Allogeneic Γδ Research Articles

Abstract C117: Preclinical characterization of allogeneic CAR γδ T Cell therapy for prostate cancer targeting a novel dimeric epitope on PSMA

Abstract 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.

Utilizing Chemotherapy to Enhance the Anti-Tumor Properties of Ex Vivo Expanded Gamma Delta (γδ) T Cells Against Acute Myeloid Leukemia

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.

Expansion, Persistence and Pharmacodynamic Profile of ADI-001, a First-in-Class Allogeneic CD20-Targeted CAR Gamma Delta T Cell Therapy, in Patients with Relapsed/Refractory Aggressive B-Cell Non-Hodgkin's Lymphoma

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.

Engineering Off-the-Shelf Gamma Delta CAR T Cells for the Treatment of Acute Myeloid Leukemia

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.

Abstract 4093: Dual targeted CAR immunotherapy for neuroblastoma using γδ T cells

Abstract 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.

Three distinct mechanisms underlying human γδ T cell-mediated cytotoxicity against malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a rare and highly aggressive thoracic tumor with poor prognosis and limited therapeutic options. Although immune checkpoint inhibitors exhibit a promising effect in some patients with unresectable MPM in clinical trials, the majority of MPM patients show only modest response rates to the currently available treatments. It is thus imperative to develop novel and innovative therapeutic modalities for MPM, including immune effector cell-based therapies. γδ T cells were expanded using tetrakis-pivaloyloxymethyl 2-(thiazole-2-ylamino) ethylidene-1,1-bisphosphonate (PTA) and interleukin-2, and the therapeutic potential of γδ T cells was examined through analyzing cell surface markers and cellular cytotoxicity against MPM in vitro using a europium chelate-based time-resolved fluorescence assay system and a luciferase-based luminescence assay system. We successfully expanded γδ T cells from peripheral blood mononuclear cells of healthy donors and MPM patients. γδ T cells expressed natural killer receptors such as NKG2D and DNAM-1 and exhibited a moderate level of cytotoxicity to MPM cells in the absence of antigens. The inclusion of PTA, (E)-4-hydroxy-3- methylbut-2-enyl diphosphate (HMBPP) or zoledronic acid (ZOL) induced a TCR-dependent cytotoxicity in γδ T cells and secreted interferon-γ (IFN-γ). In addition, γδ T cells expressing CD16 exhibited a significant level of cytotoxicity against MPM cells in the presence of an anti-epidermal growth factor receptor (EGFR) mAb, at lower concentrations than in clinical settings, whereas a detectable level of IFN-γ was not produced. Taken together, γδ T cells showed cytotoxic activity against MPM in three distinct mechanisms through NK receptors, TCRs and CD16. Since major histocompatibility complex (MHC) molecules are not involved in the recognition, both autologous and allogeneic γδ T cells could be used for the development of γδ T cell-based adoptive immunotherapy for MPM.

Large Scale Ex Vivo Expansion of γδ T cells Using Artificial Antigen-presenting Cells.

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 5573: ACE1831: A novel allogeneic αCD20-conjugated Vδ2 gamma delta T product for non-Hodgkin’s lymphoma

Abstract Introduction Autologous CAR-T has improved clinical outcomes in hematological malignancies; however need remains to improve the toxicities, relapse rates, treatment delays and costs. ACE1831 is a novel, off-the-shelf Antibody Conjugated Effector cell therapy with aCD20 linked to gd T cells by selective proprietary DNA aptamers to activation receptors without genetic modification. Data for in vitro/in vivo pre-clinical studies are presented. Methods γδ T cells were expanded from healthy donor PBMCs by cytokine stimulation and αβ T cell depletion. Both γδ T cells and αCD20 were covalently linked to selected DNA aptamers that enable conjugation, and cryopreserved. ACE1831 was evaluated for phenotype and antibody conjugation by flow cytometry, cytotoxicity and cytokine production by 4-hour co-culture with CD20+ Raji cells, and for in vivo anti-tumor activity and weight loss using SCID-Beige mice inoculated IV with 1 x 105 Raji cells, 4 doses of 1 x 107 ACE1831 on days 0, 3, 7, 10, and monitored for 123 days. All studies used cryopreserved ACE1831. Results gd T cells of ACE1831 were expanded >10,000 fold with >95% viability, >85% Vδ2 purity, and <0.03% αβ T cell impurity. γδ T cells expressed high levels of CD56, CD16, NKG2D and CD69 and low levels of PD-1 and KIRs. Following αCD20 conjugation and cryopreservation, ACE1831 drug product batches retained >90% viability and cytotoxicity against CD20+ Raji cells. In the presence of cell growth media, about 30% and 10% of αCD20 were retained on the cell surface at 24 and 48 hours, respectively, and at 24 and 48 hours, ACE1831 elicited 100% and 85% specific lysis against Raji cells. At 72 hours, specific lysis was 35%; however, in combination with obinutuzumab (1,000 ng/mL), ACE1831 provided 95% specific lysis against Raji cells. Co-cultured with Raji cells, ACE1831 produced 10 times more IFNγ and TNFα than unconjugated γδ T cells, but no measurable IL-6. When co-cultured with allogeneic PBMCs, ACE1831 showed no cytotoxicity towards PBMCs and the mixture showed no increase in IFNγ, TNFα, GM-CSF, IL-2, IL-4, IL-8, IL-6, and IL-10 levels compared to the PBMCs only group. ACE1831 showed persistence in vivo for >14 days after a single dose of 1 x 107 AC1831 in non-tumor inoculated SCID-Beige mice. Tumor inoculated mice treated with 4 doses of ACE1831 survived 123 days without weight loss or tumor relapse based on bioluminescence and necropsy, while mice treated with γδ T survived <40 days due to tumor progression. Conclusion ACE1831, a aCD20-armed allogeneic γδ T cell product, represents a novel approach for off-the-shelf treatment for B cell malignancies. Specifically, this product candidate can offer high levels of anti-tumor activity that is extended with soluble antibody using native Fc receptor expression, and may have a low risk of GvHD and IL-6 related toxicity. These data support future clinical studies in this setting. Citation Format: Tai-Sheng Wu, Hao-Kang Li, Ching-Wen Hsiao, Yi-Hung Ou, Zih-Fei Cheng, Hsiu-Ping Yang, Sen-Han Yang, Chia-Yun Lee, Yan-Liang Lin, Thorsten Graef, Sai-Wen Tang, Sonny Hsiao. ACE1831: A novel allogeneic αCD20-conjugated Vδ2 gamma delta T product for non-Hodgkin’s lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5573.

Dissecting the cellular components of ex vivo γδ T cell expansions to optimize selection of potent cell therapy donors for neuroblastoma immunotherapy trials

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.

Augmenting human gamma delta lymphocytes for cancer therapy with chimeric antigen receptors

Gamma delta lymphocytes (γδ T) sit at the interface between innate and adaptive immunity. They have the capacity to recognize cancer cells by interaction of their surface receptors with an array of cancer cell surface target antigens. Interactions include the binding of γδ T cell receptors, the ligands for which are diverse and do not involve classical major histocompatibility complex (MHC) molecules. Moreover, a variety of natural killer-like and fragment crystallizable gamma (Fcγ) receptors confer additional cancer reactivity. Given this innate capacity to recognize and kill cancer cells, there appears less rationale for redirecting specific to cancer cell surface antigens through chimeric antigen receptor (CAR) expression. Several groups have however reported research findings that expression of CARs in γδ T cells can confer additional specificity or functionality. Though limited in number, these studies collectively identify the potential of CAR-T engineering to augment and fine tune anti-cancer responses. Together with the lack of graft versus host disease induced by allogeneic γδ T cells, these insights should encourage researchers to explore additional γδ T-CAR refinements for the development of off-the-shelf anti-cancer cell therapies.

Allogeneic CD20-targeted γδ T cells exhibit innate and adaptive antitumor activities in preclinical B-cell lymphoma models.

ObjectivesAutologous chimeric antigen receptor (CAR) αβ T‐cell therapies have demonstrated remarkable antitumor efficacy in patients with haematological malignancies; however, not all eligible cancer patients receive clinical benefit. Emerging strategies to improve patient access and clinical responses include using premanufactured products from healthy donors and alternative cytotoxic effectors possessing intrinsic tumoricidal activity as sources of CAR cell therapies. γδ T cells, which combine innate and adaptive mechanisms to recognise and kill malignant cells, are an attractive candidate platform for allogeneic CAR T‐cell therapy. Here, we evaluated the manufacturability and functionality of allogeneic peripheral blood‐derived CAR+ Vδ1 γδ T cells expressing a second‐generation CAR targeting the B‐cell‐restricted CD20 antigen.MethodsDonor‐derived Vδ1 γδ T cells from peripheral blood were ex vivo‐activated, expanded and engineered to express a novel anti‐CD20 CAR. In vitro and in vivo assays were used to evaluate CAR‐dependent and CAR‐independent antitumor activities of CD20 CAR+ Vδ1 γδ T cells against B‐cell tumors.ResultsAnti‐CD20 CAR+ Vδ1 γδ T cells exhibited innate and adaptive antitumor activities, such as in vitro tumor cell killing and proinflammatory cytokine production, in addition to in vivo tumor growth inhibition of B‐cell lymphoma xenografts in immunodeficient mice. Furthermore, CD20 CAR+ Vδ1 γδ T cells did not induce xenogeneic graft‐versus‐host disease in immunodeficient mice.ConclusionThese preclinical data support the clinical evaluation of ADI‐001, an allogeneic CD20 CAR+ Vδ1 γδ T cell, and a phase 1 study has been initiated in patients with B‐cell malignancies (NCT04735471).

Clinical Safety and Efficacy of Locoregional Therapy Combined with Adoptive Transfer of Allogeneic γδ T Cells for Advanced Hepatocellular Carcinoma and Intrahepatic Cholangiocarcinoma

PurposeTo investigate the safety and efficacy of locoregional therapy plus adoptive transfer of allogeneic gamma delta (γδ) T cells for patients with hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). MethodsThirty patients with HCC and 29 patients with ICC were randomly assigned to receive locoregional therapy (HCC, Group A, n = 15; ICC, Group C, n = 15) or locoregional therapy plus γδ T cell therapy (HCC, Group B, n = 15; ICC, Group D, n = 14). Groups A and C only received locoregional ablation (cryoablation or irreversible electroporation), whereas Groups B and D received locoregional therapy followed by adoptive transfer of allogeneic γδ T cells. The primary endpoints were safety, distant progression-free survival (PFS), local PFS, and overall survival (OS). ResultsThe median distant PFS was significantly longer in the combined treatment groups than the locoregional treatment groups (HCC: 8 vs 4 months, P = .04; ICC: 8 vs 4 months, P = .021). There was no significant difference in local PFS between the 2 treatment modalities. Patients with HCC in the combined treatment group had a longer OS (median OS: 13 vs 8 months, P = .029). However, there was no significant difference in OS in patients with ICC between the 2 treatment modalities (median OS: 9.5 vs 8 months, P = .546). All adverse events were manageable with no significant difference in incidence between groups. ConclusionsThe novel combination of locoregional ablation with adoptive transfer of allogeneic γδ cells was safe, with encouraging clinical efficacy against HCC and ICC.

Expansion and Enrichment of Gamma-Delta (γδ) T Cells from Apheresed Human Product

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.

Expansion and Enrichment of Gamma-Delta (γδ) T Cells from Apheresed Human Product.

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.

Human serum albumin and chromatin condensation rescue ex vivo expanded γδ T cells from the effects of cryopreservation

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.

Characterization of Donor Variability for γδ T Cell ex vivo Expansion and Development of an Allogeneic γδ T Cell Immunotherapy.

Gamma delta (γδ) T cells recently emerged as an attractive candidate for cancer immunotherapy treatments due to their inherent cytotoxicity against both hematological and solid tumors. Moreover, γδ T cells provide a platform for the development of allogeneic cell therapies, as they can recognize antigens independent of MHC recognition and without the requirement for a chimeric antigen receptor. However, γδ T cell adoptive cell therapy depends on ex vivo expansion to manufacture sufficient cell product numbers, which remains challenging and limited by inter-donor variability. In the current study, we characterize the differences in expansion of γδ T cells from various donors that expand (EX) and donors that fail to expand, i.e., non-expanders (NE). Further, we demonstrate that IL-21 can be used to increase the expansion potential of NE. In order to reduce the risk of graft vs. host disease (GVHD) induced by an allogeneic T cell product, αβ T cell depletions must be considered due to the potential for HLA mismatch. Typically, αβ T cell depletions are performed at the end of expansion, prior to infusion. We show that γδ T cell cultures can be successfully αβ depleted on day 6 of expansion, providing a better environment for the γδ T cells to expand, and that the αβ T cell population remains below clinically acceptable standards for T cell-depleted allogeneic stem cell products. Finally, we assess the potential for a mixed donor γδ T cell therapy and characterize the effects of cryopreservation on γδ T cells. Collectively, these studies support the development of an improved allogeneic γδ T cell product and suggest the possibility of using mixed donor γδ T cell immunotherapies.

Large Scale Ex Vivo Expansion of Γδ T Cells Using Artificial Antigen Presenting Cells for the Treatment of Acute Myeloid Leukemia

Patients with relapsed or refractory acute myeloid leukemia (AML) are at increased risk of mortality. Higher γδ T cell count in a bone marrow or peripheral blood of patients with leukemia is associated with better survival. However, γδ T cells are rare in the blood and functionally impaired or exhausted 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 IL-2. Here we demonstrated that zol and IL-2, in combination with a novel genetically engineered K562 CD3/CD137L/CD28/IL15RA quadruplet artificial antigen presenting cell (aAPC), efficiently expand allogeneic donor-derived γδ T cells using a 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 co-culture with aAPC, the majority of which exhibited central (47%) and effector (43%) memory phenotypes. Additionally, >90% of the expanded γδ T cells expressed NKG2D, while they have low cell surface expression of PD1 and LAG2 inhibitory checkpoint receptors. In vitro real-time cytotoxicity analysis showed that expanded, previously cryopreserved, γδ T cells were effective in killing target cells. Our results demonstrate that large scale ex vivo expansion of donor-derived γδ T cells can be achieved with the use of CD3/CD137L/CD28/IL15RA quadruplet aAPC and zol/IL-2 for clinical application as promising antineoplastic immunotherapy. Figure 1 Disclosures Lancet: Abbvie: Consultancy; Agios Pharmaceuticals: Consultancy, Honoraria; Astellas Pharma: Consultancy; Celgene: Consultancy, Research Funding; Daiichi Sankyo: Consultancy; ElevateBio Management: Consultancy; Jazz Pharmaceuticals: Consultancy; Pfizer: Consultancy. Sallman:Celgene, Jazz Pharma: Research Funding; Agios, Bristol Myers Squibb, Celyad Oncology, Incyte, Intellia Therapeutics, Kite Pharma, Novartis, Syndax: Consultancy. Bejanyan:Kiadis Pharma: Membership on an entity's Board of Directors or advisory committees.

Irreversible electroporation plus allogenic V\u03b39V\u03b42 T cells enhances antitumor effect for locally advanced pancreatic cancer patients

Immunotherapy has limited efficacy against locally advanced pancreatic cancer (LAPC) due to the presence of an immunosuppressive microenvironment (ISM). Irreversible electroporation (IRE) can not only induce immunogenic cell death, but also alleviate immunosuppression. This study aimed to investigate the antitumor efficacy of IRE plus allogeneic γδ T cells in LAPC patients. A total of 62 patients who met the eligibility criteria were enrolled in this trial, then randomized into two groups (A: n = 30 and B: n = 32). All patients received IRE therapy and after receiving IRE, the group A patients received at least two cycles of γδ T-cell infusion as one course continuously. Group A patients had better survival than group B patients (median OS: 14.5 months vs. 11 months; median PFS: 11 months vs. 8.5 months). Moreover, the group A patients treated with multiple courses of γδ T-cell infusion had longer OS (17 months) than those who received a single course (13.5 months). IRE combined with allogeneic γδ T-cell infusion is a promising strategy to enhance the antitumor efficacy in LAPC patients, yielding extended survival benefits.ClinicalTrials.gov ID: NCT03180437.

Abstract 3588: ACTallo: A novel approach using gamma-delta T cells to allogeneic cellular therapy to treat cancer

Abstract Adoptive cellular therapy (ACT) has dramatically changed the landscape of cancer immunotherapy; however, the complex manufacture of individualized products from late-stage, heavily pre-treated cancer patients, together with high cost-of-goods, encourages the development of simpler ‘off-the-shelf' alternatives. The unique properties of γδ (gamma-delta) T cells make them a seductive candidate for effective allogeneic ACT. In contrast to αβ T cells, γδ T cells are endowed with intrinsic antitumor activity while unable to induce graft-versus-host disease. γδ T cells have a high proliferative potential before exhibiting exhaustion. Moreover, in the context of αβ TCR-engineered ACT, γδ T cells pose a lesser risk of off-target toxicity, due to the absence of expression of endogenous αβ TCR and hence, risk of mispairing with the engineered αβ TCR. Here, we will present the ACTallo® platform, which combines the unique properties of γδ T cells with Immatics' XPRESIDENT® target/TCR discovery platform. The ACTallo® process selectively expands Vγ9Vδ2 T cells that are retrovirally-transduced to co-express CD8 together with a tumor-specific αβ TCR. Over a period of 45 days or less, ACTallo® γδ T cells can expand >30,000 fold. γδ T cells obtained this way recognize tumor cells, express cytokines, degranulate and kill tumor cells specifically. In addition, ACTallo® γδ T cells show no signs of exhaustion after expansion, as evidenced by the lack of expression of PD1, TIM3 or LAG3. ACTallo® is intended to overcome the limitation of autologous cellular therapies towards off-the-shelf approach, by combining the large-scale expansion potential of allogeneic γδ T cells with novel TCRs against XPRESIDENT® mass-spectrometry guided tumor-specific targets. Citation Format: Mo Dao, Melinda Mata, Leonie Alten, Aleksandra Nowicka, Sabrina Kuttruff, Sarah Missel, Ali Mohamed, Toni Weinschenk, Yannick Bulliard, Steffen Walter. ACTallo: A novel approach using gamma-delta T cells to allogeneic cellular therapy to treat cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3588.

In Vitro Expanded γδ T Cells Derived from Cord Blood Induce Potent and Specific Cytotoxicity Against Human Acute Myeloid Leukemia Cells

Conventional chemotherapeutic regimens for acute myeloid leukemia (AML) patients have demonstrated unsurpassed efficacy in the past decades, but are far from optimal with many patients experiencing multiple disease recurrence or intolerant of the intensive chemotoxicity. Variations in the treatment scheme as well as the use of alternative, targeted agents have been pursued with limited success. This is partly ascribed to the highly heterogeneous nature of the disease comprising a dynamic repertoire of evolving leukemic clones that are both molecularly and biologically diverse, making it difficult to achieve complete disease eradication without inducing adverse off-target effects. In this regard, cellular immunotherapy has emerged as a plausible alternative, leveraging on the diversity and degeneracy of the tumor antigen-recognizing receptor complex expressed by immune cells. In particular, there is a growing interest in the specific anti-leukemia efficacy of the innate-like γδ T cells, prompted by the association of an increased number of donor derived γδ T cells (specifically the Vδ1+ subtype) in allogeneic hematopoietic stem cell transplant (HSCT) patients with improved disease control in the absence of significant graft-versus-host disease (GvHD). We therefore hypothesize that these allogeneic γδ T cells exhibit potent leukemia specific cytotoxicity and serve as an effective treatment for AML.Given the rapid availability and widespread use of cord blood (CB) as an alternative for allogeneic HSCT, we first characterized and explored the potential of expanding CB-derived γδ T cells in vitro. Compared to mobilized peripheral blood (mPB), there is a significantly lower level of γδ T cell within CB mononuclear cells (MCs) (0.61% ± 0.36% in CB vs 4.95% ± 3.83% in mPB, p<0.001). However, the fraction of Vδ1+ subset within the γδ T cells in CB is >3.5-fold higher than that in mPB (56.05% ± 9.49% in CB vs 14.54% ± 12.2% in mPB, p<0.001). Importantly, while >90% of the Vδ1+ T cells in CB are of naive or central memory phenotype, more than 40% of these cells in mPB show effector memory expression. We established that optimal in vitro expansion of CB-derived γδ T cells requires direct contact to a mixture of irradiated PBMCs and Epstein-Barr virus-transformed lymphoblastoid cell line (EBV-LCL) at a fixed ratio in the gas-permeable G-Rex culture flask. Under these conditions, we were able to achieve up to 5,200-fold expansion of the starting γδ T cells over a period of 21 days. These cells exhibit potent in vitro cytotoxicity against a range of human AML cell lines, including K562, MOLM-14, MV4-11 and NOMO-1, as well as primary patient samples in a dose dependent manner. In contrast, there is minimal in vitro cytotoxicity against CD34+ cells isolated from allogeneic CB samples even at the highest effector-to-target cell (E:T) ratio tested. Infusion of the expanded γδ T cells into NOD/SCID/IL2Rγ-/- (NSG) mice at 3 weeks post-transplantation of a FLT3-ITD+ AML patient sample (P1) resulted in a significant decrease in leukemic cell engraftment in 40% of the γδ T cells-treated mice (87.46 ± 2.25% in non-treated vs 74.85 ± 1.55% in γδ T cells-treated mice, p=0.022). In a separate experiment, infusion into NSG mouse that was engrafted with low level (0.1%) of a different FLT3-ITD+ AML patient sample (P2) maintained the leukemic cell level low at 0.1% at 4 weeks post-infusion, as opposed to the >15-fold increase in leukemic burden (1.76%) seen in the untreated mouse. Consistent with our in vitro finding, infusion of up to 5 x 108 expanded CB derived γδ T cells/kg failed to induce severe GvHD symptoms in NSG mice engrafted with allogeneic human CB cells up to 8 weeks post-infusion, with no significant effect on the level of in vivo regenerated human myeloid and lymphoid cells, as well as colony-forming cells (CFCs).In summary, our data demonstrates that in vitro expanded CB derived γδ T cells show potent AML-specific cytotoxicity both in vitro and in vivo, making it a promising alternative cell source for immunotherapy. Further investigations to enhance the mechanistic understanding would be needed to seed for future clinical translation. DisclosuresHwang:Pfizer: Honoraria, Other: Travel support; MSD: Honoraria, Other: Travel support; BMS: Honoraria, Other: Travel support; Novartis: Honoraria, Other: Travel support; Celgene: Honoraria, Other: Travel support; Roche: Honoraria, Other: Travel support; Janssen: Honoraria, Other: Travel support; Sanofi: Honoraria, Other: Travel support.