharmed in spite of TEGs persistence up to 50 days after infusion. No additional signs of off-target toxicity were observed in any other tissues.ConclusionWithin the limitations of humanized PD-X models, targeting CD277J by TEG001 is safe and efficient. Therefore, we have initiated clinical testing of TEG001 in a phase I first-in-human clinical trial (NTR6541; date of registration 25 July 2017).Electronic supplementary materialThe online version of this article (10.1186/s40425-019-0558-4) contains supplementary material, which is available to authorized users.

Backgroundγ9δ2 T cells hold great promise as cancer therapeutics because of their unique capability of reacting to metabolic changes with tumor cells. However, it has proven very difficult to translate this promise into clinical success.MethodsIn order to better utilize the tumor reactivity of γ9δ2T cells and combine this with the great potential of T cell engager molecules, we developed a novel bispecific molecule by linking the extracellular domains of tumor-reactive γ9δ2TCRs to a CD3-binding moiety, creating gamma delta TCR anti-CD3 bispecific molecules (GABs). GABs were tested in vitro and in vivo for ability to redirect T lymphocytes to a variety of tumor cell lines and primary patient material.ResultsGABs utilizing naturally occurring high affinity γ9δ2TCRs efficiently induced αβT cell mediated phosphoantigen-dependent recognition of tumor cells. Reactivity was substantially modulated by variations in the Vδ2 CDR3-region and the BTN2A1-binding HV4-region between CDR2 and CDR3 of the γ-chain was crucial for functionality. GABs redirected αβT cells against a broad range of hematopoietic and solid tumor cell lines and primary acute myeloid leukemia. Furthermore, they enhanced infiltration of immune cells in a 3D bone marrow niche and left healthy tissues intact, while eradicating primary multiple myeloma cells. Lastly, GABs constructed from natural high affinity γ9δ2TCR sequences significantly reduced tumor growth in vivo in a subcutaneous myeloma xenograft model.ConclusionsWe conclude that GABs allow for the introduction of metabolic targeting of cancer cells to the field of T cell engagers.