Novel CD19-specific γ/δ TCR-T cells in relapsed or refractory diffuse large B-cell lymphoma.

BACKGROUND: T cell receptor (TCR)-T cells possess similar effector function, but milder and more durable signal activation compared with chimeric antigen receptor-T cells. TCR-T cell therapy is another active field of cellular immunotherapy for cancer. METHODS: We previously developed a human anti-CD19 antibody (ET190L1) and generated novel CD19-specific γ/δ TCR-T cells, ET019003, by fusing the Fab fragment of ET190L1 with γ/δ TCR constant chain plus adding an ET190L1-scFv/CD28 co-stimulatory molecule. ET019003 cells were tested in preclinical studies followed by a phase 1 clinical trial. RESULTS: ET019003 cells produced less cytokines but retained comparable antitumor potency than ET190L1-CAR-T cells in vivo and in vitro. In the first-in-human trial, eight patients with relapsed or refractory DLBCL were treated. CRS of grade 1 was observed in three (37.5%) patients; ICANS of grade 3 was noted in one (12.5%) patient. Elevation of serum cytokines after ET019003 infusion was almost modest. With a median follow-up of 34 (range 6-38) months, seven (87.5%) patients attained clinical responses and six (75%) achieved complete responses (CR). OS, PFS and DOR at 3 years were 75.0%, 62.5%, and 71.4%, respectively. Notably, patient 1 with primary CNS lymphoma did not experience CRS or ICANS and got an ongoing CR for over 3 years after infusion, with detectable ET019003 cells in CSF. ET019003 showed striking in vivo expansion and persisted in 50% of patients at 12 months. Three patients received a second infusion, one for consolidation therapy after CR and two for salvage therapy after disease progression, but no response was observed. ET019003 expansion was striking in the first infusion, but poor in the second infusion. CONCLUSIONS: CD19-specific γ/δ TCR-T cells, ET019003, had a good safety profile and could induce rapid responses and durable CR in patients with relapsed or refractory DLBCL, even primary CNS lymphoma, presenting a novel and potent therapeutic option for these patients. TRIAL REGISTRATION: NCT04014894.

Therapeutic Potential of Ex Vivo Expanded γδ T Cells against Osteosarcoma Cells.

Immunotherapy is an attractive therapeutic strategy for the treatment of osteosarcoma (OS). The unique features of γδ T cells have made them popular for cancer immunotherapy. Here, we expanded γδ T cells using human peripheral blood mononuclear cells (PBMCs) and investigated their therapeutic potential against OS cells. PBMCs from healthy donors were cultured for 10 days with CON medium (unstimulated control); EX media, CON with recombinant human interleukin-2 (rhIL-2) and zoledronate; and EX28 media, CON with rhIL-2, zoledronate, and CD3/CD28 activator. The expanded γδ T cells were isolated by magnetic cell separation or fluorescence-activated cell sorting, cultured with two OS cell lines (KHOS/NP and MG-63) at various cell ratios with or without doxorubicin or ifosfamide, and analyzed for cytotoxicity and cytokine secretion. The number of CD3+γδTCR+Vγ9+ triple-positive γδ T cells and concentrations of IFN-γ and TNF-α were highest in the rhIL-2 (100 IU) and zoledronate (1 µM) supplemented culture conditions. The CD3/CD28 agonist did not show any additional effects on γδ T cell expansion. The expanded γδ T cells exhibited potent in vitro cytotoxicity against OS in a ratio- and time-dependent manner. The γδ T cells may enhance the effect of chemotherapeutic agents against OS and may be a new treatment strategy, including chemo-immunotherapy, for OS.

Current progress in chimeric antigen receptor T cell therapy for glioblastoma multiforme.

Glioblastoma multiforme (GBM) is one of the deadliest brain tumors with an unfavorable prognosis and overall survival of approximately 20 months following diagnosis. The current treatment for GBM includes surgical resections and chemo- and radiotherapeutic modalities, which are not effective. CAR-T immunotherapy has been proven effective for CD19-positive blood malignancies, and the application of CAR-T cell therapy for solid tumors including GBM offers great hope for this aggressive tumor which has a limited response to current treatments. CAR-T technology depends on the use of patient-specific T cells genetically engineered to express specific tumor-associated antigens (TAAs). Interaction of CAR-T cells with tumor cells triggers the destruction/elimination of these cells by the induction of cytotoxicity and the release of different cytokines. Despite the great promise of CAR-T cell-based therapy several challenges exist. These include the heterogeneity of GBM cancer cells, aberrant various signaling pathways involved in tumor progression, antigen escape, the hostile inhibitory GBM microenvironment, T cell dysfunction, blood-brain barrier, and defective antigen presentation. All need to be addressed before full application at the clinical level can begin. Herein we provide a focused review of the rationale for the use of different types of CAR-T cells (including FcγRs), the different GBM-associated antigens, the challenges still facing CAR-T-based therapy, and means to overcome such challenges. Finally, we enumerate currently completed and ongoing clinical trials, highlighting the different ways such trials are designed to overcome specific problems. Exploitation of the full potential of CAR-T cell therapy for GBM depends on their solution.

Adoptive Transfer of Phosphoantigen-Specific γδ T Cell Subset Attenuates Mycobacterium tuberculosis Infection in Nonhuman Primates.

The dominant Vγ2Vδ2 T cell subset recognizes phosphoantigen and exists only in humans and nonhuman primates. Despite the discovery of γδ T cells >30 y ago, a proof-of-concept study has not been done to prove the principle that the Vγ2Vδ2 T cell subset is protective against Mycobacterium tuberculosis and other infections. In this study, we used an adoptive cell-transfer strategy to define the protective role of Vγ2Vδ2 T cells in a primate tuberculosis (TB) model. Vγ2Vδ2 T cells for adoptive transfer displayed central/effector memory and mounted effector functions, including the production of anti-M. tuberculosis cytokines and inhibition of intracellular mycobacteria. They also expressed CXCR3/CCR5/LFA-1 trafficking/tissue-resident phenotypes and consistently trafficked to the airway, where they remained detectable from 6 h through 7 d after adoptive transfer. Interestingly, the test group of macaques receiving transfer of Vγ2Vδ2 T cells at weeks 1 and 3 after high-dose (500 CFU) M. tuberculosis infection exhibited significantly lower levels of M. tuberculosis infection burdens in lung lobes and extrapulmonary organs than did the control groups receiving PBLs or saline. Consistently, adoptive transfer of Vγ2Vδ2 T cells attenuated TB pathology and contained lesions primarily in the infection site of the right caudal lung lobe, with no or reduced TB dissemination to other lobes, spleen, or liver/kidney; in contrast, the controls showed widespread TB dissemination. The proof-of-concept finding supports the view that the dominant Vγ2Vδ2 T cell subset may be included in the rational design of a TB vaccine or host-directed therapy.

Stimulated γδ T cells increase the in vivo efficacy of trastuzumab in HER-2+ breast cancer.

One fourth of women with HER-2(+) metastatic breast carcinoma are treated with a combination regimen with trastuzumab, but the frequent resistance to this Ab requires definition of new means to improve its bioactivity. The mechanisms of action of trastuzumab involve several pathways including Ab-dependent cellular cytotoxicity. Because human γδ T lymphocytes mediate Ab-dependent cellular cytotoxicity and can be activated further by phosphoantigens, these cells are prone to improve the efficacy of Abs, as recently demonstrated for CD20(+) B cell lymphomas. Whether this concept applies as well with carcinomas remained to be demonstrated in vivo, however. In this study, we asked whether a combination of trastuzumab and phosphoantigen-stimulated γδ lymphocytes increases the efficacy of trastuzumab against HER-2(+) breast carcinoma cell lines in vivo. We report that repeated infusions of this combination had a better efficacy than that of trastuzumab alone against HER-2(+) mammary carcinoma xenografts in mice. In these models, reduction of tumor growth was observed together with trastuzumab opsonization of HER-2(+) cells and tumor infiltration by γδ lymphocytes. In addition in humans, the mammary carcinomas of 27 of 30 patients showed significant γδ T cell infiltrates. Altogether, these findings indicate that combination of trastuzumab and stimulated γδ cells represents a new strategy to improve the efficacy of Herceptin (trastuzumab) in HER-2(+) breast cancer.

Complete remission of lung metastasis following adoptive immunotherapy using activated autologous gammadelta T-cells in a patient with renal cell carcinoma.

BACKGROUND: gammadelta T-cells have recently attracted considerable attention in the development of novel cancer immunotherapy, and several different approaches have been designed and employed in clinical trials. CASE REPORT: A patient with lung metastasis after radical nephrectomy for renal cell carcinoma had six cycles of adoptive immunotherapy using autologous in vitro-activated gammadelta T-cells followed by low-dose interleukin-2 and zoledronic acid intravenous infusion. Complete remission was achieved which has been maintained for 2 years without any additional treatment. Immunological analysis demonstrated a high level of interferon-gamma four hours through one day following the transfer and peripheral blood gammadelta T-cells increased 10-fold from the baseline value, 7 days after the transfer. No serious adverse events were observed. CONCLUSION: Adoptive immunotherapy using gammadelta T-cells was shown here to be clinically beneficial and safe, and may become a therapeutic option for patients with advanced RCC.

Large scale expansion of gamma 9 delta 2 T lymphocytes: Innacell gamma delta cell therapy product.

gamma9delta2 T lymphocytes are non-conventional lymphocytes presenting a direct cytotoxic effect against a broad range of tumour targets. These cells also secrete inflammatory cytokines that can boost the other components of the immune system. In contrast to conventional CD8(+) T cells, the cytotoxic effect of gamma9delta2 T lymphocytes does not depend on the expression of major histocompatibility complex molecules by target tumour cells. INNACELL gammadeltatrade mark is a cell therapy product obtained by ex vivo amplification of mononuclear cells. The stimulation is achieved by a specific synthetic agonist of gamma9delta2 T lymphocytes, bromohydrin pyrophosphate (BrHPP). After a single stimulation with BrHPP, gamma9delta2 T lymphocytes are expanded for 2 weeks in a closed system in culture medium with interleukin-2 (IL-2). On day 15, cells are washed and harvested in 4% human serum albumin. In this manufacturing process, the total cell population is expanded by approximately 10-fold and gamma9delta2 T lymphocytes undergo a specific 1000-fold expansion, corresponding to a gamma9delta2 T lymphocyte enrichment of more than 70% at the end of the culture. This manufacturing process is much simpler than most current cellular therapy approaches using conventional CD8(+) T-cell lines or clones: there is no final or initial separation, no purification step and no use of feeder cells; the specific T-cell receptor-mediated signal provided by BrHPP is sufficient to trigger the IL-2-dependent expansion of the gamma9delta2 subset, which then becomes predominant in the cell culture in large amounts.

Safety profile and anti-tumor effects of adoptive immunotherapy using gamma-delta T cells against advanced renal cell carcinoma: a pilot study.

PURPOSE: Although various types of immunotherapy have been used to improve the prognosis of patients with advanced renal cell carcinoma (RCC), adoptive immunotherapy using gamma-delta (gammadelta) T cells has not yet been tried. In this study, we designed a pilot study of adoptive immunotherapy using in vitro activated gammadelta T cells against advanced RCC to evaluate the safety profile and possible anti-tumor effects of this study. EXPERIMENTAL DESIGN: Patients with advanced RCC after radical nephrectomy were administered via intravenous infusion in vitro-activated autologous gammadelta T cells every week or every 2 weeks, 6-12 times, with 70 JRU of teceleukin. Adverse events, anti-tumor effects and immunomonitoring were assessed. The anti-tumor effects were evaluated according to tumor doubling time (DT) by computed tomography (CT) and immunomonitoring was performed by flow cytometric analysis. RESULTS: Seven advanced RCC patients were entered in this study. The most common adverse events were fever, general fatigue and elevation of hepatobiliary enzymes, but no severe adverse events were seen. Prolongation of tumor DT was seen in three out of five patients; these three patients showed an increase in the number of gammadelta T cells in peripheral blood and also a high response to the antigen in vitro. CONCLUSIONS: The results indicated that adoptive immunotherapy using in vitro-activated autologous gammadelta T cells was well tolerated and induced anti-tumor effects.