Allogeneic γδ T 细胞治疗肿瘤综述

Background The tumor microenvironment (TME) poses challenges that limit the efficacy of conventional CAR-T cell therapies. Homing barriers, immunosuppressive factors, and target antigen heterogeneity can impair CAR-T cell functional activity within the TME. Alternative strategies have contemplated incorporating the use of gamma delta (γδ) T cells as a CAR-T cell approach to potentially overcome these limitations. γδ T cells possess both innate and adaptive immunity to facilitate broad tumor recognition, and their natural propensity for tissue tropism may allow for more effective tumor infiltration. Reported here is the preclinical characterization of ADI-270, an allogeneic γδ CAR-T cell product targeting CD70+ cancers, engineered with a third-generation CAR based on the natural CD27 receptor. ADI-270 is also double-armored to mitigate the immunosuppressive effects of TGFβ and reduce the potential for allogeneic rejection. Methods Vδ1 T cells engineered to express an anti-CD70 CAR and dominant negative TGFβ receptor II (dnTGFβRII) were expanded from healthy donor human PBMCs. The phenotype and functional characterization of ADI-270 were assessed with in vitro cell culture assays and in vivo tumor xenograft models. Results ADI-270 exhibited high levels of in vitro cytotoxicity against a panel of cancer cell lines and displayed a favorable inflammatory cytokine profile compared with reference scFv-based anti-CD70 CAR αβ T cells. Cytotoxicity remained potent despite low CD70 expression observed in multiple solid and hematologic tumor cell models. When armored with dnTGFβRII, ADI-270 exhibited functional resilience to TGFβ-mediated inhibition of T cell effector activity. In addition, the incorporation of potent and sensitive CD70-targeting decreased T cell-mediated alloreactive killing against ADI-270 in vitro without evidence of fratricide. Finally, ADI-270 displayed robust tumor tropism and control of primary and secondary tumor challenges in xenograft mouse models. Conclusions These results demonstrate the robust potency and capacity of ADI-270 to extend antitumor activity to cancers with heterogeneous antigen expression. The functional armoring incorporated into ADI-270 provides a mechanism to overcome the limitations of reduced efficacy and persistence within the TME. ADI-270 has the potential to target multiple CD70+ cancers with initial clinical evaluation proceeding in relapsed/refractory clear cell renal cell carcinoma. Trial registration number NCT06480565.

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Antigenic heterogeneity and limited immune cell infiltration has historically hindered immunotherapy approaches to treat the pediatric brain tumor medulloblastoma (MB). Therefore, modern MB treatment modalities rely on broadly cytotoxic chemotherapy and/or radiation, leaving survivors with side effects that diminish quality of life, stressing an urgent need for novel approaches. Gamma delta (γδ) T cells represent an emerging class of cellular immunotherapy with preclinical potency against the CNS tumor glioblastoma and pediatric solid tumors. The ability of γδ T cells to recognize tumor stress antigens, alongside their MHC independence, supports γδ T cell usage as an off-the-shelf allogeneic cellular immunotherapy for MB and other immunologically cold cancers. Our team recently identified protein tyrosine kinase 7 (PTK7) as a novel immunotherapy target highly expressed in neuroblastoma, with low pediatric healthy tissue expression. Alpha beta T cells engineered with CARs targeting PTK7 demonstrated impressive antitumor efficacy against PTK7+ neuroblastoma xenografts. In that study, we also noted MB and other pediatric solid tumors express elevated levels of PTK7 mRNA. This work therefore serves to test the susceptibility of MB to γδ T cell immunotherapy and validate if targeting PTK7 can further enhance γδ T cell potency. Six MB cell lines representing three out of the four MB molecular subgroups were chosen: DAOY, ONS-76, UW228, D341, D425 and D283. Western blot analysis showed five of six MB cell lines express PTK7 protein, and flow cytometry confirmed PTK7 membrane localization. PTK7high (DAOY) and PTK7low (ONS-76) cells were chosen to test for the antigen-specific killing potential of PTK7-targeted CAR γδ T cells against MB. To generate PTK7-targeted γδ T cells, previously cryopreserved γδ T cells were electroporated with mRNA encoding a PTK7-targeted CAR construct containing the CD28 co-stimulatory domain. After a four hour co-culture, both ONS-76 and DAOY showed moderate rates of apoptosis from exposure to mock electroporated or naïve γδ T cells at 1:1, 2:1 and 5:1 effector:target (E:T) ratios, with approximately 30-40% cell death at 5:1. There was no change in susceptibility of ONS-76 cells to PTK7-CAR γδ T cells when compared to mock electroporated or naïve γδ T cell controls. However, co-culture of DAOY cells with PTK7-CAR γδ T cells markedly increased tumor cell death by as much as 40% compared to the mock controls at E:T ratios as low as 1:1. In summary, naïve expanded γδ T cells induce pronounced MB tumor cell death. Cytotoxicity is further increased against PTK7high MB cells following the introduction of an anti-PTK7 CAR. These results provide a strong rationale for additional preclinical studies regarding the feasibility of γδ T cell-based immunotherapy against MB. Citation Format: Benjamin S. Lowry, Hunter C. Jonus, Jasmine Lee, Trent H. Spencer, Anna M. Kenney, Kelly Goldsmith. Exploring the efficacy of allogeneic gamma delta T cell adoptive cell therapy against the pediatric brain tumor medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1790.

Background Targeting epidermal growth factor receptor (EGFR) has become a strategic approach in cancer therapy, using various modalities including chimeric antigen receptor (CAR)-αβT cell therapies. Despite significant advancements in autologous CAR-αβT cell therapies in B-cell lymphoma, current cell therapies face challenges such as potential risks associated with genetic engineering, waiting time and high costs of autologous CAR-αβT cell therapies. Innovations in click chemistry and bioorthogonal chemistry have enabled the development of antibody-cell conjugation (ACC) technology, which links cancer-targeting antibodies to immune cells without genetic modifications, potentially providing a safer profile. Methods In this study, we introduce ACE2016, an innovative allogeneic cell therapy targeting EGFR. ACE2016 is generated by ACC technology to conjugate donor-derived γδ2 T cells with the EGFR-specific antibody cetuximab. Results Our preclinical studies demonstrate that ACE2016 exhibits superior cytotoxicity against various EGFR-expressing cancer cell lines and minimal cytotoxic effects on normal cells. Mechanistic studies revealed that ACE2016 enhances cytotoxicity through increased capacity towards EGFR-expressing cancer cells, enhanced levels of cytotoxic cytokines and recruitment of peripheral cytotoxic cells, reflecting significant tumor suppression and prolonged survival in ACE2016-treated groups without causing treatment-related toxicity in vivo. Conclusions These findings support the clinical potential of ACE2016 as an off-the-shelf γδ2 T-cell therapy for EGFR-expressing cancers, offering a combination of specificity, scalability, and safety in the development of solid tumor therapy.

Simple Summary CD20-targeting CAR T cells have shown remarkable clinical outcomes in patients with B-cell malignancies. However, the manufacturing processes usually involved in autologous cell collection and gene editing limit their usage and development across the board. Here, we developed an allogenic cell product ACE1831, Vγ9Vδ2 T (γδ2 T) cells equipped with anti-CD20 antibody, using antibody–cell conjugation (ACC) technology. ACE1831 elicits a specific and potent anti-tumor activity against CD20-expressing cancer cells in vitro and in vivo with no abnormal clinical observation. We further found that the ACC-linked antibody/receptor complex stimulated T cell activation upon recognizing the antigen on cancer cells. To summarize, our data show the promise of a novel combination of an ACC platform with γδ2 T cells in allogenic immunotherapy against relapsed/refractory B-cell lymphoma. Abstract Chimeric antigen receptor T cell (CAR-T) therapy has been applied in the treatment of B-cell lymphoma; however, CAR-T manufacturing requires virus- or non-virus-based genetic modification, which causes high manufacturing costs and potential safety concerns. Antibody–cell conjugation (ACC) technology, which originated from bio-orthogonal click chemistry, provides an efficient approach for arming immune cells with cancer-targeting antibodies without genetic modification. Here, we applied ACC technology in Vγ9Vδ2 T (γδ2 T) cells to generate a novel off-the-shelf CD20-targeting cell therapy ACE1831 (rituximab-conjugated γδ2 T cells) against relapsed/refractory B-cell lymphoma. ACE1831 exhibited superior cytotoxicity against B-cell lymphoma cells and rituximab-resistant cells compared to γδ2 T cells without rituximab conjugation. The in vivo xenograft study demonstrated that ACE1831 treatment strongly suppressed the aggressive proliferation of B-cell lymphoma and prolonged the survival of tumor-bearing mice with no observed toxicity. Mass spectrometry analysis indicated that cell activation receptors including the TCR complex, integrins and cytokine receptors were conjugated with rituximab. Intriguingly, the antigen recognition of the ACC-linked antibody/receptor complex stimulated NFAT activation and contributed to ACE1831-mediated cytotoxicity against CD20-expressing cancer cells. This study elucidates the role of the ACC-linked antibody/receptor complex in cytotoxicity and supports the potential of ACE1831 as an off-the-shelf γδ2 cell therapy against relapsed/refractory B-cell lymphoma.

Background Glypican-3 (GPC-3) is an oncofetal protein that is highly expressed in various solid tumors, but rarely expressed in healthy adult tissues and represents a rational target of particular relevance in hepatocellular carcinoma (HCC). Autologous chimeric antigen receptor (CAR) αβ T cell therapies have established significant clinical benefit in hematologic malignancies, although efficacy in solid tumors has been limited due to several challenges including T cell homing, target antigen heterogeneity, and immunosuppressive tumor microenvironments. Gamma delta (γδ) T cells are highly cytolytic effectors that can recognize and kill tumor cells through major histocompatibility complex (MHC)-independent antigens upregulated under stress. The Vδ1 subset is preferentially localized in peripheral tissue and engineering with CARs to further enhance intrinsic antitumor activity represents an attractive approach to overcome challenges for conventional T cell therapies in solid tumors. Allogeneic Vδ1 CAR T cell therapy may also overcome other hurdles faced by allogeneic αβ T cell therapy, including graft-versus-host disease (GvHD). Methods We developed the first example of allogeneic CAR Vδ1 T cells that have been expanded from peripheral blood mononuclear cells (PBMCs) and genetically modified to express a 4-1BB/CD3z CAR against GPC-3. The CAR construct (GPC-3.CAR/secreted interleukin-15 (sIL)-15) additionally encodes a constitutively-secreted form of IL-15, which we hypothesized could sustain proliferation and antitumor activity of intratumoral Vδ1 T cells expressing GPC-3.CAR. Results GPC-3.CAR/sIL-15 Vδ1 T cells expanded from PBMCs on average 20,000-fold and routinely reached >80% purity. Expanded Vδ1 T cells showed a primarily naïve-like memory phenotype with limited exhaustion marker expression and displayed robust in vitro proliferation, cytokine production, and cytotoxic activity against HCC cell lines expressing low (PLC/PRF/5) and high (HepG2) GPC-3 levels. In a subcutaneous HepG2 mouse model in immunodeficient NSG mice, GPC-3.CAR/sIL-15 Vδ1 T cells primarily accumulated and proliferated in the tumor, and a single dose efficiently controlled tumor growth without evidence of xenogeneic GvHD. Importantly, compared with GPC-3.CAR Vδ1 T cells lacking sIL-15, GPC-3.CAR/sIL-15 Vδ1 T cells displayed greater proliferation and resulted in enhanced therapeutic activity. Conclusions Expanded Vδ1 T cells engineered with a GPC-3 CAR and sIL-15 represent a promising platform warranting further clinical evaluation as an off-the-shelf treatment of HCC and potentially other GPC-3-expressing solid tumors.

Background Gamma-delta (γδ) T lymphocytes are primed to potently respond to pathogens and transformed cells by recognizing a broad range of antigens. However, adoptive immunotherapy with γδT cells has exhibited mixed treatment responses. Better understanding of γδT cell biology and stratifying healthy donors for allogeneic adoptive therapy is clinically needed to fully realize the therapeutic potential of γδT cells. Methods We examine 98 blood samples from healthy donors and measure their expansion capacity after zoledronate stimulation, and test the migration and cytotoxic effector function of expanded γδT cells in 2D culture, 3D tumor spheroid and patient-derived melanoma organoid assays. Results We find that γδT cell expansion capacity is independent of expansion methods, gender, age and HLA type. Basal γδT cell levels in Peripheral blood mononuclear cell (PBMC) correlate well with their expansion, migration and cytotoxic effector capacity in vitro. Circulating γδT cells with lower expression of PD-1, CTLA-4, Eomes, T-bet and CD69, or higher IFN-γ production expand better. γδT cells with central memory and effector memory phenotypes are significantly more abundant in good expanders. A cut-off level of 0.82% γδT cells in PBMC stratifies good versus poor γδT cell expansion with a sensitivity of 97.78%, specificity of 90.48% and area under the curve of 0.968 in a healthy individual. Donors with higher Vδ2 Index Score in PBMC have greater anti-tumor functions including migratory function and cytotoxicity. Conclusions Our results demonstrate that the interindividual γδT cell functions correlate with their circulating levels in healthy donors. Examination of circulating γδT cell level may be used to select healthy donors to participate in γδT-based immunotherapies.

T-cell receptor (TCR) engineered T cells have emerged as a promising cancer therapeutic. While the clinical trial using ab T cells targeting the HPV E7 protein showed promise, it also highlighted a concerning issue: tumor escape due to the acquisition of defects in peptide-HLA presentation. As an alternative cellular platform, Gamma Delta One T cells (DOT) offer several advantages over alpha/beta T cells. These include their tissue-homing abilities, their use as allogeneic cells without GVHD risk, and their potential to target cells through both TCR and NK-like receptors (which can kill HLA Class-I deficient cells). The overall purpose of this study is to establish DOTs as an effective platform for TCR-T therapy targeting solid tumors, demonstrating enhanced therapeutic efficacy in overcoming tumor escape through mutations in the antigen-processing machinery (APM). We developed a novel protocol to isolate and expand DOTs from peripheral blood using a Delta 1 TCR-specific antibody and EasySep positive selection. By day 4, DOTs reached 37.3% (SD ± 10.3%) enrichment, increasing to 91.85% (SD ± 2.15%) by the end of culture. From transduction day, cells expanded over 100-fold by day 15, demonstrating scalability for therapeutic use. Efficiently transduced with an E7-specific TCR engineered to minimize mispairing (murine constant alpha and beta chains), we achieved transduction levels comparable to alpha/beta T cells. DOTs expressed high levels of NK-like receptors, including NKG2D (94.85% SD ± 0.55%), DNAM-1 (99.1% SD ± 0.3%), and NKp30 (76% SD ± 11%), crucial for targeting HLA-negative tumors. Using HPV+ E7+ SCC90 and CaSki cell lines with varying NKG2D ligand expression, we performed CRISPR/Cas9-mediated B2M knockout to generate HLA class I-deficient variants. Cytotoxicity was evaluated using in vitro chromium release (short-term) and IncuCyte (long-term) assays, where DOTs consistently outperformed alpha/beta T cells in killing HLA-heterogeneous tumors. DOT TCR-T cells exhibited comparable cytotoxicity to alpha/beta TCR-T cells against HLA-A*02:01+ E7+ targets while excelling against HLA-negative variants. These findings establish DOTs as a robust platform for TCR-engineered therapies, capable of overcoming tumor immune evasion across diverse HLA settings. We are currently validating these results in vivo using murine models. In summary, DOTs offer a promising platform for TCR-engineered therapies, excelling in targeting HLA-heterogeneous tumor populations through their dual recognition mechanisms. This work highlights their potential for developing off-the-shelf TCR-T therapies to overcome HLA downregulation and tumor heterogeneity in solid tumors. Andres Mosquera, Diego F. Chamorro, Paul Shafer, Lauren K. Somes, Emily Madaras, Valentina Hoyos. Enhanced targeting of HPV positive cancers using gamma delta 1 T cells armored with an HPV E7-specific TCR: a novel approach to overcome HLA downregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3482.

Genetic engineering has fundamentally transformed T cell–based therapies by enabling tumor targeting capability, improving their functionality, and facilitating allogeneic use. These strategies—originally developed in αβ chimeric antigen receptor (CAR)-T cells—have become increasingly established as blueprints for enhancing the function of other immune effector cells, including gamma delta (γδ) T cells. A recent study by Nishimoto et al showcased the adaptation of these engineering approaches to Vδ1 γδ T cells (ADI-270) by coexpressing a CD70-targeted CAR and a dominant-negative TGFβRII receptor (dnTGFβRII) to target CD70+ malignancies, addressing immunosuppression and host-versus-graft rejection. This commentary explores αβ T cell-derived engineering strategies applicable to γδ T cells, while also highlighting genome-editing innovations poised to advance next-generation γδ CAR-T development.

Background: Acute myeloid leukemia (AML) is the second most common type of blood cancer in children with a high rate of treatment failure and aggressive relapse. While allogeneic HSCT offers a cure, limitations arise in achieving remission prior to transplant. Immunotherapy-based approaches can induce remission, gaining interest in AML. Here, we explore the use of gamma delta (γδ) T cells as a promising tool to fulfill this unmet need. Unlike predominant alpha beta (αβ) T cells, γδ T cells do not require antigen presentation and identify their targets in a major histocompatibility complex (MHC) independent manner. Allogeneic γδ T cells thus have minimal risk of causing graft-versus-host disease (GvHD), creating ‘off-the-shelf’ cellular therapeutics. This would be ideal to target AML, especially given the aggressive nature of relapsed disease. Cytotoxic mechanisms used by γδ T cells include recognition of cellular stress molecules such as NKG2D and DNAM-1 receptor ligands on target cells, activation of death receptor pathway through Fas-FasL and TRAIL-R interactions, as well as γδ ΤCR mediated killing through recognition of butyrophilins (BTNs) on target cells. These stress molecules are expressed on AML cells and can be further upregulated through chemotherapeutic agents such as azacitidine (a hypomethylating agent) and venetoclax (BCL-2 antagonist), drugs currently in use to treat relapsed AML. Here, we tested these drugs in combination with γδ T cells to target AML. Methods Our studies explored four different AML cell lines (Kasumi-1, Nomo-1, MV4-11 and MOLM13). Each one was respectively treated at their corresponding inhibitory concentration 25 (IC 25) and 50 (IC 50) of azacitidine and/or venetoclax for 24 hours. After treatment, AML cells were assessed by flow cytometry for expression of NKG2D and DNAM-1 ligands, death receptor pathway molecules (CD95, TRAIL-R1/R2) as well as the butyrophillins (BTN2A and BTN3A1) to elucidate which pathways are over-expressed, aiding in γδ T cell targeting and cytotoxicity. Next, we co-cultured AML cells pre-treated with azacitidine and/or venetoclax with γδ T cells and measured cytotoxicity using a flow cytometry-based assay and a bioluminescence assay. Results: Each cell line expressed a unique pattern of markers at baseline. Cell lines treated with azacitidine had a robust expression of the NKG2D ligands ULBP1, ULBP2/5/6 and ULBP3. Azacitidine also increased CD155 and TRAIL-R2 in Nomo-1 while CD112 increased in MOLM13. Most venetoclax-treated AML cells over-expressed BTN2A and BTN3A1 along with CD95 in MOLM13. Cell lines were then treated with a combination of both drugs. Dual treatment of cells resulted in a significant increase in expression of most NKG2D markers as well the death receptor Fas (CD95). Additionally, increase in CD155 as well as BTNs was seen in several cell lines. Importantly, azacitidine and venetoclax had minimal effects on the viability of γδ Τ cells when treated for 24 hours. In vitro cytotoxicity studies indicate increased cell death across multiple AML cell lines when γδ T cells were co-cultured with cells pretreated with azacitidine and venetoclax. Conclusions: Azacitidine and venetoclax treatment resulted in upregulation of several markers involved in γδ Τ cell-mediated cytotoxicity. Ex vivo expanded γδ Τ cells combined with chemotherapy-treated AML cells resulted in increased overall cytotoxicity, which is likely both additive and synergistic. Our next steps include preclinical validation of this combinatorial approach in an in-vivo mouse study.

Although Vγ9Vδ2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major histocompatibility complex (MHC)-independent manner and develop strong cytolytic effector function that makes it an ideal candidate for cancer immunotherapy. Due to the low frequency of Gamma-Delta (γδ) T cells in the peripheral blood, we developed an effective protocol to greatly expand a highly pure γδ T cells drug product for first-in-human use of allogeneic γδ T cells in patients with acute myeloid leukemia (AML). Using healthy donor apheresis as an allogenic cell source, the lymphocytes are isolated using a validated device for a counterflow centrifugation method of separating cells by size and density. The lymphocyte-rich fraction is utilized, and the γδ T cells are preferentially activated with zoledronic acid (FDA-approved) and interleukin (IL)-2 for 7 days. Following the preferential expansion of γδ T cells, a clinical-grade magnetic cell-separation device and TCRαβ beads are used to deplete contaminating T-cell receptor (TCR)αβ T cells. The highly enriched γδ T cells then undergo a second expansion using engineered artificial antigen-presenting cells (aAPCs) derived from K562 cells-genetically engineered to express single-chain variable fragment (scFv) for CD3 and CD28, 41BBL (CD137L) and IL15-RA-together with zoledronic acid and IL-2. Seeding all day-7 enriched γδ T cells in co-culture with the aAPCs facilitates the manufacture of highly pure γδ T cells with an average fold expansion of >229,000-fold from healthy donor blood.

Cancer immunotherapy using patient-derived T cells modified with chimeric antigen receptors (CAR) has shown exceptional success against lymphoid malignancies. However, these autologous cell therapies involve complex and labor-intensive cell processing. Utilizing human induced Pluripotent Stem Cells (iPSCs) to produce CAR T cells offers significant potential for developing allogeneic, off-the-shelf cancer immunotherapies. The main challenge is generating fully mature T cells from iPSCs, as iPSC-derived T cells often display characteristics of innate-like gamma-delta T cells, lacking the robust functionality of mature alpha-beta T cells derived from peripheral blood. Previously, we developed a stroma-free culture system for differentiating iPSCs into T cells that entailed genetic knockdown of the EZH1 histone methyltransferase, which functions as an epigenetic barrier to hematopoietic maturation (Jing et al, Cell Stem Cell, 2022). To facilitate cell manufacture for clinical translation, we conducted small molecule screens to identify compounds that phenocopy EZH1 deficiency in affecting lymphoid development. We discovered that inhibiting G9a/GLP similarly promotes the generation of mature T cells from iPSC-derived hemogenic endothelial cells (HECs). ATAC-seq and RNA-seq analyses revealed that G9a/GLP inhibition regulates chromatin accessibility and gene expression patterns associated with lymphoid differentiation, and influences the fate choice between myeloid and lymphoid lineages. We also showed that inhibiting G9a/GLP enhances lymphopoiesis in zebrafish, demonstrating the evolutionary conservation of G9a/GLP's role in T cell development. Most importantly, chemically-induced epigenetic reprogramming via G9a/GLP inhibition enables the generation of highly functional iPSC-derived T cells with a molecular profile similar to that of mature alpha-beta T cells from peripheral blood. Single-cell RNA-seq analysis further revealed that iPSC-derived T cells give rise to both effector and memory-like T cell subpopulations upon activation. When these epigenetically reprogrammed iPSC-derived T cells were engineered to express an anti-CD19 CAR, they showed potent effector responses and antitumor activity in vitro and in a xenograft lymphoma mouse model. We also demonstrated that mice treated with epigenetically modulated iPSC-CAR-T cells were resistant to tumor cell rechallenge, further demonstrating that iPSC-CAR-T cells derived through G9a/GLP inhibition can persist and induce sustained remission. These findings support the efficient production of clinically relevant iPSC-derived T cells for adoptive cell therapies.

Gamma delta (gd) T cells are innate immune cells that synergistically target cancer cells via NKG2D ligands (NKG2D-L) upregulated by chemotherapy. INB-200, a Phase 1 trial, demonstrated the safety and efficacy of autologously (auto) derived genetically modified drug resistant immunotherapy (DRI) gd T cells combined with maintenance temozolomide (TMZ) to treat newly diagnosed (ND) patients with GBM. Initiation of , a 3-arm phase 1b/2 study confirming the efficacy of the auto product in ND GBM and the safety and efficacy of an allogeneic (allo) product in both ND and relapsed GBM has begun. Arm A assesses the auto product in ND GBM patients with 1x107 cells delivered on Day 1 of cycles 1-6 along with TMZ maintenance therapy per the Stupp regimen. In the Phase 1b arm, the haploidentical allo product, will be assessed for safety in 6 relapsed GBM patients who will receive 1x107 cells in combination with 150mg/m2 of TMZ on Day 1 of every six 28-day cycles. Arm B will continue enrollment of relapsed patients following the Phase 1b and Arm C will test the safety of the allo derived product in ND, GBM patients. In Arm C, patients will receive 1x107 allo DeltEx DRI cells on Day 1 of cycles 1-6 in combination with TMZ maintenance. Cells are administered through a Rickham catheter in all arms. Primary endpoints for the Phase 1b define the RP2D and subject/ product characteristics to optimize manufacturing, while Phase 2 endpoints will determine the 12 mos OS rate for Arms A & C and 9 mos OS rate for Arm B. Eligible patients are adults with IDH-wt ND or relapsed GBM with no concurrent TTF or immunotherapy, willing to place an indwelling Rickham catheter. Allo arm patients must have a haploidentical donor. Fifteen US centers will conduct study.

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Induced pluripotent stem cell (iPSC)-derived cell treatment offers several competitive benefits, including consistency, homogeneity and unlimited dosage, in contrast to autologous and allogeneic donor-derived cell therapies. These attributes improve affordability and accessibility of medicines worldwide. Noteworthy, the effectiveness of T cell therapy is constrained by factors such as cell potency and persistence. Our research on iPSC-derived gamma-delta T (γδT) cells has identified a novel combinatorial KO to overcome these challenges. IL-2 administration has been approved by FDA for durable, complete, and apparently curative regressions in cancer patients. Previous studies demonstrated that IL-2 receptor beta (IL-2Rβ) chain-mediated signaling pathway is critical for killing potency and cell persistence. The suppressor of cytokine signaling (SOCS) family helps control cytokine signaling by blocking the JAK/STAT pathway. For example, cytokine-inducible SH2-containing protein (CISH) is a member of the SOCS protein which is essential for managing cytokine signaling, cell growth, differentiation, and immune system activities. Our findings indicated that iγδT cells deficient in both CISH and another SOCS family protein exhibited increased persistence and enhanced cytotoxic capabilities compared to cells with only one of the knockouts. Notably, we demonstrated the proliferative advantage of double SOCS protein KO by using a humanized mouse model. Cytokine withdrawal- and activation-induced cell death were key factors that diminish effector cell persistence post infusion. For prolonging cell survival, we identified a mediator of mitochondrial apoptotic pathway and found that this proapoptotic protein is necessary for extending the survival of iγδT cell post IL-15 withdrawal. In addition, FAS-FASL signaling was recently identified as auto-regulatory circuit for CAR-engineered lymphocyte persistence. We presented biochemical data and performed gain-of-function experiments to elucidate the mechanisms by which this proapoptotic protein contributes to the extended survival of effector cells. Furthermore, we demonstrated FAS deficiency protects functional enhanced iγδT cells from FASL-induced apoptosis and then enhances its durable killing ability against HepG2 cells. Altogether, we identified a novel combination of gene knockouts that extends longevity and profound anti-tumor efficacy without cytokine support. We achieved the goldilocks signaling balance could strengthen the efficacy and longevity of iγδT cell, while avoid the possible dysfunction, such as exhaustion, caused by introducing constitutively active cytokine receptors as many others have attempted. Building on this engineering platform, we could incorporate tumor-targeting receptors for treating different indications in the near future. Jhang-Sian Yu, Ming-Chin Ko, Chia-Hung Lin, Yi-Han Dai, Ke-Hsun Hsu, I-Ting Chen, Ting-Yi Wu, Jin-Yi Lu, Ying-Tsen Tung, Edward Po-Fan Chu, Yuan-Yu Hsia, Yo-Chuen Lin, Chieh-Teng Cheng, Tzu-Chien Kuo, Yi-Ting Lai, Wei-Ting Chen, Darien Zhing Herr Chan, Lee-Yieng Lim, Chih-Lung Chen, Chun-Yu Lin, Lin-Yu Liao, Wan-Chen Tsai, Lien-Szu Wu, Ching-Ying Huang, De-Kuan Chang, Alex Shih-Min Huang. iPSC-derived γδT with novel combinatorial KO demonstrated significant anti-tumor activity and extended longevity without cytokine support [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3479.

Allogeneic cell therapies, defined by genetically mismatched transplantation, have the potential to become a cost-effective solution for cell-based cancer immunotherapy. However, this type of therapy is often accompanied by the development of graft-versus-host disease (GvHD), induced by the mismatched major histocompatibility complex (MHC) between healthy donors and recipients, leading to severe complications and death. To address this issue and increase the potential for allogeneic cell therapies in clinical practice, minimizing GvHD is a crucial challenge. Innate T cells, encompassing subsets of T lymphocytes including mucosal-associated invariant T (MAIT) cells, invariant natural killer T (iNKT) cells, and gamma delta T (γδ T) cells, offer a promising solution. These cells express MHC-independent T-cell receptors (TCRs), allowing them to avoid MHC recognition and thus GvHD. This review examines the biology of these three innate T-cell populations, evaluates research on their roles in GvHD modulation and allogeneic stem cell transplantation (allo HSCT), and explores the potential futures for these therapies.

Background Metastatic pancreas adenocarcinoma (PDAC) is highly lethal and minimally responsive to immunotherapy. Gamma delta T cells (gDTs) are a unique T cell subset found within the PDAC microenvironment. Animal models have shown that gDTs promote oncogenic progression and ablating gDT delays cancer progression and improves survival. Understanding the activity of immune effector cells against cancer organoids provides a scalable platform to understand the impact of activation and small molecule modulation to redirect gDT killing in PDAC. Methods Patient-derived organoids (PDOs) were generated from fresh tissue sampling and exapanded using a defined, serum-free media. Upon passaging, media was collected from PDAC PDOs. Allogeneic gDTs isolated from healthy donors were cultured in PDAC PDO conditioned media from 3 separate PDOs, in 3 separate biological replicates, and then subjected to bulk RNAseq. Allogeneic gDTs were isolated and cultured with PDAC PDOs at ratio of 5:1 (gDTs:PDAC PDOs) for 48 hours. After 48 hours, staining was performed for induction of apoptosis (cleaved caspase-3/7FITC). Images were acquired by high content imaging (Cytation 5, BioTek) and quantified for response across organoid populations. Based on RNAseq nominated candidates, low density lipoprotein (LDL) was added (10 ug/mL) as well as a small molecule inverse agonist of the Liver X Receptor (LXR) beta transcription factor (SR9243) to assess the ability of these factors to modulate gDT mediated PDAC killing. Results Exposure of gDTs to PDAC PDO secreted factors results in differential regulation of cholesterol handling, including upregulation of cholesterol efflux, and downregulation of cholesterol uptake and biosynthesis related genes in conditioned T cell populations. gDTs cause marked apoptosis in PDAC PDOs, including 40% staining of % organoid area versus negligible staining in control populations of 1.9% organoid area (effect size = 9.5, p<0.001). This response was directly compared to standard chemotherapy at the time of clinical resistance to FOLFIRINOX which conferred only 2% staining of mean organoid area (effect size = 0.11, NS). Given RNAseq data demonstrating cholesterol handling changes after exposure to PDAC secreted factors, we investigated LDL supplementation in our model. Supplementation of LDL significantly improves gDT mediated killing, with induction of apoptosis seen in 52% of the PDAC PDO area (p=0.003). This effect is mimicked by the small molecule SR9243 (an LXR inverse agonist), with 56% of PDAC PDO demonstrating apoptotic induction at 48 hours. Conclusions A PDAC PDO model system can be used to investigate gDT mediated PDAC killing when applied in low-volume medium throughput screening applications.  Modulation of LXR activity and microenvironment LDL results in substantially increased immune-mediated killing of PDAC. This screening platform identifies potentially clinically actionable modulators of immune response to PDAC, and suggests potential future immunologically relevant targets that may improve the efficacy of immunotherapy. Citation Format: Johnathan D Ebben, David Turicek, Md Shahadat Hossan, Austin Stram, Ethan Lin, Nicholas Hess, Zachary Mayhew, Melissa A Kinney, Devin Burpee, Anikait Patel, Christian M Capitini, Jeremy D Kratz. Pancreas organoid immune co-culture system identifies immunomodulators in pancreas adenocarcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A071.

Gamma delta (γδ) T cells have recently emerged as an attractive candidate for cancer immunotherapy treatments due to their inherent cytotoxicity against cancer cells. Moreover, γδ T cells provide a platform for allogeneic cell therapies as they can recognize antigens independent of MHC recognition and do not cause graft-versus-host disease (GVHD). However, the lack of robust and GMP-compatible serum-free ex vivo expansion system for γδ T cells hinders the development of scalable expansion processes for advancing γδ T cell immunotherapies. In the current study, we characterize the expansion of human peripheral blood-derived γδ T cells from various donors using TheraPEAK ®T-VIVO™ Medium, a novel chemically-defined cell expansion medium which is free of undefined and animal-origin components. Peripheral blood mononuclear cells (PBMCs) stimulated with zoledronic acid resulted in more than 1,000 fold expansion of γδ T cells in only 14 days with TheraPEAK ®T-VIVO™ Medium containing 100 IU/mL human recombinant IL-2, without the need for human serum supplementation. Similarly, PBMCs stimulated with agonist antibodies targeting Vδ1 or Vδ2 T cell receptors (TCR) induced robust expansion of Vδ1 or Vδ2 T cells, respectively, in TheraPEAK ®T-VIVO™ Medium. Collectively, these data support the use of TheraPEAK ®T-VIVO™ Medium to advance the development of γδ T cell manufacturing processes.

Introduction: Autologous CD19-targeted chimeric antigen receptor (CAR)-T cell therapies have demonstrated clinical benefit for patients with R/R B-NHL; however, many patients are unable to receive these potentially life-saving therapies due to aggressive disease progression, manufacturing constraints, or limited access. A bispecific CAR-T targeting CD19 and CD20 may enhance efficacy by overcoming heterogeneous antigen expression and CD19-negative relapse. LUCAR-G39D is a first-in-class allogeneic γδ CAR-T cell therapy targeting both CD19 and CD20. (NCT06395870) Methods: This ongoing multicenter phase I clinical trial evaluates the safety and preliminary efficacy of LUCAR-G39D in adults with R/R B-NHL. Key eligibility criteria include presence of measurable lesions, expression of CD19 and/or CD20 on tumor cells, and ≥ 2 (or refractory to 1) prior lines of systemic therapy. All patients received moderate conditioning therapy with fludarabine (30mg/m2) and cyclophosphamide (500mg/m2) per day for 3-4 days followed by a single infusion of LUCAR-G39D at dose level 1 (DL1):30×106, DL2:100×106, DL3:200×106, or DL4:400×106CAR-T cells. Accelerated titration (DL1 only) followed by BOIN dose escalation was adopted. Patients who completed the 30-day DLT period were considered DLT evaluable. Treatment-emergent adverse events were graded by CTCAE v5.0; immune effector cell-associated neurologic syndrome and cytokine release syndrome (CRS) were assessed per ASTCT criteria. Objective response rates (ORR) were evaluated per Lugano 2014 criteria. CAR-T cell pharmacokinetics (PK) were assessed by qPCR. Results: As of 29 April 2025, 12 patients were dosed; 10 patients completed at least 30 days follow up or at least 1 efficacy evaluation, 10 were efficacy evaluable and 9 DLT evaluable (1 withdrawal due to PD). Of these 10 patients, the median age was 58 years (range 19-70) and 5(50%) were male. Six (60%) patients had diffuse large B-cell lymphoma, 1(10%) patient had primary mediastinal large B-cell lymphoma, and 3 (30%) had follicular lymphoma. At baseline, 10(100%) patients were CD20(+) and 6(60%) patients were CD19(+); CD19 status was unavailable for the remaining 4(40%) due to insufficient material. Four (40%) patients were IPI≥3, 3(30%) patients were triple-hit lymphoma, the median tumor burden was 1188(531-6431) mm2, and 7(70%) had stage III/IV disease. The median number of prior therapies was 3 (range 1-6). One, 4, 3, and 2 patients received DL1, DL2, DL3, or DL4, respectively. One patient at DL2 was re-dosed with the same dose level. LUCAR-G39D was well tolerated without DLT, neurotoxicity, or GvHD. CRS occurred in 4/10 (40%) patients: 2(20%) Grade 1, 1(10%) Grade 2, and 1(10%) Grade 3, with median duration 5 days (range, 3-7). Infections occurred in 5/10(50%) patients including 1(10%) Grade 1, 3(30%) Grade 2, and 1(10%) Grade 3. Of the infections, there was only 1 Grade 3 event (pneumonia); it was also the only serious adverse event and resolved post-treatment. The most common Grade 3 or Grade 4 adverse events (incidence ≥20%) included decreased neutrophil count (100%), decreased lymphocyte count (100%), decreased white blood cell (100%), and decreased platelet count (20%). No fetal adverse events occurred. PK expansion was detected in 80% (8/10) of patients for all dose levels (100% [2/2] at DL4). The median Cmax was 1999 copies/μg gDNA (range 59-164768), with a median Tmax of 5.5 days. ORR was 70% (7/10), and the complete response rate was 30%(3/10) for all patients (3 patients had deepening efficacy from Days 30-90). Both patients at DL4 achieved a response by the first efficacy evaluation. As of the cut-off date, all 7 responders were still in response and active follow up, the longest last to Day 270 (range 30-270). At a median follow-up time of 3.52 months (range 0.8-8.8), the 6-month rate of PFS was 75% (range 29.8%-93.4%). Following LUCAR-G39D, 7 of 8 patients (88%) with paired baseline and Day30 ctDNA samples had decreased ctDNA levels (median, –98%; range –23% to –100%), consistent with radiographic responses. Conclusions: LUCAR-G39D γδ CAR-T cells showed a manageable safety profile and good expansion in patients with NHL. Preliminary efficacy showed encouraging response rate and sustained durability in patients; this therapeutic strategy warrants further investigation.

Introduction ACE1831, an off-the-shelf cellular therapy comprised of allogeneic gamma delta T (γδT) cells conjugated to anti-CD20 antibody via DNA linkers using bio-orthogonal chemistry, is being studied in a first-in-human, Ph1 study (NCT05653271) in pts with B-cell R/R NHL. Methods This is an open-label, multicenter, single, ascending dose, 3+3, Phase 1 study of the safety, pharmacokinetics, pharmacodynamics and preliminary efficacy of ACE1831 at dose levels 0.3, 0.6 and 1.0 billion [B] cells administered alone or in combination with obinutuzumab. Pts with R/R NHL who had received ≥ 2 prior lines of therapy receive a single infusion of ACE1831 (w/w/o obinutuzumab) following lymphodepletion with cyclophosphamide and fludarabine. The primary endpoints include the incidence of adverse events (AEs), dose-limiting toxicities (DLTs), the determination of the recommended Phase 2 dose, as well as ACE1831 persistency, immunogenicity, biomarker induction, and preliminary efficacy per the International Working Group Response Criteria. ACE1831 persistence was evaluated by flow cytometry analysis using anti-Vδ2 TCR (γδT marker) antibody recognizing both patients' γδT cells and ACE1831 in all subjects. Results As of June 13, 2024, 5 pts received a single dose of 0.3 B cells. Four pts had DLBCL and 1 had high-grade B-cell NHL. Four pts were male and 1 female; 4 pts were White and 1 was African-American. The median age was 65 (range: 32 - 76), and pts had a median of 5 prior lines of therapy (range: 2-7), including 4 pts that had received prior CAR-T therapy. Two patients had bulky disease and 2 had extra-nodal disease. No DLTs were reported. The most frequently observed treatment emergent AEs (TEAEs) were neutrophil, platelet, and white blood cell count decreased (n=5, 4, and 3, respectively), anemia (n=3) and fatigue (n=3). Most of these AEs were considered secondary to lymphocyte depletion chemotherapy, and none were considered related to ACE1831 alone. No ACE1831 serious TEAEs were reported, and no cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS) or graft vs. host disease (GvHD) was observed. One pt with low-volume disease had a complete response that has persisted beyond 180 days; 3 pts had stable disease; and 1 patient had progressive disease. The percentage of γδT cells in CD3 + T cell population peaked between Day 5 to Day 11 post ACE1831 infusion and decreased after the peaks. Cytokines IFNγ, TNFα, IL-6, IL-8, IL-10, and IL-2 were monitored in pts treated with ACE1831 during the DLT period. The peak levels of IFNγ, TNFα, IL-6, IL-8, or IL-10 were observed between Days 3 and 22. The IL-6 peak levels in ACE1831-treated pts were markedly lower than those in CAR-T-treated pts with CRS, suggesting less CRS potential. Anti-drug antibody responses to ACE1831 were monitored by flow cytometry analysis in four pts during the DLT period with Day -5 levels as baseline and there were no increases of anti-ACE1831 antibodies observed. Conclusions Preliminary data in 5 pts showed no DLTs, ACE1831-related SAEs, as well as no CRS, ICANS, or GvHD in single dose of 0.3B cells in 5 pts. Dose escalation will continue including 0.6 and 1B cells, with updated clinical data to be provided. Clinical trial identification NCT05653271

Outcomes for relapsed/refractory (R/R) T-cell acute lymphoblastic leukemia (T-ALL) remain poor, with long-term survival less than 30%. While allogeneic hematopoietic stem cell transplantation (HSCT) offers a chance for cure, many patients are unable to achieve the necessary disease remission prior to transplantation, limiting its use. Thus, new treatment strategies are urgently needed. Though there has been great success in the implementation of chimeric antigen receptor (CAR) T cell therapy for the treatment of B-cell malignancies, similar strategies have been challenging to adapt to T-ALL due to a lack of known T-ALL specific cell-surface antigens that distinguish malignant from healthy T cells, resulting in CAR T-cell fratricide. Alternative cellular therapy platforms such as the use of allogeneic gamma delta (γδ) T cells offer a solution as a cytotoxic alternative to the traditional alpha beta (⍺β) T cell. γδ T cells form a unique class of T cell that possess the innate ability to recognize both foreign pathogens and cellular stress antigens and, importantly, can be administered across major histocompatibility complex (MHC)-barriers, permitting their use in the “off-the-shelf” setting. Indeed, our institution has developed a GMP-compliant manufacturing strategy to expand γδ T cells from healthy donor peripheral blood at clinically relevant doses, with our cellular product currently under clinical investigation in the context of neuroblastoma in combination with chemo-immunotherapy [ClinicalTrials.gov Identifier: NCT05400603]. In the leukemia setting, we have previously shown that the proteasome inhibitor bortezomib enhances γδ T cell-mediated killing of T-ALL through the upregulation of NKG2DL stress antigens (Story JY., et al. 2021), although in vivo studies proved to be inconclusive. This present study investigates the hypothesis that the hypomethylating agent azacitidine in combination with the Bcl-2 inhibitor venetoclax, a chemotherapy regimen commonly used for R/R acute leukemias, can enhance γδ T cell-mediated killing of T-ALL through similar mechanisms. Herein, we found that ex vivo expanded γδ T cells induce significant cell death against three T-ALL cell lines - Jurkat E6-1, MOLT-4, and CCRF-CEM, at increasing effector-to-target (E:T) ratios in a four-hour flow cytometry based co-culture assay examining apoptotic and cell death markers. Azacitidine and venetoclax were both highly cytotoxic against the three T-ALL cell lines, with increased cell death observed when combined with γδ T cells. To investigate mechanisms of enhanced killing, we performed a detailed multi-parametric flow cytometry analysis of T-ALL cell lines to study the effects of azacitidine and venetoclax on cell surface expression of NKG2D ligands, DNAM-1 ligands, Fas, TRAIL receptors, and BTN3A1 - all surface molecules involved in γδ T-cell mediated killing. In a T-ALL xenograft model using the Jurkat E6-1 cell line, we found that one cycle of azacitidine and venetoclax treatment significantly lowered peripheral disease burden compared to vehicle-treated mice (Student's T Test, p=0.0010) as determined by flow cytometric analysis of PBMCs. Furthermore, the addition of γδ T cells to the chemotherapy regimen resulted in an increase in median survival of nine days (37 vs. 28 days), and a greater significance in survival advantage compared to vehicle-treated mice (Mantel-Cox, p=0.007) in contrast to treatment with just chemotherapy alone (Mantel-Cox, p=0.0117). Interestingly, when mice were stratified based on sex, we see a significant survival advantage when female mice are treated with γδ T cells in combination with chemotherapy compared to chemotherapy alone (Mantel-Cox, p=0.0246). This is in contrast to male mice, where we only observe a significant survival advantage when mice are treated with γδ T cells in combination with chemotherapy compared to vehicle-treated mice (Mantel-Cox, p=0.0066), and do not see such survival advantage when males are treated with chemotherapy alone. Together, these studies demonstrate that the combination of azacitidine, venetoclax and γδ T cells results in increased killing of T-ALL cell lines both in vitro and in vivo. Ongoing studies will further explore prior observed sex-based differences in survival advantage of our xenograft T-ALL model, in addition to completion of safety studies necessary to begin clinical investigation.

Despite recent therapeutic advances, patients with relapsed/refractory acute myeloid leukemia (r/r AML) have limited treatment options and poor prognosis. Leukemic stem cells (LSCs) are considered as relapse-initiating cells and the “culprit” of treatment resistance. Allogeneic and autologous gamma delta (γδ) T cell-based therapies have been explored for a long time and proven to be safe in patients through multiple clinical trials. However, quite a few AML blasts obtained from r/r AML patients show resistance to γδ T cell-mediated cytotoxicity. Novel strategies targeted LSCs may improve efficacy of γδ T cell-mediated r/r AML therapy. Given that γδ T cells exert antitumor capacity via multiple pathways, to identify the way matters most in the resistance of γδ T cell-mediated cytotoxicity, we examined the median fluorescence intensity (MFI) of the ligands expressed in tumor cells by flow cytometry, including butyrophilin 3A (BTN3A), butyrophilin 2A1 (BTN2A1), the human MHC class I chain-related genes (MICA and MICB), the unique-long 16 binding proteins (ULBP1, ULBP2/5/6, ULBP3), Fas, the receptor of TNF-related apoptosis-inducing ligand (TRAIL) DR5, CD112 and CD155. The cytotoxicity activity of γδ T cells was measured by flow cytometry-based killing assay or bright-lite luciferase assay system after coculturing with AML cell lines and blasts at an appropriate effector to target (E:T) ratio. Then we employed a Support Vector Machine model to predict the killing efficiency and identified that BTN3A and BTN2A1 play the predominant role in γδ T cell-mediated cytotoxicity based on the Shapley additive explanation. BTN3A and BTN2A1 are the key ligands in the γδ TCR-phosphoantigen (pAg) recognition activity. PAgs, which are produced in tumor cells function as “molecular glues” to promote heteromeric association of BTN3A1 and BTN2A1 (Yuan L et al., BioRxiv 2022). To promote the γδ T cell-mediated cytotoxicity targeting r/r AML, here we synthesized a pAg prodrug, which show great plasma stability and membrane permeability. We anticipate this small molecule would lead pAg accumulation in tumor cells and cause increased γδ T cell activation. To identify the killing efficacy of pAg prodrug-induced γδ T cell activation, we select KG-1α (a human leukemia stem cell-like cell) as target cell. Zoledronate (ZOL), which is employed to stimulate γδ T cells in multiple γδ T cell-based clinical trials, is used in our killing assay as compared group. As expected, combining γδ T cells with pAg prodrug or ZOL both significantly improved the percent of specific killing when the E:T ratio is 1:1. The pAg prodrug showed more potent activity than ZOL group with EC50 value of 0.1 nM vs 17.93μM. And the specific killing percent of pAg prodrug group is 83.26±2.1 % (1 nM), comparing with negative control 15.17±3.4 % (only γδT cells). Futhermore, at relatively low E:T ratio (1:4), γδ T cells with pAg prodrug remain excellent cytotoxicity when targeting MV411 cell and OCI-AML3 cell, which are widely used in r/r AML research. After coculturing with primary leukemia stem and progenitor cells (CD34 +) obtained from r/r AML patients for 6~8 h, the apoptosis percentage of CD34 + leukemia cells was 42.34% comparing with negative control (only γδT cells) 2.53%. Then we evaluated the in vivo efficacy of combining γδT cells and pAg prodrug in NSG mice bearing MV411 cells. Compared with only γδT cells-treated group, combination with pAg prodrug presented better tumor clearing. To elucidate the pathway of pAg prodrug-mediated killing, we tested mitochondria apoptosis related events. After treated by γδ T cells with pAg prodrug, KG-1α cells showed loss of mitochondrial outer membrane potential and increasingly actived caspase 3/7. Meanwhile, pAg prodrug directly activates effector γδ T cells with increased secretion of CD107a and IFN-γ. Considering the possibility of toxicity towards normal hematopoietic stem and progenitor cells, we conducted colony-forming units (CFUs) assay. After treatment with γδT cells and pAg prodrug, the number of CFUs of CD34 + cells from cord blood was not effected compared with the CD34 + cells only group. Overall, we explored the predominant factor in γδ T cell-mediated killing and synthesized a pAg prodrug to improve the killing efficacy. We demonstrated that combining γδ T cells with pAg prodrug has effectively promoted apoptosis of AML stem and progenitor cells sparing normal hematopoietic stem and progenitor cells.

No abstract available

Bispecific T cell engagers (TCE) bring effector T cells in contact with cancer cells by binding a pan-T cell receptor sequence and a tumor-associated antigen (TAA) simultaneously, thereby initiating activation and killing properties of the T cell. One of the foremost challenges for the clinical use of TCE is tumor heterogeneity, specifically, the limited expression of specific tumor antigens on the cancer cell surface. Gamma-delta (γδ) T cells are unique in that they recognize multiple stress-associated antigens common to most malignancies regardless of origin. This function is independent of MHC restriction, enabling them to target a broad range of tumors. Here, we report initial findings from a novel γδ T cell engager platform (γδ-TCE), specifically two γδ-TCEs targeting hematologic malignancies, CD33-TCE (CD-33, AML) and CD19-TCE (CD19, B_ALL), while also enabling γδ T cells to engage tumor-associated stress ligands with innate surface receptors such as NKG2D, PVR, and others. Preliminary in vitro characterization revealed properties suggestive of pan-δ chain binding, such as competing with isoprenyl pyrophosphate activation of Vδ2+ T cells, yet efficiently driving both activation and expansion of Vδ1+ and Vδ2+ T cell subsets from peripheral blood mononuclear cells (PBMC). Robust expansion of a diverse repertoire of γδ T cells in the tumor environment is critical for continued pressure and a persistent anti-tumor response. Cytotoxicity assays show that both CD33-TCE and CD19-TCE measurably enhanced γδ T cell-mediated lysis of CD33+ AML and CD19+ ALL tumor cells, respectively, in a dose-dependent manner. In co-cultures with tumor cells, γδ-TCE- armored γδ T cells showed markedly increased degranulation and the release of pro-inflammatory cytokines, such as IFN-γ, TNF-α, granzymes, and perforin, further highlighting the activation of γδ T cells into a potent cytotoxic state and amplifying their tumor-eradicating abilities. This novel γδ T cell engager platform represents a promising new approach to cancer immunotherapy, offering an effective strategy for enhancing γδ T cell-mediated tumor cell lysis. Furthermore, the platform’s modular and flexible design suggests applications in treating a broad range of malignancies by combining the innate recognition properties of γδ T cells with the targeting of specific tumor-associated antigens, thus expanding its therapeutic potential. Lei Ding, Sadhak Sengupta, Mariska ter Haak, Kate Rochlin, Yanjie Li, Lawrence S. Lamb. A novel gamma-delta T cell engager platform for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7321.

Haematopoietic stem cell transplantation (HSCT) plays an important role in the therapy of hematological malignancies. Gamma delta T cells (ᵞᵟ T cells) are a distinct lineage of T lymphocytes that might play an important role in immune recovery and could utilize a graft –versus –leukemia effect post HSCT, furthermore, higher counts of ᵞᵟ T cells could improve clinical outcome after HSCT. This work was conducted to analyze the kinetics of gamma delta T cells recovery post HSCT, and to correlate their reconstitution with different factors that may influence the clinical outcome following HSCT. In this study, 22 consecutive allogeneic hematopoietic stem cell transplantation (HSCT) recipients were analysed during the first-year post transplantation by measuring the absolute count of CD3 T cells and percentages of gamma delta T cells subsets every month for each patient using flow cytometric technique. Statistics (means, minimal, and maximal values) were used to describe patient baseline characteristics. Results were presented as mean values of CD3+, gamma delta T cells %, and p-values. Higher gamma delta T cells percentages were significantly correlated with younger patient and donor age, sex matched transplantation, leukemic diseases, un-manipulated transplants, and in patients without chronic graft versus host disease complications. Furthermore, positive correlation between CD3 T cells counts and gamma delta T cells % was also determined. Overall survival and better clinical outcome following allogeneic HSCT could be related with proper gamma delta T cells reconstitution.

No abstract available

T cell malignancies, such as T cell lymphoma and leukemia (T-NHL/T-ALL), present substantial challenges to conventional first-line therapies, highlighting an urgent need for novel therapeutic approaches. CAR-T therapies targeting T-cell-specific antigens have emerged as promising strategies against these cancers. However, the risk of tumor contamination by patient-derived T cells underscores the necessity for developing allogeneic, blast-free CAR-T therapies. Healthy donor-derived γδ T cells are attractive candidates for allogeneic CAR-T cell therapy due to their blast-free nature and the minimal risk of inducing Graft-versus-Host Disease (GvHD). Nevertheless, the difficulties in large-scale expansion and limited in vivo efficacy remain substantial obstacles to clinical application. To overcome these limitations, we aimed to enhance both the expansion and in vivo efficacy of anti-CD5 γδ CAR-T (γδCAR5) cells by incorporating “signal-3” supplement. A membrane-bound cytokine approach was employed to ensure targeted “signal 3” supplement specifically to CAR-T cells, promoting controlled expansion and activation. Among various cytokines engineered in membrane-bound forms (IL-2, IL-12, IL-15, IL-18, IL-21, and IL-36γ), only membrane-bound IL-18 (mbIL-18) induced a more than 10-fold increase in γδCAR5 cell expansion in vitro and demonstrated robust anti-tumor efficacy in a T-ALL xenograft model. Furthermore, knockdown of CD5 and PD-1, which are associated with fratricide and immunosuppression, respectively, further maximized the anti-tumor activity of γδCAR5-mbIL-18. Consequently, this multi-armoring strategy, which integrates enhanced proliferative capacity, fratricide resistance, and immune checkpoint evasion, represents an innovative approach to maximizing the therapeutic efficacy of allogeneic cell therapies for T cell malignancies. This study provides a promising framework for the advancement of more effective CAR-T therapies against challenging T cell cancers. Yu Ri Seo, Hyeong Ji Lee, Seung Rok Yu, Hyo Bhin Lee, Je Ho Lee, Hyung Cheol Kim, Youngho Lee. Multi-armored allogeneic CD5 γδ CAR T cells enhance anti-tumor efficacy in T cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4813.

TPS4613 Background: CD70 is a type II transmembrane protein of the tumor necrosis factor superfamily normally transiently expressed in activated lymphocytes, including B, T, NK, and mature dendritic cells. CD70 is aberrantly expressed in solid and hematologic cancers and is implicated in enhanced growth, metastasis, immune evasion, and suppression. In ccRCC, CD70 expression is increased in the tumor microenvironment and on malignant cells. Despite advancements in the treatment of patients with metastatic RCC, the 5-year survival rate is 15% and there remains an unmet need. ADI-270 is an investigational, allogeneic, CD70-targeting (CD27 receptor-based) Vδ1 γδ chimeric antigen receptor (CAR) T cell product expressing a dominant negative form of the TGFβ receptor II (dnTGFβRII) to mitigate the immunosuppressive effects of TGFβ within the tumor microenvironment. γδ T cells possess innate and adaptive immunity, a natural role in immune surveillance, and the ability to home to tissues. γδ T cells are ideal for an allogeneic cell therapy as their TCR recognizes MHC-independent antigens, thereby avoiding the risk of graft versus host disease. ADI-270 has demonstrated potent preclinical activity against CD70 expressing hematological and solid tumors expressing a range of CD70 levels both in vitro and in mouse xenograft models. Furthermore, ADI-270 demonstrated superior activity against tumors expressing low levels of CD70 when compared to scFv-based αβ CAR T cell benchmarks currently in clinical development. Methods: ADI-202427001 (NCT06480565) is a multi-center, phase 1 / 2 open-label, dose-escalation and -expansion study evaluating ADI-270 in adult patients with R/R ccRCC. Selected inclusion criteria include confirmed diagnosis of R/R advanced/metastatic ccRCC, previous treatment with an immune checkpoint inhibitor and a VEGF inhibitor, and Karnofsky performance status ≥ 70. Selection exclusion criteria include receipt of CD70 targeting treatment and autoimmune disease requiring systemic immunosuppressive therapy. Objectives of phase 1 include characterizing the safety and tolerability of ADI-270, identifying the recommended phase 2 dose (RP2D), and assessing cellular kinetics (CK), immunogenicity, pharmacodynamics (PD), and anti-tumor activity. Objectives of phase 2 include characterizing the anti-tumor activity, safety, immunogenicity, CK, and PD profile of ADI-270 at the RP2D. The totality of data from Phase 1 will be used to determine the RP2D for the Phase 2 part of the study. Responses will be evaluated per the RECIST 1.1 criteria. Additional efficacy analyses include duration of response, progression-free survival, and overall survival. Enrollment in study ADI-202427001 is ongoing. Clinical trial information: NCT06480565 .

Highlights What are the main findings? Provision of a phenotypic analysis of γδ T cells at the time of isolation and after ten days of stimulation using a protocol based on zoledronate and interleukin 2. Identification of potential phenotypic markers predictive of donor suitability for allogeneic cell therapies. What are the implications of the main findings? Contribution to the understanding of γδ T cell biology and their therapeutic potential. Basis for future clinical trials. Abstract Due to their anti-tumor activity and non-major histocompatibility complex (MHC) binding T cell receptor, γδ T cells are suitable candidates for allogeneic cellular immunotherapy in cancer. Recently, we developed a new protocol called Ko-Op for stimulation of γδ T cells (specifically Vy9Vδ2 T cells) that generates a cell product consisting mainly of γδ T cells with preserved anti-tumor activity targeted for clinical-grade application. In this study, we investigated the phenotype of stimulated γδ T cells and correlated this with results of functional assays to obtain a deeper understanding of the characteristics of stimulated γδ T cells. Additionally, an intensive analysis of surface molecules of unstimulated and stimulated γδ T cells is presented. Since heterogeneous results regarding the response to therapy with γδ T cells observed in earlier clinical trials could be a consequence of various extents of γδ T cell adhesion and migration ability, we addressed surface molecules associated with cellular activity and adhesion and migration functions as well. By investigating correlations between the phenotype of unstimulated γδ T cells and cellular cytotoxicity, as well as the degranulation ability of stimulated γδ T cells, we could draw conclusions about optimal donors for further allogeneic cellular therapies. Finally, we demonstrated that the phenotype varies over the time of culture and is clearly modifiable by changing the stimulation protocol.

Simple Summary Classic CAR-αβ T cells are a milestone in cancer immunotherapy. However, αβ T cells have a poor invasion of solid tumors, and the recognition of antigens is limited by a major histocompatibility complex, which is limited to autologous therapy. γδ T cells recognize antigens independent of the presentation of major histocompatibility complex molecules and can be used for allogeneic therapy. In this study, we designed a novel type of CAR-γδ T cell targeting Claudin18.2 by infection of primary γδ T cell with lentivirus. The CAR-CLDN18.2-γδ T cells show superior cytotoxicity against solid tumors than classical CAR-CLDN18.2-αβ T cells. Our results provide a new idea for the allogeneic CAR-T treatment strategy for CLDN18.2-positive solid cancer.

Despite significant progress in treating hematological malignancies with chimeric antigen receptor (CAR) T cell therapies, their application in solid tumors remains challenging. The use of autologous T cells in solid tumor patients is often limited by prior chemotherapy and advanced disease stage at treatment initiation. Unlike hematologic cancers targeted by CARs against well-defined antigens, solid tumors present limited suitable targets, underscoring the need for combination approaches that engage tumor cells through multiple mechanisms. Gamma delta (γδ) T cells have emerged as a promising platform for allogeneic CAR therapies due to their innate antitumor activity, MHC-independent tumor antigen recognition, and multiple tumor-sensing mechanisms, all combined with a low risk of graft-versus-host disease (GvHD). Vγ9Vδ2 T cells, the main γδT cell subset in peripheral blood has been preferred for initial engineering given their robust expansion via bisphosphonates and cytokines. Zoledronate, a third-generation amino-bisphosphonate inhibits the farnesyl pyrophosphate synthase enzyme which metabolizes isopentenyl pyrophosphate (IPP), leading to increased intracellular IPP levels in monocytes that activates Vγ9Vδ2 T cells through butyrophilin family molecules. This activation mechanism resembles the γδ T cell response to intracellular bacterial infections, where microbial phosphoantigens like (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) drive γδ T cell expansion. However, IPP in human cells normally does not activate this activation mechanism except under malignant transformation where phosphoantigens accumulate. We expanded Vγ9Vδ2 T cells with zoledronate, IL-2, and IL-15, followed by transduction with a lentiviral vector (LV) manufactured with a baboon endogenous virus envelope protein (BaEV) pseudotyped LV encoding a B7H3 (CD276)-specific third generation CAR. This approach achieved consistent CAR expression at extremely low multiplicity of infection (MOI), while preserving an appropriate γδ T cell phenotype. We evaluated the activity of gamma-delta (γδ) CAR-T cells expanded using this method against tumor spheroids derived from various CD276+ cancer cell lines: MDA-MB-468 (triple-negative breast cancer), U-87MG (glioblastoma), SK-OV-3 (ovarian carcinoma), and RD (rhabdomyosarcoma) at multiple effector-to-target (E:T) ratios. Motivated by prior evidence that bisphosphonates activate γδ T cells in cancer patients, we pre-sensitized target cells for 16 hours with 500 ng/mL zoledronate prior to co-culture with effector cells. Untransduced γδ T cells displayed low baseline cytotoxicity, which significantly increased following zoledronate treatment of target cells. CAR-modified γδ T cells demonstrated potent cytotoxicity early in culture, with a modest but sustained enhancement observed upon zoledronate sensitization. Furthermore, comparison of wild-type U87MG cells with a B7H3 knockout variant revealed comparable cytotoxic effects, indicating that target antigen loss did not significantly diminish antitumor activity. From this we can conclude that armoring γδ T cells imparts antigen specificity, but this must be analyzed in the context of the strong cytotoxic activity mediated by the innate immune receptors present on activated γδ T cells. These findings support further investigation into the combined use of pre infusion of zoledronate and CAR γδ T cells as a strategy to potentiate cytotoxic responses against human solid tumors.

Summary The primary challenges of CAR-T cells for solid tumor treatment involve the efficient delivery and infiltration of CAR-T cells into the tumor site, as well as overcoming the immunosuppressive tumor microenvironment. Combining chemokine-guided trafficking and delivery with the clearance of immunosuppressive barriers represents a promising strategy. An EGFR-targeted CAR-γδ T, CAR-E276 that co-expresses CCR6 and secretes PD1 blockade was in vivo validated using a non-small cell lung cancer (NSCLC) CDX model. The results from CAR-E276 indicated that integrating chemokines, checkpoint blockades, and γδ T cell properties into CAR-T cells facilitated their efficient trafficking into tumor tissues, ensured prolonged persistence, and achieved robust tumor-killing effects without inducing GvHD. Notably, CAR-E276 also exhibited potential for off-the-shelf and allogeneic applications. These findings are expected to offer valuable insights and drive the development of CAR-T therapies targeting solid tumors.

Introduction: Relapsed or refractory (R/R) acute myeloid leukemia (AML) has a dismal prognosis. Due to the highly heterogeneous nature of AML blasts, Chimeric Antigen Receptor (CAR) -T cells targeting specific antigens, including CD123, CLL-1 and CD33, have demonstrated limited efficacy across leukemic cells and certain off-target toxicity to normal cells. On the other hand, γδ T cells are immune cells with inherent anti-tumor activity through multiple receptors such as γδ TCRs, NKRs, DNAM-1 and they can be developed into allogeneic cell therapy. Unmodified γδ T cells can be efficiently expanded in vitro and show killing of AML cell lines. In clinical trials, they show certain activity toward AML yet efficacy would still need to be improved. It is likely that γδ T based cell therapy may offer solution to address the heterogenic nature of AML if its natural killing mechanism can be further enhanced. To this end, we have designed various constructs with different targeting moieties and tested them for in vitro killing. Our top candidate Super-γδ T is armed with an NKG2D based CAR and is manufactured by non-viral site-specific integration technology that we have developed in-house (PrecisionGENE, PrecisionGENetic Engineering). This Super-γδ T showed enhanced killing toward AML and several tumor cell lines from various blood and solid tumors, with no obvious toxicity toward normal human cells. Methods: In the current study, a novel NKG2D based CAR was inserted into a target gene locus by the non-viral site-specific integration technology. Specific killing activities of Super-γδ T cells to AML cell lines (HL60, THP1) and several tumor cell lines (including NSCLCs, HCCs, RCCs and OCs) were measured by a luciferase-based method. To investigate the killing activity for CLL1 negative tumor cells, a CLL1 knocked out tumor cell line-THP1-CLL1-KO was generated, and the specific killing of unmodified γδ T, CLL1 CAR-αβ T, and Super-γδ T to THP1- CLL1- KO were studied in vitro. To evaluate the toxicity of Super-γδ T cells to PBMCs, unmodified γδ T, CLL1 CAR-αβ T, and Super-γδ T were co-cultured with human PBMC, respectively, and PBMC composition changes were measured by a flow-based assay. Results: To minimize off-target editing, we used site-specific integration technology PrecisionGENE for the manufacturing of Super-γδ T cells and have achieved over 70% integration efficiency. Cytotoxicity of the newly developed Super -γδ T cells to AML cell lines was tested in vitro. Notably, Super-γδ T cells showed almost 100% killing to AML cell lines (HL60 cells) in vitro at effector-to-target ratio of 3:1, which is comparable to CLL1 CAR-γδ T, but over 5-fold more potent than unmodified γδ T with 20%-30% killing. In addition, there was significantly increased cytokine secretion from Super -γδ T cells when co-cultured with AML tumor cells compared to γδ T. To evaluate whether Super -γδ T cells could overcome cancer cell heterogenicity, a CLL1 negative target cell THP1-CLL1-KO cell line was used for comparison. Importantly, Super-γδ T cells showed almost 80% killing efficiency against the THP1-CLL1-KO at a E:T ratio of 1:3, while CLL1 CAR- αβ T's killing was minimal. Moreover, normal PBMCs and fibroblast cells were used to study for the off-target cytotoxicity of Super-γδ T cells. It is worth mentioning that while there was no obvious off-target toxicity by Super-γδ T for monocyte when co-culturing with PBMC, CLL1 CAR-αβ T showed killing toward monocytes. To further investigate the killing potential toward different tumor cell lines, cytotoxicity of Super-γδ T cells to various blood and solid tumor cell lines was studied in vitro. Super-γδ T showed enhanced cytotoxicity to a broad-spectrum of tumor cell lines including NSCLC, HCC, RCC and OC cell lines, independent of their mutation background. In vivo efficacy for Super-γδ T is under-going and the result will be presented. Conclusions: Taken together, Super-γδT showed much enhanced anti-tumor functionality and superior safety profile in vitro. Super-γδT demonstrated anti-tumor activity against both blood and solid tumor cell lines independent of their mutation background. Therefore, Super-γδ T has the potential to be used as a novel cellular therapeutic agent for treating R/R AML and multiple solid tumors, and it could be efficacious toward cancers of various types, stages and forms.

Background: Adoptive cellular therapy (ACT) has been transformative in the treatment of hematological malignancies. Limitations of ACTs include difficulty identifying suitable tumor antigens for solid tumors, the ability to target only one antigen per construct which commonly results in secondary resistance due to antigen escape, and largely autologous approaches which are challenging in heavily pre-treated patients with poor marrow function and those with rapidly progressive disease who may not receive their personalised cellular products in time. This is a phase I study evaluating a novel off-the-shelf allogenic mRNA-electroporated NKG2D ligand-targeting CAR-grafted γδ T cell therapy. NKG2DLs are a group of 8 types of stress-induced cancer antigens which are preferentially and widely expressed on tumor cells from diverse tissue origins but are not typically present on normal tissue. NKG2DL-targeting γδ T cells will be manufactured from peripheral blood mononuclear cells isolated from healthy donors enrolled on the donor protocol of the ANGELICA trial. ANGELICA is a phase I study evaluating the safety, tolerability and recommended phase 2 dose (RP2D) of NKG2DL-targeting CAR-grafted γδ T cells in patients with treatment refractory tumors. Methods: Patients will be enrolled in a 3+3 design. Dose escalation will be performed at 3 dose levels: 1x108, 3x108 and 1x109 per infusion (adjusted for body weight). Lymphodepletion with fludarabine 25mg/m2/day and cyclophosphamide 250mg/m2/day will given for 3 days and completed at least 2 days prior to the first cycle of treatment. Patients will receive 4 doses of weekly infusions for the first cycle, with up to 5 subsequent infusions every 2 months as maintenance. Patients will receive intravenous zoledronic acid 1mg prior to each cellular infusion and subcutaneous IL-2 1x106 IU/m2 within 2 hours of each cellular infusion as pre-clinical data demonstrated cancer cell sensitisation and prolonged γδ T cell survival with these adjuncts. Dose-limiting toxicities will be assessed over the first 8 weeks. Adverse events, response rates (RECIST v1.1), survival outcomes and immunomonitoring (immune cell phenotyping and serum cytokine analysis) will be assessed. The trial is currently enrolling healthy donors; enrolment of patients at the first dose level will begin in February 2024. NCT05302037 TABLE 1: NAND Dose Level and Schedule Dose Level Dose per infusion Infusion schedule Number of patients 1 Weight 65kg and above: 1x108.Weight less than 65kg: 1.5x106. Cycle 1: weekly x 4 infusions. Maintenance phase: 2-monthly x 5 infusions. 3-6 2 Weight 65kg and above: 3x108.Weight less than 65kg: 4.6x106. 3-6 3 Weight 65kg and above: 1x109.Weight less than 65kg: 1.5x107. 3-6 Citation Format: Joan Choo, Wee Kiat Tan, Lucas Luk, Jieming Zeng, Teck Guan Soh, Sou Yen Soon, Jedidah Lieow, Calista Wong, Mei Yan Pang, Sudipto Bari, Michelle Poon, Liang Piu Koh, Wee Joo Chng, Anand Jeyasekharan, Lip Kun Tan, Esther Chan, Raghav Sundar. A phase I trial to evaluate allogeneic NKG2DL-targeting chimeric antigen receptor-grafted γδ T cells in subjects with advanced solid tumors or hematological malignancies (the ANGELICA Trial) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT160.

γδ T cells (GDTs) represent a promising allogeneic immune cell platform due to their MHC-unrestricted cytotoxicity. In this study, we developed a novel B7-H3-specific CAR vector (CD3ζ-CD28-based) and evaluated the therapeutic efficacy of B7-H3-directed γδ CAR-T (B7H3-GD CAR-T) cells in preclinical glioblastoma models. We constructed a lentiviral vector encoding a B7-H3-specific CAR and transduced it into human allogeneic GDT cells. B7-H3 expression was assessed in glioblastoma cell lines (U87, U373, U138) and 10 patient-derived primary cell lines. Formalin-fixed paraffin-embedded (FFPE) tumor specimens were analyzed using immunohistochemistry. Additionally, TCGA data were analyzed for B7-H3 expression and survival correlation. In vitro cytotoxicity and cytokine secretion were evaluated. In vivo efficacy was tested in an orthotopic U87 glioblastoma model in NOG mice. Bioinformatic analysis of the TCGA database confirmed that higher B7-H3 expression correlates with poorer overall survival in glioma patients. IHC of FFPE specimens demonstrated increased B7-H3 expression in high-grade gliomas, surpassing HER2 and GD2 expression. We validate that B7-H3 is overexpressed in high-grade gliomas and associated with poor prognosis, suggesting its value as a therapeutic target. In vitro, B7H3-GD CAR-T cells exhibited markedly enhanced cytotoxicity and IFN-γ release compared to unmodified GDTs, with killing strongly correlating with B7-H3 expression and abrogated by B7-H3 antibody blockade. In vivo, a single intratumoral injection of 1×10⁶ B7H3-GD CAR-T cells resulted in complete tumor regression in >80% of mice. Furthermore, intraventricular injection of B7H3-GD CAR-T cells also demonstrated comparable efficacy, indicating the feasibility of clinically applicable delivery routes. This is the first study to demonstrate potent, target-dependent antitumor activity of B7H3-targeted γδ CAR-T cells in glioblastoma. Our results validate B7-H3 as a clinically relevant target and support the use of allogeneic GDTs as a next-generation platform for glioblastoma immunotherapy.

Introduction: PSMA is a transmembrane glycosylated homodimer overexpressed in >80% of prostate cancers, with increased expression in advanced stages of the disease. Formation of homodimeric PSMA is necessary for enzymatic function and displays epitopes that are potentially more distinct from alternative PSMA-like proteins. Clinically, autologous anti-PSMA αβ CAR T cells have shown initial efficacy with limited therapeutic index. Compared to αβ T cells and other innate cells, γδ T cells are capable of multifunctional innate and adaptive targeting and infiltrate into tumor-associated tissues, including associated tropism for prostate tumor tissues. Additionally, γδ CAR T cells have demonstrated enhanced tumoricidal activity and activation-induced cytokine profiles that may decrease CRS-associated toxicities. We characterized γδ T cells modified from a set of novel scFv-based CARs targeting PSMA for prostate cancer and mapped unique epitopes for binders associated with the most potent CARs. Methods: Phage panning was used to identify anti-PSMA scFv sequences, which were reformatted into IgGs and characterized for binding to both cells expressing endogenous PSMA and recombinant PSMA. Binders with favorable profiles were reformatted into CARs in VH-VL and VL-VH orientations and transduced into Vδ1 T cells, a primarily tissue-resident subset, activated from healthy donor PBMCs. We identified lead CARs based on anti-tumor efficacy both in vitro in coculture assays and in vivo in tumor xenograft models and compared their activity to Vδ1 CAR T cells transduced with a clinically validated benchmark, J591. Epitope mapping of binders in the lead CARs was performed using a funnel of molecular assays including dot blots, competition assays, ELISAs on recombinant PSMA from different species and cross-linking mass spectrometry (XL-MS). Results: High-affinity PSMA binders yielded potent CARs. Lead PSMA CARs demonstrated robust in vitro cytotoxicity and antigen-specific binding to PSMA-expressing cell lines in a manner comparable to, or greater than the J591-based CAR T cells. Additionally, lead PSMA CARs showed anti-tumor control in 22Rv1 human prostate cancer xenografts, a highly resistant model with heterogenous PSMA expression. Epitope mapping studies on binders in lead PSMA CARs identified conformational, membrane-distal epitopes that are distinct from the predicted linear epitope of J591 and relevant comparators. Conclusion: The epitope for a lead PSMA antibody includes residues in a helix involved in PSMA dimer formation. The γδ CAR T construct derived from this binder demonstrates enhanced preclinical performance when compared to the J591 reference CAR T. Combining the features of this novel target and the potential of the γδ-based CAR T approach, the lead PSMA γδ CAR T cells described here are candidates for further clinical development in the context of additional armoring technologies and CAR architecture modifications designed to complement therapeutic potential. Citation Format: Nitya S. Ramadoss, Elizabeth Speltz, Tonya Capillo, Erika Meaddough, Katherine Wang, Natalia Friedland, Jonathan Wong, Alexander G Teague, Aruna Azameera, Smitha Rao Yelaguli Gundurao, Ramandeep Kaur, Morgan Smith-Boeck, Michael Salum, Yvan Chanthery, Shon Green, Marissa Herrman, Kevin P Nishimoto, Blake T Aftab, Arun Bhat. Preclinical characterization of allogeneic CAR γδ T Cell therapy for prostate cancer targeting a novel dimeric epitope on PSMA [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C117.

γδ T cells represent a promising cell platform for adoptive cell therapy. Their natural anti-tumor reactivity and HLA-independent target cell recognition make them an attractive platform for allogeneic adoptive immunotherapy clinical interventions. Initial clinical trials exploring allogeneic γδ T-cell therapies have demonstrated encouraging safety profiles. However, their therapeutic efficacy, especially against solid tumors, remains limited. This highlights the need for further optimization of γδ T cell products to improve anti-tumor potency, such as the increased targeting induced by the expression of a chimeric antigen receptors (CAR). However, a critical challenge in the development of CAR-γδ T cell therapies has been optimizing transduction efficiency with standard vector formats allowing for optimal CAR transgene expression that then produces an optimal therapeutic product. Here we present an effective method for enhancing CAR transgene expression in γδ T cells using a Baboon-pseudotyped lentiviral vector (BaEV-LV), comparing it to the conventional vesicular-stomatitis-virus-G protein (VSV-G) LVs. BaEV-LV significantly enhanced the transduction efficiency of γδ T cells with CARs, while conserving the beneficial cell product composition and phenotype of untransduced γδ T cells. The γδ T cells transduced with BaEV-LV CARs demonstrated significantly enhanced cytotoxicity against B7H3-expressing tumor cells in both 2D and 3D in vitro models. Our findings represent a significant advancement in CAR-γδ T cell engineering, offering a promising new avenue for cancer immunotherapy that combines the unique properties of Vγ9Vδ2 T cells with the targeted specificity of CAR technology. This method is compatible with automated closed-system platforms such as the CliniMACS Prodigy®, facilitating Good Manufacturing Practice (GMP)-compliant production for clinical trials. This feature significantly enhances the translational potential of engineered γδ T cells, paving the way for the development of next-generation γδ T cell-based immunotherapies.

γδ T cells are unconventional T cells which kill tumors independently of antigen presentation by MHC class I, making them a promising candidate for allogeneic cell therapy. While Vδ2 γδ T cells are the most prominent in blood, the less prevalent Vδ1 subset has shown superior tumor killing. However, difficulties in expanding Vδ1 T cells has limited their clinical use. Here, we evaluated the expansion and activation of Vδ1 T cells using K562 feeder cells expressing membrane-bound IL-21. We first expanded γδ T cells from PBMCs long-term, and assessed their anti-tumor functions against breast and ovarian cancer cell lines and a xenograft model of human ovarian cancer. We also tested their metabolic function within the suppressive ovarian cancer ascites tumor microenvironment (TME) to assess metabolic fitness. Lastly, we generated Vδ1 cells with stable anti-HER2 chimeric antigen receptor (CAR) expression and assessed their cytotoxicity against HER2+ tumor cells. We found that expanded γδ T cells were primarily Vδ1 and displayed higher cytotoxicity than unexpanded γδ T cells. Expanded Vδ1 cells significantly reduced tumor burden in vivo and retained their cytotoxicity and metabolism in the ascites TME. Anti-HER2 CAR-Vδ1 T cells displayed enhanced cytotoxicity and degranulation against HER2+ breast cancer cells. Overall, we demonstrate the anti-tumor potential of HER2 CAR-expressing expanded Vδ1 cells as a metabolically fit, off-the-shelf cell therapy for hard-to-treat solid tumors. Supported by the Canadian Institutes of Health (CIHR) Research Doctoral Scholarship Award; CIHR Project Grant. Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Cellular immunotherapy for aggressive pediatric solid tumors like neuroblastoma (NB) has focused on autologous products of αβ T cells, that so far, have been uniformly unsuccessful. γδ T cells offer a potentially superior, off-the-shelf therapy that is directly cytotoxic towards tumors without alloreactivity. Furthermore, γδ T cell infiltration of the hostile, solid tumor microenvironment is a prognostic marker of favorable disease outcome. Our team recently opened a first-in-child evaluation of unengineered allogeneic γδ T cells in combination with dinutuximab, an anti-GD2 antibody, and chemotherapy (NCT05400603). Our focus now is to optimize a second-generation γδ T-cell therapy by engineering the expression of tumor targeting chimeric antigen receptors (CAR). We hypothesize that CAR-targeting will further enhance γδ T-cell homing and antitumor potency. While anti-GD2 antibodies have been clinically successful immunotherapies for NB, GD2-targeted cell therapies need improvement. Our team recently validated a novel immunotherapy target for NB, protein tyrosine kinase 7 (PTK7), an inactive tyrosine kinase expressed highly amongst all NBs with low to no normal pediatric tissue expression. We designed a dual-targeting platform directed against both GD2 and PTK7 using γδ T cells, where CARs are separately encoded and dually expressed. Previously optimized transgenes containing GD2 or PTK7 scFv targeting domains followed by a CD8 hinge region, CD28 co-stim/trans-membrane domain, and CD3ζ signalling domain were inserted under the T7 promoter for mRNA production. Purified mRNA was electroporated into γδ T cells following their thaw from cryopreservation. Electroporation titrations were performed to optimize CAR expression to be detected up to 72 hours post-modification, which is compatible with the approximate in vivo lifespan γδ T cells in mice. Simultaneous expression of both CARs appears highest 24 h after electroporation, with ~70% of the target γδ T cell population modified. Anti-GD2/PTK7 γδ T cells are potent against the NB cell line IMR5 (GD2+PTK7+) in a 4 h cytotoxicity assay at effector:target ratios as low as 0.5:1. Importantly, specificity is also shown against NB cell lines genetically engineered to represent the clinical heterogeneity of GD2 and PTK7 expression that may be observed, where the dual CAR therapy is effective against GD2+PTK7+, GD2+PTK7−, and GD2−PTK7+, but not GD2−PTK7− NBs. In conclusion, we developed a dual CAR-based cellular therapy for NB using γδ T cells as an effector population in place of classical αβ T cells. Early studies demonstrate feasibility for innovative dual CAR expression and show promise for strong anti-NB potency. Future work will optimize CAR signaling domains for maximal efficacy in solid tumors, as well as confirm efficacy and safety in vivo with results rapidly translatable into our established γδ T cell clinical trial pipeline. Citation Format: Hunter C. Jonus, Jasmine Y. Lee, Jordan A. Silva, H. Trent Spencer, Kelly C. Goldsmith. Dual targeted CAR immunotherapy for neuroblastoma using γδ T cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4093.

Summary Adoptive cell therapy using allogeneic γδ-T cells is a promising option for off-the-shelf T cell products with a low risk of graft-versus-host disease (GVHD). Long-term persistence may boost the clinical development of γδ-T cell products. In this study, we found that genetically modified Vγ9+Vδ2+ T cells expressing a tumor antigen-specific αβ-TCR and CD8 coreceptor (GMC) showed target-specific killing and excellent persistence. To determine the mechanisms underlying these promising effects, we investigated metabolic characteristics. Cytokine secretion by γδ-TCR-stimulated nongene-modified γδ-T cells (NGMCs) and αβ-TCR-stimulated GMCs was equally suppressed by a glycolysis inhibitor, although the cytokine secretion of αβ-TCR-stimulated GMCs was more strongly inhibited by ATP synthase inhibitors than that of γδ-TCR-stimulated NGMCs. Metabolomic and transcriptomic analyses, flow cytometry analysis using mitochondria-labeling dyes and extracellular flux analysis consistently suggest that αβ-TCR-transduced γδ-T cells acquire superior mitochondrial function. In conclusion, αβ-TCR-transduced γδ-T cells acquire superior mitochondrial function with promising persistence.

ABSTRACT γδ T lymphocytes represent an emerging class of cellular immunotherapy with preclinical promise to treat cancer, notably neuroblastoma. The innate-like immune cell subset demonstrates inherent cytoxicity toward tumor cells independent of MHC recognition, enabling allogeneic administration of healthy donor-derived γδ T cell therapies. A current limitation is the substantial interindividual γδ T cell expansion variation among leukocyte collections. Overcoming this limitation will enable realization of the full potential of allogeneic γδ T-based cellular therapy. Here, we characterize γδ T cell expansions from healthy adult donors and observe that highly potent natural killer (NK) lymphocytes expand with γδ T cells under zoledronate and IL-2 stimulation. The presence of NK cells correlates with both the expansion potential of γδ T cells and the overall potency of the γδ T cell therapy. However, the potency of the cell therapy in combination with an antibody-based immunotherapeutic, dinutuximab, appears to be independent of γδ T/NK cell content both in vitro and in vivo, which minimizes the implication of interindividual expansion differences toward efficacy. Collectively, these studies highlight the utility of maintaining the NK cell population within expanded γδ T cell therapies and suggest a synergistic action of combined innate cell immunotherapy toward neuroblastoma.

No abstract available

Relapse is the most frequent cause of treatment failure after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Natural killer (NK) cells and γδ T cells reconstitute early after allo-HSCT, contribute to tumor immunosurveillance via major histocompatibility complex-independent mechanisms and do not induce graft-versus-host disease. Here we performed a quantitative and qualitative analysis of the NK and γδ T cell repertoire in healthy individuals, recipients of HLA-matched sibling or unrelated donor allo-HSCT (MSD/MUD-HSCT) and umbilical cord blood-HSCT (UCB-HSCT). NK cells are present at high frequencies in all allo-HSCT recipients. Immune reconstitution (IR) of vδ2+ cells depended on stem cell source. In MSD/MUD-HSCT recipients, vδ2+ comprise up to 8% of the total lymphocyte pool, whereas vδ2+ T cells are barely detectable in UCB-HSCT recipients. Vδ1+ IR was driven by CMV reactivation and was comparable between MSD/MUD-HSCT and UCB-HSCT. Strategies to augment NK cell mediated tumor responses, similar to IL-15 and antibodies, also induced vδ2+ T cell responses against a variety of different tumor targets. Vδ1+ γδ T cells were induced less by these same stimuli. We also identified elevated expression of the checkpoint inhibitory molecule TIGIT (T cell Ig and ITIM domain), which is also observed on tumor-infiltrating lymphocytes and epidermal γδ T cells. Collectively, these data show multiple strategies that can result in a synergized NK and γδ T cell antitumor response. In the light of recent developments of low-toxicity allo-HSCT platforms, these interventions may contribute to the prevention of early relapse.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an immunosuppressive tumor microenvironment with a dense desmoplastic stroma. The expression of β-galactoside-binding protein galectin-3 is regarded as an intrinsic tumor escape mechanism for inhibition of tumor-infiltrating T cell function. In this study, we demonstrated that galectin-3 is expressed by PDAC and by γδ or αβ T cells but is only released in small amounts by either cell population. Interestingly, large amounts of galectin-3 were released during the co-culture of allogeneic in vitro expanded or allogeneic or autologous resting T cells with PDAC cells. By focusing on the co-culture of tumor cells and γδ T cells, we observed that knockdown of galectin-3 in tumor cells identified these cells as the source of secreted galectin-3. Galectin-3 released by tumor cells or addition of physiological concentrations of recombinant galectin-3 did neither further inhibit the impaired γδ T cell cytotoxicity against PDAC cells nor did it induce cell death of in vitro expanded γδ T cells. Initial proliferation of resting peripheral blood and tumor-infiltrating Vδ2-expressing γδ T cells was impaired by galectin-3 in a cell-cell-contact dependent manner. The interaction of galectin-3 with α3β1 integrin expressed by Vδ2 γδ T cells was involved in the inhibition of γδ T cell proliferation. The addition of bispecific antibodies targeting γδ T cells to PDAC cells enhanced their cytotoxic activity independent of the galectin-3 release. These results are of high relevance in the context of an in vivo application of bispecific antibodies which can enhance cytotoxic activity of γδ T cells against tumor cells but probably not their proliferation when galectin-3 is present. In contrast, adoptive transfer of in vitro expanded γδ T cells together with bispecific antibodies will enhance γδ T cell cytotoxicity and overcomes the immunosuppressive function of galectin-3.

CD70 is a type II transmembrane protein of the tumor necrosis factor superfamily normally transiently expressed in activated lymphocytes, including B, T, NK, and mature dendritic cells. CD70 is aberrantly expressed in solid and hematologic cancers and is implicated in enhanced growth, metastasis, and immune evasion and suppression. In ccRCC, CD70 expression is increased in the tumor microenvironment and on malignant cells. Despite advancements in the treatment of patients with metastatic RCC, the 5 year survival rate is 15% and there remains an unmet need. ADI-270 is an investigational, allogeneic, CD70 targeting (CD27 receptor based) Vδ1 γδ CAR T cell product expressing a dominant negative form of the TGFβ receptor II (dnTGFβRII) to provide resistance against an immunosuppressive tumor microenvironment. γδ T cells possess innate and adaptive immune response functions and a natural role in immune surveillance and anti tumor immunity, including the ability to home to tissues and recognize tumors through multiple antigens. γδ T cells are ideal for an allogeneic cell therapy as their TCR recognizes MHC independent antigens and avoids the risk of graft versus host disease without the need for gene editing. ADI-270 has demonstrated potent preclinical activity against CD70 expressing hematological and solid tumors expressing a range of CD70 levels both in vitro and in mouse xenograft models. Furthermore, ADI-270 demonstrated superior activity against tumors expressing low levels of CD70 when compared to scFv based αβ CAR T cell benchmarks currently in clinical development. ADI-202427001 (NCT06480565) is a phase 1 / 2 open label, dose escalation and dose expansion study evaluating ADI-270 in adult pts with R/R ccRCC. Inclusion criteria include confirmed diagnosis of R/R advanced/metastatic ccRCC, previous treatment with an immune checkpoint and VEGF inhibitor; exclusion criteria include previous CD70 targeting treatment and autoimmune disease requiring systemic immunosuppressive therapy. Objectives of phase 1 include characterizing the safety and tolerability of ADI-270, identifying the RP2D for phase 2, and assessing cellular kinetics, immunogenicity, pharmacodynamics (PD), and efficacy; objectives of phase 2 include characterizing the efficacy, safety, immunogenicity, and PD profile of ADI-270 at the RP2D. Safety, efficacy, CK, and PD data from Phase 1 will determine the RP2D for Phase 2. Response will be evaluated per RECIST 1.1 criteria. Additional efficacy analyses include duration of response, progression free survival, and overall survival. Enrollment in study ADI-202427001 is ongoing. Sumanta K. Pal, Benjamin Garmezy, Helen Budworth, Xiaoyan Du, Shon Green, Kevin Nishimoto, Jackie Kennedy-Wilde, Blake T. Aftab, Julia Maltzman, Gregory S. Vosganian, Brian Rini. A phase 1/2 first in human study of ADI-270, an armored allogeneic anti-CD70 chimeric antigen receptor (CAR)γδT cell therapy, in patients (pts) with relapsed or refractory (R/R) clear cell renal cell carcinoma (ccRCC) (trial in progress) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr CT187.

Immune checkpoint inhibitors have played a pivotal role in cancer treatment, and various combination therapies are being developed. However, their add-on effects remain limited, highlighting the urgent need for novel therapeutic modalities. Among various approaches, the adoptive transfer of immune effector cells has attracted particular attention, especially the use of allogeneic immune effector cells due to the frequently immunosuppressed status of cancer patients. We have previously focused on Vδ2 γδ T cells and NK cells, developing methods for their large-scale expansion. These innate immune effector cells are highly suitable for allogeneic adoptive transfer, as they exhibit tumor cytotoxicity independently of the major histocompatibility complex. In this study, we evaluated the cytotoxic properties of these innate immune effector cells, including innate cytotoxicity, antigen-specific cytotoxicity, antibody-dependent cellular cytotoxicity (ADCC), and cytokine production. After obtaining informed consent, peripheral blood mononuclear cells (PBMCs) were collected from healthy adults, lung cancer patients, and pleural mesothelioma patients to prepare PBMCs. The PBMCs were stimulated with tetrakis-pivaloyloxymethyl 2-(thiazole-2-ylamino) ethylidene-1, 1-bisphosphonate (PTA), a bisphosphonate prodrug, along with IL-2 to enable the expansion of Vδ2 γδ T cells. The expanded Vδ2 γδ T cells were then co-cultured with human lung cancer cell lines to evaluate their effector functions in vitro. Additionally, CD3-negative PBMCs were cultured with IL-2 and IL-18 to expand NK cells, which were subsequently co-cultured with human pleural mesothelioma cells to assess the effector functions of NK cells in vitro. PTA was used as the antigen, and anti-EGFR antibody was used for ADCC assays. Vδ2 γδ T cells exhibited PTA-dependent cytotoxicity against lung cancer cells. Moreover, Vδ2 γδ T cells with high CD16 expression demonstrated ADCC and IFN-γ production against lung cancer cells. NK cells displayed innate cytotoxicity against pleural mesothelioma cells, and this cytotoxic activity increased in an antibody-dependent manner. The use of PTA/IL-2 enabled the high-purity expansion of Vδ2 γδ T cells, which exhibited robust immune effector functions against lung cancer cells via T cell receptor and CD16-dependent mechanisms. Similarly, IL-2/IL-18 facilitated the high-purity expansion of NK cells, which demonstrated strong innate cytotoxicity and ADCC against pleural mesothelioma cells. Moving forward, in vivo studies are planned to advance the potential for infusion therapy with Vδ2 γδ T cells and NK cells. Hiromi Tomono, Kazumasa Akagi, Yasuhiro Umeyama, Midori Matsuo, Hirokazu Taniguchi, Hiroshi Gyotoku, Shinnosuke Takemoto, Yoshimasa Tanaka, Hiroshi Mukae. Innate immune cell-mediated anti-tumor activity against lung cancer and pleural mesothelioma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 881.

The benefits of CAR-T therapy could be expanded to the treatment of solid tumors through the use of derived autologous αβ T cell, but clinical trials of CAR-T therapy for patients with solid tumors have so far been disappointing. CAR-T therapy also faces hurdles due to the time and cost intensive preparation of CAR-T cell products derived from patients as such CAR-T cells are often poor in quality and low in quantity. These inadequacies may be mitigated through the use of third-party donor derived CAR-T cell products which have a potent anti-tumor function but a constrained GVHD property. Vγ9Vδ2 TCR have been shown to exhibit potent antitumor activity but not alloreactivity. Therefore, in this study, CAR-T cells were prepared from Vγ9Vδ2 T (CAR-γδ T) cells which were expanded by using a novel prodrug PTA. CAR-γδ T cells suppressed tumor growth in an antigen specific manner but only during a limited time window. Provision of GITR co-stimulation enhanced anti-tumor function of CAR-γδ T cells. Our present results indicate that, while further optimization of CAR-γδ T cells is necessary, the present results demonstrate that Vγ9Vδ2 T cells are potential source of ‘off-the-shelf’ CAR-T cell products for successful allogeneic adoptive immunotherapy.

The induced pluripotent stem cell (iPSC)-derived cell therapy bears unparallel competitiveness in the value proposition across the cellular medicine landscape. iPSC allows a given cellular therapeutic to be produced from a single cell clone that harbors all the desired genetic manipulations, thus ensures absolute homogeneity of the gene edits at the single cell level. The subsequent uniformed differentiation and expansion methodologies further safeguard the consistency of the therapeutic products and eliminate patient-to-patient or donor-to-donor manufacturing inconsistencies commonly seen in the autologous and allogeneic donor-derived cell therapies. These features will not only dramatically reduce the cost-of-goods but also enhance the affordability and accessibility, hence democratizing the application of the cell therapy globally. The gamma delta (γδ) T cell possesses numerous unique characteristics that are advantageous as a cellular therapeutic in the clinic, such as manifesting minimal CRS, GvHD, and ICANS etc. Moreover, activated γδT cells could target a broad range of tumor cells and demonstrate the capacity for tumor reduction in murine xenotransplant tumor models. However, it is well known that the responses of the allogeneic T cell therapeutics are hampered by the lack of persistence and durable anti-tumor activity, which could dim the prospects of utilizing the γδT cell as an effective cancer therapeutic. Here, to overcome these shortcomings, through a hypothesis-driven, combinatorial KO of SOCS family genes, we successfully enhanced the durable killing capacity of iγδT by creating a “conditional” activation scenario that allows protracted and amplified signal transduction events induced by activating cytokines. We also lengthened the longevity of the iγδT by depleting pro-apoptotic genes to bolster effector's intrinsic survival capability, while eliminating B2M/CIITA and increasing the expression of HLA-E to evade host immune surveillance. Further, a proprietary signal converter was designed to transform the soluble factors generated during tumor-effector engagement into the inducers of productive signal events that drive iγδT proliferation and activation. Altogether, these genetic engineering efforts that aim to achieve the goldilocks signaling balance could strengthen the efficacy and longevity of the iγδT, while avoid the possible dysfunction, like exhaustion, caused by introducing constitutively active cytokine receptors as many others have attempted. Building on this engineered platform, through incorporating chimeric antigen receptor (CAR) constructs against specific tumor associated antigens (TAAs), we further demonstrated that these engineered CAR-iγδTs exhibited robust long-term durable killing (more than 2 weeks) without any supporting cytokines in the tumor rechallenging studies in vitro, accompanied by the significantly increased tumor lysis enzyme secretion. In addition, these CAR-iγδT cells, either single dosed or repeated, eliminated the engrafted tumor cells in vivo with persisted cellular kinetics and boosted expansion in the selected B cell- and myeloid-malignancy models. Altogether, the summation of aforementioned data elevated our confidence of applying BeiGene's engineered CAR-iγδT in the clinic in the near future. Combining with the robust iPSC differentiation and expansion methodologies to derive γδTs, we believe that arriving to an inflection point of turning cell therapy into a massively produced medicine should not be far.

Vγ9Vδ2 T cells are promising candidates for cellular tumor immunotherapy. Due to their HLA-independent mode of action, allogeneic Vγ9Vδ2 T cells can be considered for clinical application. To apply allogeneic Vγ9Vδ2 T cells in adoptive immunotherapy, the methodology used to obtain adequate cell numbers with optimal effector function in vitro needs to be optimized, and clinical safety and efficacy also need to be proven. Therefore, we developed a novel formula to improve the expansion of peripheral γδ T cells from healthy donors. Then, we used a humanized mouse model to validate the therapeutic efficacy of expanded γδ T cells in vivo; furthermore, the expanded γδ T cells were adoptively transferred into late-stage liver and lung cancer patients. We found that the expanded cells possessed significantly improved immune effector functions, including proliferation, differentiation, and cancer cell killing, both in vitro and in the humanized mouse model. Furthermore, a phase I clinical trial in 132 late-stage cancer patients with a total of 414 cell infusions unequivocally validated the clinical safety of allogeneic Vγ9Vδ2 T cells. Among these 132 patients, 8 liver cancer patients and 10 lung cancer patients who received ≥5 cell infusions showed greatly prolonged survival, which preliminarily verified the efficacy of allogeneic Vγ9Vδ2 T-cell therapy. Our clinical studies underscore the safety and efficacy of allogeneic Vγ9Vδ2 T-cell immunotherapy, which will inspire further clinical investigations and eventually benefit cancer patients.

Background Chimeric antigen receptor (CAR)-T cells have emerged as a breakthrough treatment for relapse/refractory hematological tumors, showing impressive complete remission rates. However, around 50% of the patients relapse before 1-year post-treatment. T-cell ‘fitness’ is critical to prolong CAR-T persistence and activity. Allogeneic T cells from healthy donors are less dysfunctional or exhausted than autologous patient-derived T cells; in this context, Delta One T cells (DOTs), a recently described cellular product based on MHC/HLA-independent Vδ1+γδ T cells, represent a promising allogeneic platform. Methods Here we generated and preclinically validated, for the first time, 4-1BB-based CAR-DOTs directed against the interleukin-3α chain receptor (CD123), a target antigen widely expressed on acute myeloid leukemia (AML) blasts. Results CD123CAR-DOTs showed vigorous, superior to control DOTs, cytotoxicity against AML cell lines and primary samples both in vitro and in vivo, even on tumor rechallenge. Conclusions Our results provide the proof-of-concept for a DOT-based next-generation allogeneic CAR-T therapy for AML.

Background: The T cell antigen coupler (TAC) is a novel, proprietary chimeric receptor that facilitates the redirection of T cells to tumor cells and activates T cells by co-opting the endogenous T cell receptor complex with the goal to elicit safe and durable anti-tumor responses. TAC01-HER2, a first-in-class, autologous TAC T cell product targeting HER2 (ERBB2), has entered a phase I/II clinical trial in patients with HER2-positive solid tumors. Here, we describe the development of an allogeneic HER2-TAC T cell product based on Vγ9Vδ2 (γδ) T cells which belong to a subset of T cells that recognize target cells in a human leukocyte antigen (HLA) independent manner. Thus, γδ T cells do not cause GvHD and have the potential for allogeneic cell therapy applications. Materials and Methods: A variety of in vitro and in vivo assays were used to evaluate the potency and safety of HER2-TAC γδ T cells generated from multiple donors. In vitro assays included flow cytometric analysis determining the γδ T cell phenotype, intracellular cytokines, CD69 upregulation, and T cell proliferation. Anti-tumor cytotoxicity was assessed via real-time microscopy-based co-culture assays. Mixed lymphocyte reactions (MLR) were performed to measure cytokine production and proliferation of HER2-TAC γδ T cells in response to HLA mismatches between unrelated donors. In vivo studies examined the anti-tumor effect of HER2-TAC γδ T cells against established solid HER2-expressing tumors. Results: HER2-TAC γδ T cells selectively reacted to HER2-expressing tumor cells in co-culture, as demonstrated by CD69 upregulation, intracellular cytokine production, increase in proliferation, and cytotoxicity. In contrast, HER2-TAC γδ T cells failed to show activity in MLR assays, potentially indicating that HER2-TAC γδ T cells are free of GvH reactivity. These MLR assays comprised dendritic cells that represent the major HLA subtypes found in North America. In addition, HER2-TAC γδ T cells showed strong anti-tumor efficacy in HER2-positive tumor xenograft models without signs of toxicity. Conclusions: The in vitro and in vivo data confirm strong and specific activity of HER2-targeted TAC γδ T cells against HER2-expressing tumor models and highlights the potential of the TAC platform in the development of an allogeneic product for therapeutic applications in solid tumors. Citation Format: Suzanna L. Prosser, Stacey X. Xu, Ling Wang, Ritu R. Randhawa, Sailaja Pirati, Laura Ravensbergen, Seungmi Yoo, Miyoung Jung, Laurentia Gheorghiu, Angel Gomez, Gurleen Sandhu, Chris Ayers, Donna Rill, Christopher W. Helsen, Andreas G. Bader. Preclinical characterization of allogeneic Vγ9Vδ2 HER2-TAC T cells for the treatment of HER2-positive solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1773.

Chimeric antigen receptor T cell therapy (CART) therapy has shown remarkable success in the treatment of B cell acute lymphoblastic leukemias (B-ALL) and lymphomas. However, CART therapies for acute myeloid leukemia (AML), where 5-year survival rates are significantly lower than for B-ALL, are only in their infancy. CD33-CART have potent activity against AML in preclinical models and a first-in-child/first-in-human phase 1/2 CD33-CART clinical trial for AML is ongoing in the Pediatric Oncology Branch of the National Cancer Institute (NCT03971799). Nonetheless, published outcomes suggest a modest efficacy of approximately 50% (Shahzad et al., Front Immunol 2023), highlighting the critical need to develop new strategies to improve CART accessibility and a more robust anti-AML response. We hypothesized that off-the-shelf gamma delta (γδ) CD33 CART cells could potentially overcome current challenges for the treatment of AML. γδ lineage T cells are unconventional lymphocytes whose functions are not restricted to MHC-mediated antigen presentation; they are primed for immediate responses, including tumor killing. Furthermore, allogeneicγδ T cells have the potential to induce robust anti-tumor cytotoxicity without causing graft versus host disease (GVHD). Here, we generated γδ CAR T cells from healthy donor elutriated lymphocytes by activation with zoledronic acid and IL-2 for 7-14 days. Within 9 days post stimulation, the vast majority of lymphocytes were Vδ2+ and 30-40% were successfully transduced with a lentiviral CD33 CAR construct harboring the 4-1BB costimulatory domain. Importantly, and unlike conventional alpha beta (ab) T lymphocytes, >98% of these γδ CD33CAR T cells expressed IFNγ under basal conditions. This characteristic likely accounted for the efficient in vitro killing of AML cell lines by untransduced γδ T lymphocytes under conditions of high effector/target (E/T) ratios. While untransduced γδ T cells did not exhibit cytotoxicity following repeat AML stimulations, γδ CD33CAR T lymphocytes exhibited proficient in vitro cytotoxicity, with killing rates that were more rapid than those initiated by ab CD33 CART (Figure 1). These characteristics were associated with a prolonged metabolic activity of γδT cells; γδ CD33 CART expressed high levels of the GLUT1 glucose transporter for >14 days post activation whereas GLUT1 levels on ab CD33 CART returned to resting within 10 days. High GLUT1 levels were linked to efficient killing under conditions of basal glucose levels. Most notably, γδ CD33CAR T lymphocytes achieved high in vivo cytotoxicity, assessed using bioluminescent AML cell line xenografts in humanized NSG mice. Together, these data highlight the feasibility of generating allogeneic γδ CD33CART with a strong anti-AML cytotoxic response.

Background: Gamma delta(γδ) T cells have been associated with improved graft-versus-leukemia effect, leukemia-free-survival, and reduced risk of relapse in acute myeloid leukemia (AML). Variable delta 2 (Vδ2) cells are a subset of γδ T cells that are known to have a strong anti-cancer effect in a variety of malignancies, and increased Vδ2 cells have also been associated with better outcomes in chronic lymphocytic leukemia (CLL) and B cell acute lymphocytic leukemia. Earlier studies have indicated that AML blasts can disrupt T cell development. However, the effect of AML on γδ T cell memory phenotypes is not known. Naïve γδ cells are double-positive for CD27 and CD45RA and can differentiate into CD45RA-negative central memory (CM) γδ T cells in response to pAg stimulation. CM γδ T cells have the greatest potential for proliferation, and additional pAg stimulation causes them to further differentiate into effector memory (EM) γδ T cells. EM γδ T cells do not express CD27 or CD45RA, and though they are less proliferative than CM γδ T cells, they express more cytokines. EM γδ T cells can further differentiate into terminally differentiated effector memory (TEMRA) γδ T cells, which exhibit the highest cytotoxicity but the lowest proliferation. TEMRA γδ T cells also regain CD45RA expression. Imbalances of these γδ T cell memory phenotypes have been associated with poorer treatment responses and outcomes in CLL and myeloma. In this study, we have investigated γδ T cell memory phenotypes in AML patients. Additionally, we have tested ex-vivo expanded donor-derived Vδ2 γδ T cells for their effect on AML cells. We hypothesized that AML alters the memory phenotypes of γδ T cells and that ex-vivo expanded donor-derived Vδ2 γδ T cells induce a potent apoptotic effect on AML cell lines and primary AML cells. Methods: To examine γδ T cell memory phenotypes in AML patients, we have developed a multi-parameter flow cytometry assay (8-color panel) using a Miltenyi Biotec MACSQuant16. Peripheral blood mononuclear cells from healthy donors (n=10) and AML patients (n=14) were used in this study. To investigate the potential of donor-derived γδ T cell therapy for AML, we co-cultured freshly thawed Vδ2 γδ T cells (GMP-grade frozen γδ T cells were obtained from CytoMed Therapeutics Limited, Singapore) overnight at effector-to-target cell ratios (E:T) of 2:1, 5:1, and 10:1 with 8 AML cell lines (OCI-AML2, OCI-AML3, U937, THP1, Kasumi-1, MV4-11, Molm-13, and Molm-14), as well as primary AML cells derived from patient samples. We analyzed the effect of the γδ T cells on apoptosis induction in the AML cells using flow cytometry. Results: We did not observe any significant changes in the percentages of naïve or EM γδ T cell memory phenotype populations between samples from AML patients and healthy donors. However, we found significantly lower CM and significantly higher TEMRA γδ T cell populations in AML patients than in healthy individuals, with the mean CM percentage dropping from 52.47% ± 13.62% to 22.09% ± 10.07% (p<0.0001) and the mean TEMRA percentage increasing from 15.61% ± 10.08% to 35.55% ± 24.07% (p<0.05). As CM cells are the most proliferative memory phenotype and TEMRA cells are the least, this decrease indicates that γδ T cell populations have less capacity to proliferate in AML patients than in healthy individuals. This also suggests that adoptive transfer of donor-derived γδ T cells may provide a therapeutic opportunity for AML. Next, we determined the cytotoxic effect of donor-derived Vδ2 γδ T cells. When co-cultured with AML cells, the donor-derived γδ cells exerted a potent E:T-dependent apoptotic effect on all leukemic cell lines tested, including the TP53-mutant Kasumi-1 and FLT3-mutant Molm-13, Molm-14, and MV4-11 cells. The mean percentage of apoptotic AML cells was 41.30% ± 22.74% for the 2:1 E:T ratio, 67.52% ± 22.94% for the 5:1 ratio, and 77.54% ± 14.07% for the 10:1 ratio. One-way ANOVA confirmed an E:T ratio-dependent increase in apoptotic AML cells (p<0.0001). Experiments investigating the effect of Vδ2 γδ T cells on primary AML cells and in vivo studies are ongoing. Conclusion: γδ T cell memory phenotype profiles from AML patients showed a marked decrease in CM γδ T cells and increase in TEMRA γδ T cells, suggesting a more limited proliferative capacity in AML. Furthermore, expanded donor-derived Vδ2 γδ T cells induced E:T-dependent apoptosis in AML cell lines and may be a promising off-the-shelf treatment option for AML patients.

Although autologous cell therapy has shown remarkable clinical success and brought forth a medicinal revolution, significant hurdles remain. These include, but not limited to cell supply scarcity, prohibitively high costs stemming from the source material and manufacturing variabilities, lengthened and complex patient journey before receiving treatment, and the restrictive sites of manufacturing and therapeutic administration, creating formidable accessibility and affordability challenges. Many of the aforementioned issues central to successfully democratizing cell therapy could be ameliorated by developing off-the-shelf cell products that are uniformed, universal, and massively scalable. While donor-based allogeneic cell therapy could tackle certain aspects of the issues, allogeneic induced pluripotent stem cell (iPSC)-derived cell therapy offers an unequalled solution to answer most of the challenges. iPSC-derived cell therapy will ride on a stable, unlimited cell supply, a uniformed manufacturing process, and a homogeneously edited cellular product manufactured at a low cost. However, there are three conditions to fulfill before one can take the full advantage of the iPSC-derived cell therapy. First is the acquisition of the well-characterized GMP-grade iPSC lines that are free of pathogenic genetic alterations. The publicly or commercially available iPSC banks are usually populated with uncharacterized and sometimes dubious iPSC clones. It has been published that 20% of the publicly available iPSC lines harbor at least one cancer-related mutations. Therefore, obtaining iPSC clones with very high confidence of genomic integrity could not be underestimated. Second, not one iPSC clone is the same, and each will offer certain degree of differentiation and expansion efficiency into a given cell type due to the unknown donor genetics and epigenetics composition. Therefore, a survey of the most suitable iPSC clones will be required. Third, the tailored optimization of the differentiation and expansion methodologies is needed to exploit the maximum productivity of the selected iPSC clones. It is the perfect marriage between finding the right clone and establishing the most optimal differentiation and expansion approaches pertinent to a given iPSC clone that will ultimately realize the full potential of the iPSC-derived cell therapies and bring the next wave of cell therapy revolution. To meet the aforementioned requisites, a stringent donor recruitment process compliant with the regulations of multiple key regions was implemented. Eligible donors' blood cells were reprogrammed to clinical-grade T cell-derived iPSC lines (αβT-derived iPSC and γδT-derived iPSC). Selected T-iPSC clones underwent rigorous characterization through various methodologies, including pluripotent marker expression, in vitro and in vivo differentiation assays, karyotyping, and comprehensive genome integrity assessments. Ten clinical-grade T-iPSC lines successfully passed all tests and were found to be free of likely-pathogenic variants present in the OncoKB, ClinVar, and COSMIC databases. Subsequently, unique protocols were created to maximize the differentiation and explosion potential of the selected iPSC clones. Here, we describe industry-leading feeder-free differentiation and expansion platforms using our GMP-grade iPSCs. Our platforms are capable of producing 95% CD8αβ+ mature T cells and over 99% γδT cells without enrichment process. Remarkably, both iPSC-derived αβT and γδT cells can be expanded more than 10 million-fold from a single iPSC. These protocols represent the most competitive approach in the field, offering unparalleled scalability and efficiency for the iPSC-derived T cell production. Moreover, both iPSC-derived αβT and γδT cells demonstrated cytotoxic activity in functional assays. To achieve commercial-scale expansion, the differentiation and expansion process were reproduced in a closed system, enabling large-scale production of functional T cells in a single manufacturing batch. These advancements could significantly enhance the accessibility and affordability of iPSC-based therapies. By leveraging iPSC-derived therapy platforms, BeiGene Cell Therapy represents a substantial advancement in the field, providing a powerful resource for developing allogeneic T cell therapies and paving the way for future innovations in cell therapy.

Introduction: Chimeric antigen receptor T cell (CAR-T) therapy has shown remarkable efficacy against B cell malignancies for patients who have limited treatment options. Successful advancement of CAR-T therapy to myeloid hematopoietic and solid malignancies, however, has been limited by the potential of life-threatening on-target, off-tumor toxicities against healthy tissues, the natural tumor heterogeneity and consequent selection pressure leading to relapse. Addressing these limitations to widen the therapeutic index is crucial as we seek safer and more effective CAR-T therapies. In this regard, γδ T cells as first responders of immunity hold promise as they can directly identify and eliminate malignant cells through the recognition of multiple tumor-associated stress antigens not commonly expressed on normal tissues. We sought to combine the tumor-sensing capabilities of γδ T cells and the localization enhancement of CAR-T by excluding the CD3ζ domain in a non-signaling CAR (nsCAR) to allow the targeted killing against tumors, while sparing healthy tissues. Methods: Standard and nsCAR constructs against CD19, CD33 and CD123 were cloned into separate second-generation lentiviral vectors. The CAR constructs contained either a scFv targeting the respective antigen (anti-CD19/anti-CD33) or an IL-3 zetakine (IL-3z) targeting the CD123 followed by a flag-tag, a hinge, a transmembrane domain and a costimulatory domain + CD3ζ. IL-15 co-expression was incorporated to enhance γδ T cell fitness and persistence. CAR activation was determined by Jurkat T cells co-cultured with target ALL or AML cell lines followed by lentivector transduction of activated and expanded Vδ2 + γδ T cells. Cellular cytotoxicity was assessed at multiple E:T ratios against respective leukemia cell lines and healthy peripheral blood cells expressing the target antigen. Results: We observed significant upregulation of CD69 surface expression indicating activation of CD3ζ+ CD19CAR/CD33CAR/IL-3z Jurkat cells (3x, 20x, and 2x respectively) following culture with Nalm6 (CD19) and KG-1 (CD33 & IL-3z). CD3ζ- ns19CAR/ns33CAR/nsIL-3z Jurkat cells did not upregulate CD69 in parallel cultures. We also observed a time-dependent reduction of the CD3ζ+ CD33 CAR + population over 7 days (43%) in Jurkat cells following extended coculture with KG-1 cells, while the nsCAR + Jurkat population remained unchanged, indicating the nsCAR may mitigate activation induced cell death (AICD). Ex vivo activated γδ T cells from healthy donors (N=4), were transduced with nsCAR lentiviral vectors with high efficiency (up to 80%). The ns19CAR cells effectively killed CD19 + Nalm6 cells and demonstrated enhanced (>1.5x) cytotoxicity compared to untransduced γδ T cells (UTD). After 48hr co-culture (E:T=2:1), the ns19CAR cells killed 79.7%+6.6% Nalm6 cells compared to 46.1%+7.2%) with UTD. Minimal cytotoxicity was observed for ns19CAR and UTD γδ T cells against B cells from healthy donor PBMC 5.2%+6.6% for ns19CAR vs. to -4.6%+7.7% with UTD. No significant difference in cytotoxicity against the CD19- K562 cells between the UTD or ns19CAR cells (73.5% vs. 71.5%, E:T=2:1) suggesting the enhanced cytotoxicity is ns19CAR directed. Similarly, ns33CAR and nsIL-3z cells, although requiring higher E:T ratios for similar killing efficiency, continued to demonstrate enhanced (up to 2.0x) killing against AML cells (HL-60, KG-1 and MOLM13) and CML cells K-562 compared to UTD in cytotoxicity assay with 24hr co-culture. Meantime, minimal cytotoxicity (<10%) was observed for ns33CAR, nsIL-3z, or UTD γδ T cells against CD33+ cells isolated from healthy donor PBMC. Conclusions: Ex vivo activated nsCAR cells efficiently recognize and kill leukemia cell lines while sparing peripheral blood cells bearing the same target antigen. The nsCAR cells also show increased cytotoxicity against leukemias over unmodified activated γδ T cells suggesting improvement in tropism and/or binding efficiency. In summary, our findings showed that the combination of nsCAR on γδ T cells may increase the therapeutic index to allow expansion of CAR-T therapy to cancers with unacceptable target expression on critical healthy cell populations. Further optimization of the nsCAR constructs may potentially enhance both the efficacy and safety profile of the next generation adoptive cell therapies against wider selection of cancers.

Chimeric antigen receptor (CAR) T cells have transformed the field of cancer immunotherapy. Other approaches, such as the use of Fc gamma chimeric receptor (Fc γ -CR)-T cells have further expanded the applicability of such therapies in both solid and liquid tumours with the added benefit of tackling some of the hurdles associated with CAR-T therapies. Previous studies have generated Fc γ -CR-T cells from autologous cells

Acute myeloid leukemia (AML) remains an elusive disease to treat, let alone cure, even after highly intensive therapies such as stem cell transplants. Adoptive cell therapeutic strategies based on conventional alpha beta (αβ)T cells are an active area of research in myeloid neoplasms given their remarkable success in other hematologic malignancies, particularly B-cell-derived acute lymphoid leukemia, myeloma, and lymphomas. Several limitations have hindered clinical application of adoptive cell therapies in AML including lack of leukemia-specific antigens, on-target-off-leukemic toxicity, immunosuppressive microenvironments, and leukemic stem cell populations elusive to immune recognition and destruction. While there are promising T cell-based therapies including chimeric antigen receptor (CAR)-T designs under development, other cytotoxic lymphocyte cell subsets have unique phenotypes and capabilities that might be of additional benefit in AML treatment. Of particular interest are the natural killer (NK) and unconventional T cells known as invariant natural killer T (iNKT) and gamma delta (γδ) T cells. NK, iNKT, and γδT cells exhibit intrinsic anti-malignant properties, potential for alloreactivity, and human leukocyte-antigen (HLA)-independent function. Here we review the biology of each of these unconventional cytotoxic lymphocyte cell types and compare and contrast their strengths and limitations as the basis for adoptive cell therapies for AML.

Tumor antigen escape limits the durability of antigen-specific immunotherapies, particularly chimeric antigen receptor (CAR)-based treatments. Malignant cells evade detection through six routes: antigen mutation or alternative splicing, impaired antigen processing, lineage switching, membrane redistribution, trogocytic epitope masking, and CAR-induced shielding during autologous manufacture. First noted in blood cancers, these tactics increasingly appear in solid tumors, where heterogeneity and immune suppression exacerbate escape. Emerging countermeasures broaden or restore antigen recognition: multi-specific modalities (dual/tandem CARs, bispecific engagers, adaptor CARs), logic-gated synNotch circuits, antigen-upregulating mRNA vaccines and epigenetic drugs, and non-conventional effectors such as invariant natural killer T (iNKT), gamma delta T (γδ T), and mucosal-associated invariant T (MAIT) cells. Collectively, these advances signal a shift toward adaptable, off-the-shelf, biomarker-guided platforms designed to keep pace with tumor evolution and achieve escape-resistant immunity.

Conventional immunotherapy, including immune checkpoint blockade and chimeric antigen receptor (CAR)-T cells, has revolutionized cancer therapy over the past decade. Yet, the efficacy of these therapies is limited by tumor resistance, antigen escape mechanisms, poor persistence, and T-cell exhaustion, particularly in the treatment of solid tumors. The emergence of unconventional immunotherapies offers novel opportunities by leveraging diverse immune cell subsets and synthetic biologics. This review explores various immunotherapy platforms, including gamma delta T cells, invariant natural killer T cells, mucosal-associated invariant T cells, engineered regulatory T cells, and universal CAR platforms. Additionally, it expands on biologics, including bispecific and multispecific antibodies, cytokine fusions, agonists, and oncolytic viruses, showcasing their potential for modular engineering and off-the-shelf applicability. Distinct features of unconventional platforms include independence from the major histocompatibility complex (MHC), tissue-homing capabilities, stress ligand sensing, and the ability to bridge adaptive and innate immunity. Their compatibility with engineering approaches highlights their potential as scalable, efficient, and cost-effective therapies. To overcome translational challenges such as functional heterogeneity, immune exhaustion, tumor microenvironment-mediated suppression, and limited persistence, novel strategies will be discussed, including metabolic and epigenetic reprogramming, immune cloaking, gene editing, and the utilization of artificial intelligence for patient stratification. Ultimately, unconventional immunotherapies extend the therapeutic horizon of cancer immunotherapy by breaking barriers in solid tumor treatment and increasing accessibility. Continued investments in research for mechanistic insights and scalable manufacturing are key to unlocking their full clinical potential.

Chimeric antigen receptor (CAR) therapies have revolutionized cancer treatment, particularly with the success of CAR-T cells in hematologic malignancies. However, their application to solid tumors remains limited by major challenges, including cytokine release syndrome (CRS), neurotoxicity, poor tumor infiltration, antigen heterogeneity, and high manufacturing costs. These limitations have prompted growing interest in alternative immune effector cells. Innate immune cells - such as natural killer (NK) cells, macrophages, invariant natural killer T (iNKT) cells, gamma delta (γδ) T cells, dendritic cells (DCs) and neutrophils - offer distinct advantages. They are associated with a lower risk of graft-versus-host disease (GvHD), possess intrinsic tumor-homing and cytotoxic properties, and are suitable for off-the-shelf therapeutic platforms. This review explores the biological rationale and clinical potential of CAR-engineered innate immune cells, highlighting key findings from preclinical and clinical studies. Finally, we discuss combinatorial strategies and future directions that could shape the next generation of CAR-based therapies for solid tumors.

Chimeric antigen receptor-cell therapies have demonstrated remarkable success in haematological malignancies but face significant hurdles in solid tumours. The hostile tumour microenvironment, antigen heterogeneity, limited tumour infiltration, and CAR-cell exhaustion contribute to reduced efficacy. Additionally, toxicity, off-target effects, and manufacturing challenges limit widespread clinical adoption. Overcoming these barriers requires a multifaceted approach that enhances CAR-cell persistence, trafficking, and tumour-specific targeting. Recent advancements in alternative cellular therapies, such as CAR-natural killer cells, CAR-macrophages, gamma delta CAR-T cells, and CAR-natural killer T cells, provide promising avenues for improving efficacy. These strategies leverage distinct immune cell properties to enhance tumour recognition and persistence. Furthermore, combination therapies, including chemotherapy, radiotherapy, antibodies, small molecule inhibitors, cancer vaccines, oncolytic viruses, and multi-CAR cell combination therapy, offer synergistic potential by modulating the TME and improving CAR-cell functionality. This review explores the challenges of CAR-based cellular therapies in solid tumours and highlights emerging strategies to overcome therapeutic limitations. By integrating novel cellular platforms and combination approaches, we seek to provide insights into optimising CAR-cell therapies for durable responses in solid malignancies.

Background: Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disorder (PTLD) is a life-threatening complication following hematopoietic stem cell and solid organ transplantation. Although CD20-targeted antibodies and EBV-specific T cells have shown promise, many patients remain refractory to these therapies. Gamma delta T (γδ T) cells are emerging as a potent and universal platform for adoptive cell therapy due to their MHC-independent antiviral and antitumor activity. However, their clinical efficacy is limited by poor tumor-targeting efficiency. To address this limitation, conjugating γδ T cells with CD20-specific antibodies may offer a viable therapeutic strategy for EBV-PTLD. Methods: We utilized antibody–cell conjugation (ACC) technology based on metabolic glycan labeling (MGL), which modifies cell-surface sialic acids with bioorthogonal handles, enabling rapid and efficient conjugation of CD20 antibodies to γδ T cell surface sialoglycans via click chemistry. In vitro, the CD20 antibody-conjugated γδ T (CD20-γδ T) cells were co-cultured with EBV and CD20-positive targets, including EBV-transformed lymphoblastoid cell lines (EBV-LCLs), Raji cells, rituximab-resistant Raji cells, and primary EBV-PTLD patient-derived cells. For in vivo evaluation, NPG mice bearing Raji-luciferase tumors were treated intravenously with CD20 antibody, γδ T cells or CD20-γδ T cells every three days for a total of six doses. Mice were monitored for tumor development by bioluminescence imaging, body weight and survival. In vivo kinetics of the infused CD20-γδ T cells were also evaluated. Results: CD20 antibodies were efficiently conjugated to γδ T cell surfaces within 2 hours, achieving ~100% conjugation efficiency, a high cell-surface antibody density (~10⁶ antibodies per γδ T cell), and no detectable impairment of cell function. In vitro experiments showed that CD20-γδ T cells exhibited superior cytotoxicity against CD20+ cell lines and primary EBV-PTLD patient-derived primary cells, compared to CD20 antibody alone or γδ T cells without CD20 antibody conjugation. The in vivo mice model demonstrated that CD20-γδ T cells treatment strongly suppressed the aggressive proliferation of Raji tumor with no observed toxicity, while γδ T cells or CD20 antibody showed moderate inhibition. The median survival time of the tumor-bearing mice were extended from 16 days (untreated) to 20 days (γδ T cells), 28 days (CD20 antibody), and 48 days (CD20-γδ T cells). Conclusions: We developed a novel off-the-shelf CD20-γδ T cell therapy using MGL-based ACC technology, effectively combining the innate antitumor activity of γδ T cells with the precision of CD20 targeting. This approach exhibits potent efficacy against EBV-PTLD in vitro and in vivo, offering a promising allogeneic immunotherapy for refractory EBV-associated diseases.

Gamma delta (γδ) T cells are defined by their unique ability to recognize a limited repertoire of non-peptide, non-MHC-associated antigens on transformed and pathogen-infected cells. In addition to their lack of alloreactivity, γδ T cells exhibit properties distinct from other lymphocyte subsets, prompting significant interest in their development as an off-the-shelf cellular immunotherapeutic. However, their low abundance in circulation, heterogeneity, limited methods for ex vivo expansion, and under-developed methodologies for genetic modification have hindered basic study and clinical application of γδ T cells. Here, we implement a feeder-free, scalable approach for ex vivo manufacture of polyclonal, non-virally modified, gene edited chimeric antigen receptor (CAR)-γδ T cells in support of therapeutic application. Engineered CAR-γδ T cells demonstrate high function in vitro and and in vivo. Longitudinal in vivo pharmacokinetic profiling of adoptively transferred polyclonal CAR-γδ T cells uncover subset-specific responses to IL-15 cytokine armoring and multiplex base editing. Our results present a robust platform for genetic modification of polyclonal CAR-γδ T cells and present unique opportunities to further define synergy and the contribution of discrete, engineered CAR-γδ T cell subsets to therapeutic efficacy in vivo.

Simple Summary Induced pluripotent stem cells (iPSCs) that can be genetically engineered and differentiated into different types of immune cells, providing an unlimited resource for developing off-the-shelf cell therapies. Here, we present a comprehensive review that describes the current stages of iPSC-based cell therapies, including iPSC-derived T, nature killer (NK), invariant natural killer T (iNKT), gamma delta T (γδ T), mucosal-associated invariant T (MAIT) cells, and macrophages (Mφs). Abstract Cell-based immunotherapy, such as chimeric antigen receptor (CAR) T cell therapy, has revolutionized the treatment of hematological malignancies, especially in patients who are refractory to other therapies. However, there are critical obstacles that hinder the widespread clinical applications of current autologous therapies, such as high cost, challenging large-scale manufacturing, and inaccessibility to the therapy for lymphopenia patients. Therefore, it is in great demand to generate the universal off-the-shelf cell products with significant scalability. Human induced pluripotent stem cells (iPSCs) provide an “unlimited supply” for cell therapy because of their unique self-renewal properties and the capacity to be genetically engineered. iPSCs can be differentiated into different immune cells, such as T cells, natural killer (NK) cells, invariant natural killer T (iNKT) cells, gamma delta T (γδ T), mucosal-associated invariant T (MAIT) cells, and macrophages (Mφs). In this review, we describe iPSC-based allogeneic cell therapy, the different culture methods of generating iPSC-derived immune cells (e.g., iPSC-T, iPSC-NK, iPSC-iNKT, iPSC-γδT, iPSC-MAIT and iPSC-Mφ), as well as the recent advances in iPSC-T and iPSC-NK cell therapies, particularly in combinations with CAR-engineering. We also discuss the current challenges and the future perspectives in this field towards the foreseeable applications of iPSC-based immune therapy.

Background T cell-based immunotherapies using chimeric antigen receptors (CAR) or bispecific antibodies (BsAb) have produced impressive responses in hematological malignancies. However, major hurdles remained, including cytokine release syndrome, neurotoxicity, on-target off-tumor effects, reliance on autologous T cells, and failure in most solid tumors. BsAb armed T cells offer a safe alternative. Methods We generated ex vivo armed T cells (EATs) using IgG-[L]-scFv-platformed BsAb, where the anti-CD3 (huOKT3) scFv was attached to the light chain of a tumor-binding IgG. BsAb density on EAT, in vitro cytotoxicity, cytokine release, in vivo trafficking into tumors, and their antitumor activities were evaluated in multiple cancer cell lines and patient-derived xenograft mouse models. The efficacy of EATs after cryopreservation was studied, and gamma delta (γδ) T cells were investigated as unrelated alternative effector T cells. Results The antitumor potency of BsAb armed T cells was substantially improved using the IgG-[L]-scFv BsAb platform. When compared with separate BsAb and T cell injection, EATs released less TNF-α, and infiltrated tumors faster, while achieving robust antitumor responses. The in vivo potency of EAT therapy depended on BsAb dose for arming, EAT cell number per injection, total number of EAT doses, and treatment schedule intensity. The antitumor efficacy of EATs was preserved following cryopreservation, and EATs using γδ T cells were safe and as effective as αβ T cell-EATs. Conclusions EATs exerted potent antitumor activities against a broad spectrum of human cancer targets with remarkable safety. The antitumor potency of EATs depended on BsAb dose, cell number and total dose, and schedule. EATs were equally effective after cryopreservation, and the feasibility of third-party γδ-EATs offered an alternative for autologous T cell sources.

Background: A major challenge to off-the-shelf allogeneic cell therapies is the HLA-targeted elimination of genetically dissimilar cells. To combat this, HLA Class I can be deleted through transgene insertion at the beta-2 microglobulin (B2M) locus, while HLA Class II can be deleted by disruption of the gene encoding MHC Class II Transactivator (CIITA). Lack of HLA Class I expression, however, marks cells for elimination by natural killer (NK) cells, which normally receive inhibitory signals from this protein family. HLA-E and HLA-G are non-classical HLA Class I molecules that are far less polymorphic than HLA-A/B/C. They can inhibit natural killer cell-mediated activation by engaging NKG2A, KIR2DL4, ILT2, and ILT4. The objective of this study was to assess allo-evasion from NK cells by iPSC-derived cells engineered to overexpress HLA-E and -G. Methods: Allo-evasion by HLA-E, -G or both was first assessed in the chronic myelogenous leukemia K562 cell line, which expresses very low HLA Class I and is highly susceptible to NK-mediated killing. K562 cells were engineered using CRISPR/CAS9 to express chimeric molecules encoding either HLA-E or HLA-G heavy chains tethered to a defined nominal self-peptide and B2M, or HLA-E/peptide/B2M-P2A-HLA-G/peptide/B2M. Along with parental K562, these were used as targets in co-cultures with allogeneic PBMCs (n=22 donors) at different E:T ratios. Target killing was observed using flow cytometry and normalized to parental K562. Phenotyping of effector PBMCs for NK frequency and expression of HLA-E and -G receptors (NKG2A, KIR2DL4, ILT2, and ILT4) was performed to study correlations with killing efficiency and allo-evasion. IPSCs with endogenous B2M knocked out were also engineered to overexpress HLA-E and -G with the constructs described above. These were then differentiated into NK cells and gamma delta T cells using proprietary processes. Allo-evasion of these cells from killing by the same PBMC cohort was evaluated by flow cytometry. Results: While NK cells across donors expressed heterogeneous combinations of HLA-E and -G receptors, the strongest correlation with PBMC allo-responsiveness was NK frequency. K562 and iPSC-derived cells lacking HLA were susceptible to killing by PBMCs. Overexpression of HLA-E and -G offered protection to K562 and iPSC-derived cells against all tested donors. HLA-E offered more protection than HLA-G, and the combination of both HLA-E and -G was most potent. Conclusion: When genetically dissimilar HLA Class I protein family must be deleted to prevent graft rejection, expression of the more-conserved HLA-E and -G can effectively protect allogeneic drug products from elimination. These Allo-evasionTM edits may increase the in vivo persistence and enable multi-dosing of our iPSC-derived allogeneic cell therapies. Citation Format: Andriana Lebid, Dae Hwan Kim, Gabrielle Greco, Heidi Jessup, Alyssa Suarez, Shelby Brown, Nicholas Alexander, Mitan Desai, Buddha Gurung, Barry Morse, Daniel J. Perry, Michael F. Naso, Hyam Levitsky. Engineered expression of HLA-E and HLA-G protects iPSC-derived cells from killing by primary NK cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1320.

Despite positive outcomes in some hematological oncology patients, several barriers to current autologous CAR T-cell therapies exist that prevent broader clinical success. These include tumor antigen escape, on-target off-tumor toxicities, and limited patient access to treatment. To increase safety and efficacy of CAR T-cell therapies, we are developing a Universal CAR (UCAR) that is activated through a soluble tumor targeting protein that pairs specifically with the UCAR. The UCAR approach enables a single CAR T-cell to target many different antigens through alternative targeting proteins to address antigen escape and tumor heterogeneity. Additionally, greater control of the activity of the cells within a patient is possible since effector activity is regulated through the dosing of the soluble protein. UCAR platforms are particularly interesting for inclusion into allogeneic cell therapy products, as the combination of these technologies enables a fully universal and “off-the-shelf” cell therapy cell line. Here, we describe the development and functional assessment of the Universal Targeting Receptor-Adaptor Platform (UTRAP), an adaptable, highly specific, and developable system comprised of a novel UCAR and a soluble Bispecific Anti-Idiotype Targeting (BAIT) protein. We demonstrate that T-cells engineered with UTRAP have tunable activity that elicits robust cytotoxicity and tumor control, equivalent to a conventional CAR T-cell in vitro and in vivo. Our results show the flexibility of the antibody-based platform including multi-specific targeting and antigen swapping. Finally, we demonstrate that UTRAP is fully functional in allogeneic iPSC-derived gamma delta T-cells. These results pave the way for UTRAP to be used in any cell type advancing towards a truly universal, off-the-shelf cell therapy product that can adapt to target any tumor type.

Clinical applications of gamma delta (γδ) T cells have advanced from initial interest in expanding γδ T cells in vivo to the development of a manufacturing process for the ex vivo expansion. To develop an "off-the-shelf" allogeneic γδ T cell product, the cell manufacturing process must be optimized to include cryopreservation. It is known that cryopreservation can dramatically reduce viability of primary cells and other cell types after thawing, although the exact effects of cryopreservation on γδ T cell health and functionality have not yet been characterized. Our aim was to characterize the effects of a freeze/thaw cycle on γδ T cells and to develop an optimized protocol for cryopreservation. γδ T cells were expanded under serum-free conditions, using a good manufacturing practice (GMP) compliant protocol developed by our lab. We observed that cryopreservation reduced cell survival and increased the percentage of apoptotic cells, two measures that could not be improved through the use of 5 GMP compliant freezing media. The choice of thawing medium, specifically human albumin (HSA), improved γδ T cell viability and in addition, chromatin condensation prior to freezing increased cell viability after thawing, which could not be further improved with the use of a general caspase inhibitor. Finally, we found that cryopreserved cells had depolarized mitochondrial membranes and reduced cytotoxicity when tested against a range of leukemia cell lines. These studies provide a detailed analysis of the effects of cryopreservation on γδ T cells and provide methods for improving viability in the post-thaw period.

No abstract available

The intricacy of diseases, shaped by intrinsic processes like immune system exhaustion and hyperactivation, highlights the potential of immune renormalization as a promising strategy in disease treatment. In recent years, our primary focus has centered on γδ T cell-based immunotherapy, particularly pioneering the use of allogeneic Vδ2^+ γδ T cells for treating late-stage solid tumors and tuberculosis patients. However, we recognize untapped potential and optimization opportunities to fully harness γδ T cell effector functions in immunotherapy. This review aims to thoroughly examine γδ T cell immunology and its role in diseases. Initially, we elucidate functional differences between γδ T cells and their αβ T cell counterparts. We also provide an overview of major milestones in γδ T cell research since their discovery in 1984. Furthermore, we delve into the intricate biological processes governing their origin, development, fate decisions, and T cell receptor (TCR) rearrangement within the thymus. By examining the mechanisms underlying the anti-tumor functions of distinct γδ T cell subtypes based on γδTCR structure or cytokine release, we emphasize the importance of accurate subtyping in understanding γδ T cell function. We also explore the microenvironment-dependent functions of γδ T cell subsets, particularly in infectious diseases, autoimmune conditions, hematological malignancies, and solid tumors. Finally, we propose future strategies for utilizing allogeneic γδ T cells in tumor immunotherapy. Through this comprehensive review, we aim to provide readers with a holistic understanding of the molecular fundamentals and translational research frontiers of γδ T cells, ultimately contributing to further advancements in harnessing the therapeutic potential of γδ T cells.

Tafasitamab is an Fc-modified monoclonal antibody that binds to CD19, a cell-surface antigen that is broadly expressed on various types of B-cell non-Hodgkin’s lymphoma (NHL). Antibody-dependent cellular cytotoxicity (ADCC), a key mode of action of tafasitamab, is mediated through the binding of tafasitamab’s Fc region to FcγRIIIa receptors on immune effector cells and results in antitumor activity. Despite the proven clinical activity of tafasitamab in combination with lenalidomide in the treatment of diffuse large B-cell lymphoma (DLBCL), a higher number of immune cells in cancer patients may improve the activity of tafasitamab. Here, we characterized two ex vivo-expanded FcγRIIIa receptor—expressing cell types—γδ T and MG4101 natural killer (NK) cells—as effector cells for tafasitamab in vitro, and found that in the presence of these cells tafasitamab was able to induce ADCC against a range of NHL cell lines and patient-derived cells. We also explored the concept of effector cell supplementation during tafasitamab treatment in vivo by coadministering MG4101 NK cells in Raji and Ramos xenograft models of NHL. Combination treatment of tafasitamab and allogeneic MG4101 NK cells in these models demonstrated a survival benefit compared with tafasitamab or MG4101 monotherapy (Raji: 1.7- to 1.9-fold increase in lifespan; Ramos: 2.0- to 4.1-fold increase in lifespan). In conclusion, adoptive cell transfer of ex vivo-expanded allogeneic NK or autologous γδ T cells in combination with tafasitamab treatment may potentially be a promising novel approach to increase the number of immune effector cells and enhance the antitumor effect of tafasitamab.

Adoptive T-cell therapy has shownpromise in recent years, particularly in blood cancers using autologous CAR αβ T cells that target CD19.1 However, γδ T-cell therapies have many advantages compared to their αβT-cell counterparts. Tumour infiltration by γδ T cells is one of the best predictors of improved prognosis in numerous solid tumours.2,3 In addition, γδ T cells are equipped with an array of innate-like receptors that recognize several phosphoantigens and stress ligands, theoretically reducing the chance of immune evasion by tumour cells.4 When expanded in the presence of the prototypic tumour-associated immunosuppressive cytokine, transforming growth factor-ß, γδ T cell intrinsic anti-tumour activity is further enhanced, accompanied by resistance to suppressive effects of this cytokine.5 Furthermore, the lack of HLA restriction of γδ T cells negates their ability to induce graft versus host disease, when used in the allogeneic setting.6 The γδ T-cell subset that has been most frequently studied for use as a cell therapy is blood derived, the majority of which express a Vγ9Vδ2 T-cell receptor. However, the anti-tumour activity of these cells when tested in 2D culture models has not been replicated in clinical trials.7 Cancer cells cohabit and interact with mesenchymal stroma and leukocytes, collectively creating a highly immunosuppressive tumour microenvironment (TME). Immunosuppressive cells found in this ecosystem include cancerassociated fibroblasts (CAFs), regulatory T cells and a range of suppressive myeloid cell types. This complexity is not recapitulated in simple 2D cancer model systems. While some immunocompetentmousemodelsmaymimic attributes of the human TME, Vγ9Vδ2 T cells are not found

The recent successes of chimeric antigen receptor T cells in the treatment of hematological malignancies have clearly led to an explosion in the field of adoptive cell therapy for cancer. Current efforts are focused on the translation of this exciting technology to the treatment of solid tumors and the development of allogeneic ‘off-the-shelf’ therapies. γδ T cells are currently gaining considerable attention in this field as their unique biology and established role in cancer immunosurveillance place them in a unique position to potentially overcome these challenges in adoptive cell therapy. Here, we review the relevant aspects of the function of γδ T cells in cancer immunity, and summarize clinical observations and clinical trial results that highlight their emerging role as a platform for the development of safe and effective cancer immunotherapies.

The transformation of chronic lymphocytic leukemia to an aggressive lymphoma, called Richter transformation, is often accompanied by resistance to chemotherapy and high mortality. Thus, novel therapeutic strategies are required for the successful treatment of these patients. One possibility is cellular immunotherapy with chimeric antigen receptor T cells. However, the time delay until cells are available and the limited number of effector cells due to the impaired immune system of these patients potentially compromises the efficacy of this approach. Another promising attempt might be the therapy with γδ T cells. Once activated, they exhibit various antitumor effects against several types of malignancies. Furthermore, they can be safely used in an allogeneic setting and can be multiplied in vivo as already demonstrated in clinical studies. In vitro data, in addition, show that the cytotoxicity of γδ T cells can be significantly enhanced by monoclonal antibodies. Here we present a patient, who suffered from Richter transformation and did not respond to several lines of immunochemotherapy. Due to the lack of further therapy options, we conducted an individual therapy with adoptive transfer of haploidentical γδ T cells combined with the application of the monoclonal antibody obinutuzumab. A histologically confirmed complete remission was achieved through this therapy approach, whereby relevant side effects were not seen. This case highlights the potential of γδ T cells and the feasibility of this therapeutic approach for further clinical trials.

Abstract In hepatocellular carcinoma (HCC), γδ T cells participate in mediating the anti‐tumour response and are linked with a positive prognosis. However, these cells can become pro‐tumoural in the tumour microenvironment (TME). We aimed to decipher the immune landscape and functional states of HCC‐infiltrating γδ T cells to provide fundamental evidence for the adoptive transfer of allogeneic Vδ2+ γδ T cells in HCC immunotherapy. We performed single‐cell RNA sequencing (scRNA‐seq) on γδ T cells derived from HCC tumours and healthy donor livers. Confocal microscopy, flow cytometry and a Luminex assay were applied to validate the scRNA‐seq findings. The γδ T cells in the HCC TME entered G2/M cell cycle arrest, and expressed cytotoxic molecules such as interferon‐gamma and granzyme B, but were functionally exhausted as indicated by upregulated gene and protein LAG3 expression. The γδ T cells in the HCC TME were dominated by the LAG3+Vδ1+ population, whereas the Vδ2+ γδ T population was greatly depleted. Moreover, glutamine metabolism of γδ T cells was markedly upregulated in the glutamine‐deficient TME. Both in vitro and in vivo experiments showed that glutamine deficiency upregulated LAG3 expression. Finally, our results indicated that ex vivo‐expanded Vδ2+ γδ T cells from healthy donor could complement the loss of T cell receptor clonality and effector functions of HCC‐derived γδ T cells. This work deciphered the dysfunctional signatures of HCC‐infiltrating γδ T cells in the HCC TME, providing scientific support for the use of allogeneic Vδ2+ γδ T cells in HCC cellular therapy.

Higher γδ T cell counts in patients with malignancies are associated with better survival. However, γδ T cells are rare in the blood and functionally impaired in patients with malignancies. Promising results are reported on the treatment of various malignancies with in vivo expansion of autologous γδ T cells using zoledronic acid (zol) and interleukin-2 (IL-2). Here we demonstrated that zol and IL-2, in combination with a novel genetically engineered K-562 CD3scFv/CD137L/CD28scFv/IL15RA quadruplet artificial antigen-presenting cell (aAPC), efficiently expand allogeneic donor-derived γδ T cells using a Good Manufacturing Practice (GMP) compliant protocol sufficient to achieve cell doses for future clinical use. We achieved a 633-fold expansion of γδ T cells after day 10 of coculture with aAPC, which exhibited central (47%) and effector (43%) memory phenotypes. In addition, >90% of the expanded γδ T cells expressed NKG2D, although they have low cell surface expression of PD1 and LAG3 inhibitory checkpoint receptors. In vitro real-time cytotoxicity analysis showed that expanded γδ T cells were effective in killing target cells. Our results demonstrate that large-scale ex vivo expansion of donor-derived γδ T cells in a GMP-like setting can be achieved with the use of quadruplet aAPC and zol/IL-2 for clinical application.

ABSTRACT Cancer cell therapies have primarily focused on engineering autologous αβ T cells with chimeric antigen receptors (CARs), achieving clinical success against hematologic malignancies. However, their effectiveness against solid tumors is limited by challenges such as antigen escape, suppression by the metabolically hostile tumor microenvironment (TME), and manufacturing difficulties. γδ T cells are unconventional T cells with innate tumor-targeting capabilities independent of MHC class I, making them an emerging candidate for allogeneic cell therapy. While the Vδ1 T cell subset has shown promising anti-tumor killing their clinical application has been hindered by difficulties in achieving robust expansion for therapeutic use. Here, we evaluated the potential of K562 feeder cells expressing membrane-bound IL-21 (K562-mb-IL-21) to expand and activate γδ T cells from peripheral blood. Our findings show that this method preferentially expands Vδ1 T cells, resulting in an activated phenotype characterized by enhanced expression of NK cell activation receptors, innate cytotoxicity against breast and ovarian cancer cells, and sustained metabolic function in patient-derived ascites TME. When engineered with a CAR, Vδ1 T cells exhibited further enhanced anti-tumor efficacy in an immunodeficient NRG xenograft model of human ovarian cancer. These findings highlight K562-mb-IL-21 expanded peripheral blood Vδ1 T cells as a promising ‘off-the-shelf’ allogeneic therapy for solid tumors.

γδ T cells and natural killer (NK) cells have attracted much attention as promising effector cell subsets for adoptive transfer for use in the treatment of malignant and infectious diseases, because they exhibit potent cytotoxic activity against a variety of malignant tumors, as well as virus-infected cells, in a major histocompatibility complex (MHC)-unrestricted manner. In addition, γδ T cells and NK cells express a high level of CD16, a receptor required for antibody-dependent cellular cytotoxicity. Adult T-cell leukemia–lymphoma (ATL) is caused by human T-lymphotropic virus type I (HTLV-1) and is characterized by the proliferation of malignant peripheral CD4+ T cells. Although several treatments, such as chemotherapy, monoclonal antibodies, and allogeneic hematopoietic stem cell transplantation, are currently available, their efficacy is limited. In order to develop alternative therapeutic modalities, we considered the possibility of infusion therapy harnessing γδ T cells and NK cells expanded using a novel nitrogen-containing bisphosphonate prodrug (PTA) and interleukin (IL)-2/IL-18, and we examined the efficacy of the cell-based therapy for ATL in vitro. Peripheral blood samples were collected from 55 patients with ATL and peripheral blood mononuclear cells (PBMCs) were stimulated with PTA and IL-2/IL-18 for 11 days to expand γδ T cells and NK cells. To expand NK cells alone, CD3+ T-cell-depleted PBMCs were cultured with IL-2/IL-18 for 10 days. Subsequently, the expanded cells were examined for cytotoxicity against ATL cell lines in vitro. The proportion of γδ T cells in PBMCs was markedly low in elderly ATL patients. The median expansion rate of the γδ T cells was 1998-fold, and it was 12-fold for the NK cells, indicating that γδ T cells derived from ATL patients were efficiently expanded ex vivo, irrespective of aging and HTLV-1 infection status. Anti-CCR4 antibodies enhanced the cytotoxic activity of the γδ T cells and NK cells against HTLV-1-infected CCR4-expressing CD4+ T cells in an antibody concentration-dependent manner. Taken together, the adoptive transfer of γδ T cells and NK cells expanded with PTA/IL-2/IL-18 is a promising alternative therapy for ATL.

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Introduction ADI-001 is a first-in-class allogeneic gamma delta (γδ) CAR T cell therapy targeting the B-cell antigen, CD20. Expansion and persistence of cell therapy products and release of functional cytokines have historically correlated with patient outcomes. Here we report cellular kinetics and pharmacodynamic correlates from a phase 1, multicenter, open-label, dose escalation study to evaluate ADI-001 in R/R B cell NHL. Methods Cellular kinetics of ADI-001 were measured using three orthogonal methods, including quantitative SNP profiling of cell product (AlloCell), flow-cytometry for CAR+ Vδ1 γδ T cells, and droplet digital PCR (ddPCR) quantification of CAR transgene copies. Using these methods, expansion of ADI-001 was assessed in the peripheral blood for 24 evaluable patients across four dose levels in association with a phase 1 dose-escalation trial (NCT04735471). Relationships between ADI-001 cellular kinetics and radiographic clinical responses were also examined. Serum biomarkers related to host immune cell recovery during lymphodepletion and cytokine release were monitored for pharmacodynamic purposes. Humoral immunogenicity was assessed by Luminex™-based anti-HLA antibody screening. Other correlative characteristics were also evaluated, including degree of HLA mismatching between patient and ADI-001 product in relation to response and/or ADI-001 expansion and persistence. Results As of the May 2023 cutoff for reporting, ADI-001 was detected at all dose levels using ddPCR and flow cytometry to quantify CAR transgene copies and CAR+ Vδ1 γδ T cells, respectively. Treatment at the highest dose level, achieved a notable mean C max of 201,666 copies/µg or 483 CAR+ cells/µl, and mean day 28 persistent exposure of 16,553 copies/µg or 21 CAR+ cells/µL. Additional measures of ADI-001 exposure (AlloCell) further demonstrated a robust dose-dependent expansion profile of ADI-001 in the peripheral blood. Subjects with a BOR of CR or PR were observed to have a mean peak of 180,107 CAR copies/µg versus a mean peak of 20,950 CAR copies/µg in subjects with a BOR of SD or PD. Additionally, as expected following lymphodepletion, a transient increase in the homeostatic cytokine IL-15, coinciding with ADI-001 expansion and host-mediated immune cell recovery, was observed. Using high-resolution HLA typing, the degree of HLA mismatch between allogeneic ADI-001 product and patients did not associate with degree of cellular expansion, day 28 persistence, or response. Finally, emergence of donor-specific anti-drug antibodies following treatment was not observed. Conclusions Using three orthogonal measures of exposure, we show for the first time robust dose-dependent expansion and persistence of an allogeneic CAR γδ T cell therapy, ADI-001, irrespective of extensive degree of HLA mismatch. ADI-001 C max and D28 persistent exposure were comparable to, or exceeded, those demonstrated by alternative allogeneic CAR T therapies and approved autologous CD19 CAR T therapies. Favorable clinical responses were also associated with higher mean and median peak of ADI-001 cells.

HLA‐G is considered as an immune checkpoint protein and a tumor‐associated antigen. In the previous work, it is reported that CAR‐NK targeting of HLA‐G can be used to treat certain solid tumors. However, the frequent co‐expression of PD‐L1 and HLA‐G) and up‐regulation of PD‐L1 after adoptive immunotherapy may decrease the effectiveness of HLA‐G‐CAR. Therefore, simultaneous targeting of HLA‐G and PD‐L1 by multi‐specific CAR could represent an appropriate solution. Furthermore, gamma‐delta T (γδT) cells exhibit MHC‐independent cytotoxicity against tumor cells and possess allogeneic potential. The utilization of nanobodies offers flexibility for CAR engineering and the ability to recognize novel epitopes. In this study, Vδ2 γδT cells are used as effector cells and electroporated with an mRNA‐driven, nanobody‐based HLA‐G‐CAR with a secreted PD‐L1/CD3ε Bispecific T‐cell engager (BiTE) construct (Nb‐CAR.BiTE). Both in vivo and in vitro experiments reveal that the Nb‐CAR.BiTE‐γδT cells could effectively eliminate PD‐L1 and/or HLA‐G‐positive solid tumors. The secreted PD‐L1/CD3ε Nb‐BiTE can not only redirect Nb‐CAR‐γδT but also recruit un‐transduced bystander T cells against tumor cells expressing PD‐L1, thereby enhancing the activity of Nb‐CAR‐γδT therapy. Furthermore, evidence is provided that Nb‐CAR.BiTE redirectes γδT into tumor‐implanted tissues and that the secreted Nb‐BiTE is restricted to the tumor site without apparent toxicity.

Background Patients with 2017 European LeukemiaNet (ELN) adverse risk acute myeloid leukemia (AML) are at 40% risk of relapse leading to poor survival after allogeneic hematopoietic cell transplantation (allo-HCT) (Hansen TCT 2021, Jimenez BMT 2021). Ex vivo donor-derived gamma delta T cells (GDT) expanded with artificial antigen presented calls (aAPC) are a novel therapy with potent MHC-independent antineoplastic cytotoxicity. An ongoing phase 1/1b study is evaluating the safety and potential efficacy of ex vivo expanded donor GDT after reduced-intensity conditioning (RIC) allo-HCT in patients with ELN adverse risk AML ( NCT05015426). We report on manufacturing, safety, and correlative biology of expanded donor GDT after allo-HCT in the first 5 treated patients (Table 1). Methods The primary endpoint is the maximum-tolerated dose (MTD) of allogeneic donor GDT as a single infusion at day 60 after RIC allo-HCT. Next generation sequencing was used for measurable residual disease (MRD) assessment pre-HCT and post-GDT infusion. Donor GDT collected from the same donor of the allo-HCT were enriched from non-mobilized leukapheresis product by stimulation with zoledronic acid and IL-2 for 7 days followed by alpha beta T cell depletion using the Miltenyi CliniMACS. Enriched GDT (>90% TCRVδ2) were then co-cultured with irradiated aAPCs in Wilson Wolf G-Rex 100MCS over 10 days (Boucher J. Immunother 2023). This single center, investigator-initiated phase 1 trial is testing 3 distinct dose cohorts of infused GDT (5 x 10 6 cells/kg, 2.5 x 10 7 cells/kg, and 1 x 10 8 cells/kg) with acceptable range of 25% margin. A Bayesian optimal interval design is used to guide dose escalation/de-escalation decisions. Results First 3 patients enrolled in dose level 1 (DL1) cohort received GDT dose of 6.25 x 10 6 cells/kg, while last 2 patients enrolled in DL2 cohort received 3.13 x 10 7 cells/kg dose. Dose-limiting toxicities (DLT) were evaluated for 42 days following GDT infusion and no DLT were observed. None of the treated patients experienced cytokine release syndrome (CRS), Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS) or graft-versus-host disease (GVHD) during DLT period. Among patients treated on DL1, patient #01 had ASXL1 AML with MRD pos (VAF 43.9%) prior to RIC haploidentical (Haplo) HCT. He is in ongoing complete remission (CR) 9 months post-HCT and has had no GVHD. Patient #02 had MRD pos (VAF 61.7%) AML with TP53 mutation/complex karyotype prior to RIC matched sibling donor allo-HCT. He is in CR and has mild chronic GVHD at 7 months post-HCT. Patient #03 with pre-HCT MRD negRUNX1 AML is in ongoing CR 7 months post-RIC Haplo HCT with no GVHD. At day 28 and day 120 post-GDT, all 3 patients were MRD neg in bone marrow and had 100% bone marrow and peripheral blood leukocyte and granulocyte chimerism. In DL2 cohort, patient #04 with RUNX1 AML and patient #05 with t(9;22) AML both were MRD neg prior to RIC Haplo HCT and remain in CR with no GVHD at 5 and 4 months post-HCT, respectively. At day 28 post-GDT, both patients were MRD neg and had 100% chimerism. Patient #05 had a fall during DLT period in a setting of orthostatic hypotension requiring hospitalization for 24 hours for hydration. This was the only grade 3 adverse event observed in this trial. Serum cytokines analyzed included IL2, IL6, IL15, IFNγ, TNFα, Angiopoietin 1&2, and GM-CSF (Figure 1). We compared results prior and up to 90 days post-GDT infusion and found no significant differences, which can explain no CRS or ICANS events yet observed on this trial. Immunophenotype (TCRVδ2, TCRVδ1, PD1, CTLA4, TIGIT and CD28) of GDT was analyzed in samples of donor apheresis, pre- and post-enrichment, infused GDT product and at days 28 and 90 post-infusion. GDT (TCRVδ2) persisted at day 28 and day 90 post-infusion and expressed low levels of cell surface inhibitory receptors. Conclusion This preliminary interim report demonstrates favorable safety and tolerability of donor GDT therapy with no CRS, ICANS or GVHD in first 5 patients treated in this trial. All these patients with adverse risk AML are in ongoing MRD neg CR following RIC allo-HCT, including 2 pre-HCT MRD pos cases with TP53 AML and AXSL1 AML. Early results of this trial are promising, which provides validation for continued testing of adoptive transfer of ex vivo expanded donor GDT. Updated results of this trial will be presented at the annual conference.

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TO THE EDITOR: Cancers derived from the malignant transformation of gamma–delta (γδ) T cells carry very poor prognosis. The major pathologies recognised are γδ T acute lymphoblastic leukaemia (γδ T-ALL), and two lymphoma subtypes: hepatosplenic T cell lymphoma (HSTL) and primary cutaneous γδ T cell lymphoma (PCγδ-TCL) [1]. γδ T-ALL represents approximately 10% of cases of T-ALL and is associated with high rates of induction failure, relapse and excess mortality [2]. HSTL is a rare (approximately 3% of cases of T cell lymphoma [1]) but highly aggressive disorder, which typically presents in males in the 2nd or 3rd decade of life, often in association with immunosuppressive therapy [3]. It carries the worst prognosis of all lymphoma subtypes, with a median survival of only 6–8 months [4] and only isolated cases of long-term survival [5]. PCγδ-TCL is also rare (approximately 1% of skin lymphomas [1]) and presents with cutaneous involvement, typically associated with visceral and/or bone marrow disease. Again, outcomes are poor, with 75% 1-year mortality in the largest published case series [6]. Treatment for γδ malignancies is with cytotoxic chemotherapy, with no tumour-specific therapies currently available. By contrast, in analogous B-cell malignancies, highly effective immunotherapies, including monoclonal antibodies, bispecific T cell engagers and chimeric antigen receptor (CAR)-T cells [7] are available. These therapies have revolutionised the treatment and outcome of advanced B-cell malignancies. CAR-T cells against CD19 in particular have demonstrated the potential to induce longlasting complete remissions even in patients with advanced and refractory cancers [7]. For γδ malignancies, the defining immunophenotypic characteristic is expression of the γδ T cell receptor (TCR), present in >95% of cases of HSTL and in all PCγδ-TCL and γδ T-ALL [3]. Importantly, in normal tissues expression is limited to γδ T cells, where it functions as the antigen recognition receptor. Here, we developed CAR-T cells targeting the γδ TCR and demonstrate in vitro and in vivo efficacy against γδ T cell malignancies. Our data offers proof-of-concept for γδ TCR-targeting with CAR-T cells as a potential approach to bring highly potent immunotherapy to the treatment of γδ malignancies. Primary αβ T cells were retrovirally transduced to express anti-γδ TCR CAR or control anti-CD19 CAR (Fig. 1a). Following transduction with anti-CD19 CAR, a small proportion of γδ T cells persisted in the culture, including some which expressed antiCD19 CAR. By contrast, for anti-γδ TCR CAR, no γδ T cells were detected in the culture, suggesting ‘purging’ of these cells by the transduced population (Fig. 1b). CAR-T cells were then co-cultured with T cell lines which natively express (Loucy – Vγ9Vδ2, BE13 – Vγ8Vδ1, MOLT13 – Vγ3Vδ1 [8]) or are negative for surface γδ TCR (SupT1-CD19). While control anti-CD19 CAR lysed only SupT1CD19 cells, anti-γδ TCR CAR-T lysed only γδ TCR-positive cell lines (Fig. 1c). In addition, anti-γδ TCR CAR-T cells demonstrated specific secretion of cytokines including interferon-γ, IL-2, IL-13 and TNF-α (Fig. 1d). Next, we co-cultured anti-CD19 or anti-γδ CAR-T cells with normal autologous γδ T cells. At a high E:T ratio (1:1), target normal γδ T cells were partially lysed (Fig. 1e), with concomitant expansion of anti-γδ CAR-T cells (Fig. 1f). A marked downregulation of γδ TCR expression was noted on surviving γδ T cells (Fig. 1g). Interestingly, by contrast, at lower E:T ratios (1:2 and 1:4), paradoxical γδ T cell expansion was instead observed (Fig. 1e), associated with reduction in numbers of anti-γδ CAR-T cells (Fig. 1f). This suggests lysis of anti-γδ CAR-T by target normal γδ T cells. To assay the in vivo potency of anti-γδ TCR CAR-T cells, we utilised the Loucy murine model of disseminated γδ TCR-positive leukaemia (Fig. 2a, d). NSG mice were intravenously injected on CAR D-12 with 4 × 10 Loucy cells, engineered to stably express Firefly luciferase. Tumour engraftment was confirmed by bioluminescence imaging (BLI) at D-1 (data not shown), then mice were treated on D0 with 8 × 10 anti-γδ TCR or control anti-CD19 CAR-T cells. Mice receiving antiγδ TCR CAR demonstrated reduction of tumour burden, as assessed by flow cytometry of bone marrow and spleen at necropsy on D14 (Fig. 2b, c, Supplementary Fig. 1), BLI (Fig. 2e, f) and bleed at D30 (Fig. 2g). Prolonged survival (Fig. 2h) was seen in anti-γδ TCR CAR recipients compared to CD19 CAR-treated animals, although all animals eventually died of progressive γδ TCR-positive disease, with no evidence of antigen downregulation. In common with other NSG models, CAR-T cell persistence was limited, with no detectable cells in the peripheral blood at D30 (data not shown). Despite success in B-cell malignancies, a lack of acceptable targets means targeted immunotherapy is rarely applied to T cell malignancies. The anti-CD30 antibody-drug conjugate

Engagement between the natural killer group 2, member D (NKG2D) receptor and its ligands is one of the main mechanisms used by immune cells to target stressed cells for cell death. NKG2D ligands are known markers of cellular stress and are often upregulated on tumor cells. Certain drugs can further increase NKG2D ligand levels, thereby making tumor cells more susceptible to immune cell detection and destruction. However, the effectiveness of this approach appears to be limited with drug treatment alone, possibly due to immune dysregulation in the setting of malignancies. We hypothesized that a more effective approach would be a combination of NKG2D ligand-inducing drugs, such as the proteasome inhibitor bortezomib, and ex vivo-expanded peripheral blood γδ T cells (i.e., Vγ9Vδ2 T cells). Acute myeloid leukemia (AML) is a high-risk hematologic malignancy, and treatment has shown limited benefit with the addition of bortezomib to standard chemotherapy regimens. Two AML cells lines, Nomo-1 and Kasumi-1, were treated with increasing concentrations of bortezomib, and changes in NKG2D ligand expression were measured. Bortezomib treatment significantly increased expression of the NKG2D ligand UL16 binding protein (ULBP) 2/5/6 in both cell lines. Vγ9Vδ2 T cells were expanded and isolated from peripheral blood of healthy donors to generate a final cellular product with a mean of 96% CD3+/γδ T-cell receptor-positive cells. Combination treatment of the AML cell lines with γδ T cells and bortezomib resulted in significantly greater cytotoxicity than γδ T cells alone, even at lower effector-to-target ratios. Based on the positive results against AML and the generalizable mechanism of this combination approach, it was also tested against T-cell acute lymphoblastic leukemia (T-ALL), another high-risk leukemia. Similarly, bortezomib increased ULBP 2/5/6 expression in T-ALL cell lines, Jurkat and MOLT-4 and improved the cytotoxicity of γδ T cells against each line. Collectively, these results show that bortezomib enhances γδ T-cell-mediated killing of both AML and T-ALL cells in part through increased NKG2D ligand-receptor interaction. Furthermore, proof-of-concept for the combination of ex vivo-expanded γδ T cells with stress ligand-inducing drugs as a therapeutic platform for high-risk leukemias is demonstrated.

After hematopoietic stem cell transplantation (HSCT), successful engraftment and immune recovery is necessary to protect the patient from relapse and infection. Many studies highlight the importance of conventional αβ T cell recovery after HSCT, but the impact of γδ T cell recovery has not been well described. Here, we investigate the recovery of γδ T cells in 102 pediatric patients with acute leukemia in first clinical remission who underwent allogeneic HSCT at St. Jude Children's Research Hospital from 1996 to 2011. Mean patient age was 10.5 ± 5.9 years (range, .6 to 25.2), and mean survivor follow-up was 2.7 ± 1.8 years (range, .12 to 6.0). Diagnoses included 59% patients with acute lymphoblastic leukemia and 41% patients with acute myelogenous leukemia. Multivariate analysis demonstrated significant impact of the maximum number of CD3(+), CD4(+), and CD8(+) T cells and donor source on the γδ T cell recovery (P < .0001, P < .0001, P < .0001, and P < .004, respectively). Univariate and multivariate models found the number of γδ T cells after HSCT to be associated with infections (P = .026 and P = .02, respectively). We found the probability of infections for patients with an elevated number of γδ T cells was significantly lower compared with patients with low or normal γδ T cells after HSCT (18% versus 54%; P = .025). Bacterial infections were not observed in patients with elevated γδ T cells. Finally, event-free survival was significantly higher in patients with enhanced γδ T cell reconstitution compared with patients with low/normal γδ T cell reconstitution after HSCT (91% versus 55%; P = .04). Thus, γδ T cells may play an important role in immune reconstitution after HSCT.

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Background: Gamma-delta (γδ) T cells engage heme malignancies via innate sensing of multiple ligands such as PVR, B7-H6, DNAM-1, and the NKG2DL family. These MHC unrestricted cells do not initiate graft-versus-host disease (GvHD), are anti-leukemic and may address the ~50% incidence of relapse following haplo-reduced intensity conditioning (RIC) stem cell transplantation. Infusion of donor-derived allogeneic ex vivo activated γδ T cells (EAGD) post-transplant may reduce relapse without severe GvHD. We present updated clinical and correlative data from dose-escalation and recommended phase 2 dose (RP2D) cohort. Methods: Adults with newly diagnosed or relapsed ALL, CML, AML, or MDS undergoing first HSCT with RIC received EAGD cells after neutrophil engraftment. Three dose levels (DL) of 1x106, 3x106 and 1x107 cells/kg were planned. Primary endpoints included dose-limiting toxicities (DLT), grade (G) 3-4 adverse events (AE's) including GvHD, with secondary endpoints of relapse-free (RFS) and overall survival. Immunophenotyping and cytokine analysis were conducted over the one year post-HSCT. Results: 14 subjects enrolled, 10 treated (4 treated at DL1, 6 treated at the RP2D of DL2). Untreated subjects included a screen failure, manufacturing failure, one subject who died prior to dosing, and a subject who received an out of study specification product. Dosed subjects were 60% male, 90% white, median age 68, and primarily AML subjects in CR1. RP2D was defined by an acceptable toxicity profile, no DLT's, prolonged RFS and elevated γδ levels. Most common AE's included platelet count decrease, anemia, decreased ANC (92% each), WBC count decrease (83% ), hypomagnesemia (75%), maculopapular rash (67%) and blood creatinine increased, hypokalemia, nausea, diarrhea and decreased lymphocyte count (58% each). EAGD related toxicities were primarily G1-2, included maculopapular rash (50% incidence with 10% G3 events), acute GvHD (G2 in 70%) and 31% chronic GVHD. Treatment related serious AE's in 23% of patients included G3 nausea (8%), G2 rash (15%). No DLTs, neurotoxicity, cytokine release syndrome or treatment related deaths were reported. At median follow-up of 19.0 months (mos), 15.3 mos median duration of morphologic complete remission (CR) was observed in 10 evaluable subjects. Seven of 10 evaluable subjects remain in CR with one subject receiving intermittent hypomethylating therapy for emergence of recipient chimerism. Of the seven, three subjects remain relapse free beyond 3 years, including 2 with trisomy 8 and del 7 along with IDH and/or FLT-3 mutation. One patient died relapse free due to idiopathic pulmonary syndrome unrelated to EAGD cells at 15.5 mos, and 2 subjects with TP53 mutations (an MDS/MPN overlap and an ALL subject) relapsed at 12.5 and 14.7 mos, respectively, despite the ALL subject having received 7 prior induction regimens. Peripheral lymphodepletion persisted through d100 post-transplant followed by T cell recovery, where CD8+ T cells shift from central memory to effector memory and terminal effector memory T cells (TEMRA) CD45RA/CD27 phenotypes. Continued γδ T cell expansion is observed in DL2 at 60-365d post-BMT vs. DL1 and historical haplo/PTCy patients, p = <0.05 suggesting durability of the anti-leukemic effect and continued leukemic surveillance. Summary: EAGD cells demonstrated manageable safety with no severe acute GvHD. Durable RFS ≥ 12mos was achieved in 100% of subjects followed for a year despite historical ~50% relapse rates at 1 year. This, along with evidence of γδ T cells expansion post-HSCT suggests this therapy may be effective in mitigating relapse after haplo-HSCT and warrants on-going study continuation. Based on preliminary data, a registrational trial in AML patients is forthcoming. NCT03533816

Predicting the transplant outcome of Acute Lymphoblastic Leukemia patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a challenge. It is necessary to have prognostic biomarkers that can be monitored in these patients. In the present single center, long term follow-up study, immune cell composition or the immunophenotype of Acute Lymphoblastic Leukemia patients (n = 14) was analyzed at various intervals following allogeneic HSCT and correlated with clinical outcomes. Long-term survivors post-HSCT showed distinct immune cell profiles compared to non-survivors. Long term survivors exhibited higher expression of Vδ2+CD69+ (p = 0.0126), and Vδ1+CD16b+ T cells (p = 0.0485) at baseline, compared to non-survivors. The analysis of donor immune cell composition showed difference at baseline, donors with higher levels of NKT+ cells and lower Vδ1+perforin+ T cells led to longer survival of respective recipients. Earlier WBC engraftment and incidence of chronic GVHD were other independent clinical factors associated with higher overall survival post- allogeneic HSCT. Furthermore, donors with higher baseline percentages of γδ T cells, CD19+ B cells, CD4 T cells, Vδ2 Naïve, Vδ2 TemRA, CD3+CD25+, and Vδ2+CD25+; and low Effector Memory CD3+ and Vδ1+ T cells were associated with fewer Cytomegalovirus (CMV) reactivation in their recipients. The present study underscores the importance of compositional analysis of immune cells in both recipients and donors as a discerning predictive tool for anticipating long-term clinical outcomes following allogeneic HSCT and highlights the need for future validation in larger cohorts.

Background: Gamma-delta (gd) T cells are MHC unrestricted lymphocytes that recognize and lyse malignant cells in allogeneic settings. Although haploidentical transplant with PTCy has reduced the risk of graft-versus-host disease (GvHD); the incidence of relapse remains up to 50% at year 1. Early post-transplant infusion of haploidentical EAGD cells may decrease relapse risk through a graft-versus-leukemia (GvL) effect without severe GvHD. We present updated clinical and correlative data from our Phase I trial using our recommended phase 2 dose (RP2D). Methods: Adults with newly diagnosed or relapsed ALL, CML, AML undergoing first haploidentical transplant with reduced-intensity flu/cy/TBI conditioning received EAGD intravenously within 7 days of neutrophil engraftment. Peripheral blood was collected at EAGD infusion and monthly through day 90, with additional collections every 6 months through 1 year. Primary endpoints include dose-limiting toxicities (DLT), grade (G) 3-4 adverse events including GvHD with secondary endpoints of relapse and overall survival. Biologic parameters included multiparameter flow cytometric immunophenotyping and additional serum cytokine analysis using the Olink ® 48 target panel. Results: 14 subjects were enrolled with 4 treated at Dose Level (DL) 1 of 1 x 10 6 EAGD/kg and 6 subjects treated at the RP2D of DL2 (3 x 10 6 EAGD/kg). Untreated subjects included: One screen failure, a manufacturing failure, one subject who died prior to dosing, and one subject who received an out of study specification product. Treated subjects were 60% male, median age of 68, and primarily AML subjects in CR1. RP2D was defined by an acceptable toxicity profile, no DLT's, prolonged RFS and elevated gd levels. Peripheral lymphodepletion persisted through the first 100 days post-BMT followed by T cell recovery from a CD45+CD27-effector phenotype to central and effector memory phenotype. Notably, DL2 showed significant increases in gd T cell count recovery at days 60, 100 and 180 post-BMT vs. DL1 and historically untreated Haplo/PTCy patients, p = <0.05. NK cells remained within the low normal range and <3% of circulating T cells were Tregs. Preliminary serum cytokine analysis revealed elevated IL-6 and IL-15 in serum post- transplant prior to EAGD infusion, but dropped to normal median levels at day 60, 20-30 days after EAGD infusion. One subject received intermittent hypomethylating therapy for emergence of recipient chimerism. Treatment emergent AE's included transient WBC and ANC decreases (100% each), platelet count decrease and anemia (88.9% each) and maculopapular rash, hypomagnesemia and blood creatine increased (55.6% each). EAGD related toxicities were primarily G1-2, included G1-2 skin acute GvHD and gastrointestinal GvHD, and one case of G3 platelet count decrease. Treatment related serious AE's of G3 nausea and G2 rash were reported. No DLTs, neurotoxicity, cytokine release syndrome or treatment related deaths were reported. Conclusions: Post-BMT infusion of donor-derived, EAGD cells has demonstrated manageable safety with no infusional toxicity or ≥G3 acute GvHD or extensive chronic GvHD. Durable relapse free survival at a maximum of >3 years in subjects with poor risk cytogenetics and adverse clinical risk factors combined with ongoing homeostatic reconstitution of increasing gd T cell effectors post-BMT suggests this therapy may be an effective measure in mitigating relapse after HaploBMT. NCT03533816

T-cell receptor (TCR) γδ cells are perceived as innate-like effector cells with the possibility of mediating graft-vs. -tumor (GVT) without causing graft-vs.-host disease (GVHD) in the setting of hematopoietic allogeneic stem cell transplantation (HSCT). We conducted a prospective study to assess the clinical impact of TCR γδ cell immune reconstitution on overall survival, relapse-free-survival, relapse and GVHD. The impact of CD3, CD4, and CD8 T cells together with NK cells including subtypes were analyzed in parallel. A total of 108 patients with hematological malignancies transplanted with HLA-matched, T cell replete stem cell grafts were included for analyses of absolute concentrations of CD3, CD4, and CD8 positive T cells and NK cells together with a multi-color flow cytometry panel with staining for TCRαβ, TCRγδ, Vδ1, Vδ2, CD3, CD4, CD8, HLA-DR, CD196, CD45RO, CD45RA, CD16, CD56, CD337, and CD314 at 28, 56, 91, 180, and 365 days after transplantation. Immune reconstitution data including subsets and differentiation markers of T and NK cells during the first year after transplantation was provided. Patients with TCR γδ cell concentrations above the median value of 21 (0–416) × 106 cells/L 56 days after transplantation had significantly improved overall survival (p = 0.001) and relapse-free survival (p = 0.007) compared to patients with concentrations below this value. When day 56 cell subset concentrations were included as continuous variables, TCR γδ cells were the only T cell subsets with a significant impact on OS and RFS; the impact of TCR γδ cells remained statistically significant in multivariate analyses adjusted for pre-transplant risk factors. The risk of death from relapse was significantly decreased in patients with high concentrations of TCR γδ cells 56 days after transplantation (p = 0.003). Also, the risk of acute GVHD was significantly lower in patients with day 28 TCR γδ cell concentrations above the median of 18 × 106 cells/L compared to patients with low concentrations (p = 0.01). These results suggest a protective role of TCR γδ cells in relapse and GVHD and encourage further research in developing adaptive TCR γδ cell therapy for improving outcomes after HSCT.

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The purpose of this study was to examine the ability of gamma(delta) T cells to cause graft-vs.-host disease (GVHD) after allogeneic bone marrow transplantation (BMT) and to determine whether these cells offered any therapeutic advantages relative to alphabeta T cells. Due to the paucity of naive gamma(delta) T cells in mice and humans, gamma(delta), T cells (obtained from alpha(beta) T cell-deficient murine donors) were ex vivo activated and expanded in interleukin (IL)-2 so as to achieve sufficient cell numbers and to serve as a more clinically feasible strategy. After transplantation into lethally irradiated hosts, donor gamma(delta) T cells were detected in target organs of GVHD such as the spleen and intestines 2 weeks after BMT and constituted the primary T cell subpopulation. Large doses (150 x 10(6)) of activated gamma(delta) T cells, which we have previously shown capable of facilitating engraftment in MHC-disparate recipients, failed to cause fatal GVHD in lethally irradiated recipients of MHC-incompatible donor marrow grafts (C57BL/6 [H-2b]-->B10.BR [H-2k] and C57BL/6 [H-2b]-B6D2F1[H-2b/d]). The absence of GVHD was confirmed by histologic analysis of target organs, splenic B cell reconstitution, and appropriate negative selection in the thymus, that were all comparable to those observed in mice transplanted with T cell-depleted BM only. While early splenic reconstitution was attributable to donor gamma(delta) T cells, analysis of durably engrafted chimeras 2 months posttransplant revealed that the vast majority of donor splenic T cells expressed the alpha(beta) T cell receptor. The results of secondary adoptive transfer assays showed that these cells were tolerant of recipient alloantigens in vivo, demonstrating that gamma(delta) T cells did not prevent the subsequent development of donor anti-host tolerance in BM-derived alpha(beta) T cells. When comparatively evaluated, the minimal number of naive alpha(beta) T cells necessary for donor engraftment caused significantly more fatal GVHD than the corresponding minimal dose of activated gamma(delta) T cells and thus had a superior therapeutic index. These studies indicate that doses of activated gamma(delta) T cells that are able to promote alloengraftment do not cause lethal GVHD in mice transplanted with MHC-incompatible marrow grafts.

No abstract available

BackgroundThe immune modulatory effect of granulocyte colony-stimulating factor (G-CSF) on T cells resulted in an unexpected low incidence of graft-versus-host disease (GVHD) in allogeneic peripheral blood stem cell transplantation (allo-PBSCT). Recent data indicated that gamma delta+ T cells might participate in mediating graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effect after allogeneic hematopoietic stem cell transplantation. However, whether G-CSF could influence the T cell receptors (TCR) of gamma delta+ T cells (TRGV and TRDV repertoire) remains unclear. To further characterize this feature, we compared the distribution and clonality of TRGV and TRDV repertoire of T cells before and after G-CSF mobilization and investigated the association between the changes of TCR repertoire and GVHD in patients undergoing G-CSF mobilized allo-PBSCT.MethodsThe complementarity-determining region 3 (CDR3) sizes of three TRGV and eight TRDV subfamily genes were analyzed in peripheral blood mononuclear cells (PBMCs) from 20 donors before and after G-CSF mobilization, using RT-PCR and genescan technique. To determine the expression levels of TRGV subfamily genes, we performed quantitative analysis of TRGV I~III subfamilies by real-time PCR.ResultsThe expression levels of three TRGV subfamilies were significantly decreased after G-CSF mobilization (P = 0.015, 0.009 and 0.006, respectively). The pattern of TRGV subfamily expression levels was TRGV II > TRGV I > TRGV III before mobilization, and changed to TRGV I > TRGV II > TRGV III after G-CSF mobilization. The expression frequencies of TRGV and TRDV subfamilies changed at different levels after G-CSF mobilization. Most TRGV and TRDV subfamilies revealed polyclonality from pre-G-CSF-mobilized and G-CSF-mobilized samples. Oligoclonality was detected in TRGV and TRDV subfamilies in 3 donors before mobilization and in another 4 donors after G-CSF mobilization, distributed in TRGV II, TRDV 1, TRDV 3 and TRDV 6, respectively. Significant positive association was observed between the invariable clonality of TRDV 1 gene repertoire after G-CSF mobilization and low incidence of GVHD in recipients (P = 0.015, OR = 0.047).ConclusionsG-CSF mobilization not only influences the distribution and expression levels of TRGV and TRDV repertoire, but also changes the clonality of gamma delta+ T cells. This alteration of TRGV and TRDV repertoire might play a role in mediating GVHD in G-CSF mobilized allo-PBSCT.