Drug Resistant Immunotherapy Research Articles

INB-200: Fully enrolled phase 1 study of gene-modified autologous gamma-delta (γδ) T cells in patients with newly diagnosed glioblastoma multiforme (GBM) receiving maintenance temozolomide (TMZ).

2042 Background: γδ T cells can target NKG2D ligands that are upregulated on tumor cells after alkylating chemotherapy exposure. IN8bio’s DeltEx drug resistant immunotherapy (DRI) are genetically engineered γδ T cells expressing methylguanine-DNA methyltransferase (MGMT), which conveys TMZ resistance to enable concomitant therapy and continued surveillance against tumor cells. Updated results from the Phase 1 trial which fully enrolled adult newly diagnosed GBM patients with adequate organ function, KPS ≥ 70% follow. Methods: Cohorts (C) 1, 2 and 3 received 1, 3 or 6 doses (1 x 107 DRI cells/dose) into the resection cavity with 150 mg/m2 of IV TMZ on Day (D) 1 of each Stupp maintenance cycle. The primary endpoint is safety and secondary endpoints include survival; immunologic correlative analyses are included. Dose limiting toxicities (DLTs) are defined as treatment related ≥ grade (G) 3 cardiopulmonary or hepatic toxicity, G4 toxicity exceeding 72 hours or neurologic deterioration that exceeds 2 weeks. Results: 23 patients were enrolled, with 11 dosed and 2 awaiting dosing (61% male; median age 68 (range: 21-74); 92% IDH-WT, 54% MGMT unmethylated). No DLTs, cytokine release syndrome (CRS) or neurotoxicity (ICANS) are reported. Most common adverse events were decreased WBC/platelet count, asthenia, fatigue, hydrocephalus, headache, decreased appetite, urinary tract infection, thrombosis and balance disorder. Conclusions: γδ T cells successfully infused with peripheral TMZ-based lymphodepletion evidenced with near or below normal range T, B, and NK subsets for up to 1 year. The majority of dosed patients who received DRI exceeded the expected median PFS of 7 months (5.8-8.2 months) with Stupp alone and had manageable toxicity with a continued encouraging trend in PFS. Long-term follow-up for durability of PFS and OS continue. Clinical trial information: NCT04165941 . [Table: see text]

CTIM-35. INB-400 PHASE 1B/2 DRUG RESISTANT IMMUNOTHERAPY WITH ACTIVATED, GENE MODIFIED ALLOGENEIC OR AUTOLOGOUS ΓΔ T CELLS IN COMBINATION WITH MAINTENANCE TEMOZOLOMIDE RECURRENT OR NEWLY DIAGNOSED GLIOBLASTOMA

Abstract 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.

CTIM-42. INB-200: PHASE 1 STUDY OF GENE MODIFIED AUTOLOGOUS GAMMA-DELTA (ΓΔ) T CELLS IN NEWLY DIAGNOSED GLIOBLASTOMA MULTIFORME (GBM) PATIENTS RECEIVING MAINTENANCE TEMOZOLOMIDE (TMZ)

Abstract γδ T cells target NKG2D ligands that are upregulated on tumor cells after chemotherapy exposure. IN8bio’s DeltEx drug resistant immunotherapy (DRI) are γδ T cells genetically engineered to express MGMT, conveying TMZ resistance to allow concomitant therapy. We present updated results from this Phase 1 trial, which enrolled adult newly diagnosed glioblastoma patients with adequate organ function, KPS ≥ 70%. Cohorts (C) 1, 2 and 3 received 1, 3 or 6 doses (1 x 107) of DRI cells intratumorally with 150 mg/m2 of TMZ on Day (D) 1 of Stupp maintenance. The primary endpoint is safety and secondary endpoints include survival; immunologic and genomic correlative analyses are included. Dose limiting toxicities (DLTs) are defined as treatment related ≥ grade (G) 3 cardiopulmonary or hepatic toxicity, G4 toxicity exceeding 72 hours or neurologic deterioration that exceeds 2 weeks. 21 patients were enrolled, with 11 dosing (57% male; median age 68 (range: 21-76); 86% IDH-WT, 52% MGMT unmethylated). No DLTs, cytokine release syndrome (CRS) or neurotoxicity (ICANS) reported. Most common adverse events were WBC/platelet count decreased, asthenia, headache, hydrocephalus, decreased appetite and balance disorder. C1 pts had PFS of 8.3, 11.9, 7.4 months (m) and OS of 15.6, 17.7 and 9.6m, respectively. In C2, 1 pt remains progression free at 26.4m, while one progressed at 22m and another died without relapse at 8.7m. C3 pt had a PFS of 7.1 and OS of 13.11m with other pts in dosing. γδ T cells successfully infused with peripheral TMZ-based lymphodepletion evidenced with near or below normal range T, B, and NK subsets for up to 1 year. DRI T cells have manageable toxicity with encouraging trend in PFS with enriched γδ T cell remaining despite peripheral lymphodepletion.

Abstract 4202: Dual chlorotoxin and methylguanine-DNA methyltransferase (MGMT) γδ-T cells for drug resistant immunotherapy

Abstract High-grade gliomas are the most aggressive and refractory of brain tumors. Historical approaches to treating glioblastoma (GBM) using chimeric antigen receptor modified T cells (CAR-T) have been challenging, without a lasting benefit to patients, often due to tumor heterogeneity and a lack of tumor specific antigens. We report preliminary in vitro design and optimization of a γδ T cell-based CAR-T using Chlorotoxin (CLTX-CAR), a peptide isolated from the venom of the Israeli Deathstalker Scorpion that efficiently binds to GBM and other tumor cells with limited cross reactivity to normal tissues. This CLTX-CAR (CTLX-CAR-noZ) omits a CD3z signaling domain to maintain the natural innate targeting and tumor recognition properties of the γδ T cell through its endogenous T cell and NKG2D receptors, and CD16-based antibody-dependent cellular cytotoxicity (ADCC). This construct also includes our Drug Resistant Immunotherapy (DRI) platform that incorporates an additional p140K-MGMT encoding gene into the construct. This confers resistance to alkylating agents such as temozolomide (TMZ) that allows our DRI γδ T cells to remain viable and functional when combined with chemotherapy to force high-expression of natural killer group D ligands (NKG2DL) on the surface of tumor cells. As expected, the non-signaling, CLTX-CAR-noZ modified Jurkat cells did not result in T cell activation, as compared to the highly activated CD69 corresponding to signaling CLTX-CAR-CD3z transduced Jurkat cells. Conversely, CTLX-CAR-noZ transduced γδ T cells demonstrated higher cytotoxicity against U87MG and U251MG GBM cell lines over unmodified control γδ T cells. Dual-CLTX-CAR constructs lacking CD3z and using 2x CLTX peptides as the targeting domain (dCLTX-CAR-noZ) exhibited up to 90% lentiviral transduction efficiency into γδ T cells. When co-cultured in suspension with GBM cells in vitro, dCLTX-CAR-noZ γδ T cells demonstrated an average cytotoxicity of 62.1% against U87MG-GFP cells at an E:T ratio of 4:1 in comparison to 35.4% with non-transduced γδ T control cells. Time-lapsed microscopic recording over a period of 15hrs also verified killing of U251MG-GFP cells by dCLTX-CAR-noZ γδ T cells when cocultured with GBM cells at an E:T ratio of 2:1. Serial-killing of tumor cells by γδ T cells was documented. We are advancing this approach to xenograft animal models and as a clinical development candidate (INB-300) for both GBM and other extracranial tumors. Citation Format: Lei Ding, Yanjie Li, Evan Klumpp, Lawrence Lamb. Dual chlorotoxin and methylguanine-DNA methyltransferase (MGMT) γδ-T cells for drug resistant immunotherapy [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 4202.

Abstract 1173: Maintenance-phase Temozolomide as a lymphodepletion platform for intracranialadoptiveγδ T cell-basedtherapy in primary high-grade gliomas

Abstract Introduction: Lymphodepletion is a key factor for responses in both autologous and allogeneic cell therapies. We are currently evaluating in a Phase I trial, the combination of Temozolomide (TMZ) chemotherapy with methylguanine-DNA methyltransferase (MGMT) modified γδ T cells (Drug Resistant Immunotherapy, or DRI) during maintenance TMZ in patients receiving the Stupp protocol. In this report, we examine both DRI graft and circulating lymphocyte phenotype and function during the treatment phase. Methods: Patients meeting enrollment criteria undergo tumor resection, placement of a Rickham catheter in the tumor cavity, and an apheresis collection immediately prior to standard chemoradiotherapy (CRT). DRI follows TMZ on Day 1 of maintenance and consists of 150mg/m2 IV TMZ and concurrent intracranial injection of 1.0 x 107 DRI γδT cells. Peripheral blood (PB) was collected at apheresis and immediately prior to the start of each 28-day maintenance cycle. Flow cytometric (FACS) analysis of PB and DRI grafts using antibodies to CD3, CD4, CD8, CD16/56, CD19, TCR-γδ, Vδ1, Vδ2, CD197, CD27, CD28, CD57, CD45RA, Th1/Th2/Th17 PB serum cytokines, and DRI cytotoxic graft function were obtained. When applicable, FFPE sections of recurrent resected GBM tissues were examined for immune infiltration. Results: DRI grafts contained 74-93% activated γδ T cells with 0.3-0.5 MGMT copies/cell. Cytotoxicity against K562 ranged from 30% to 75% at 40:1 (E:T). Peripheral lymphodepletion was evident throughout TMZ treatment in the Stupp protocol. T cell counts fell precipitously after CRT and cycle 1 (1647+774 vs 609+214 cells/mL) and remained low throughout maintenance phase (range 214-1450 T cells/mL). NK counts remained normal and showed uneven recovery through the first three cycles but failed to recover for the remainder of maintenance phase. γδ T cells also recovered modestly through the first two cycles but failed to recover after additional cycles. Interestingly, CD45RA+CD27- effector T cells showed only slight increases for each patient throughout TMZ maintenance. Cytokine analysis did not show a clear trend except for consistent T cell expression of perforin and IP-10. One patient with recurrent GBM was successfully resected 110d following DRI. Histopathology revealed widespread necrosis with significant infiltration of CD4+ and CD8+ T cells and γδ T cells in the tumor parenchyma. Conclusions: Standard of care TMZ is lymphodepleting and DRI therapy can be conducted in an environment favorable to T cell persistence and sustained immune response. One treated patient demonstrated infiltration of both αβ and γδ T cells 110 days following a single dose of DRI γδ T cells. TMZ maintenance during the Stupp regimen prolongs lymphodepletion, thereby presenting a favorable setting for adoptive cell therapy. Citation Format: Lawrence S. Lamb, Lei Ding, Ryan C. Miller, Mariska ter Haak, Caitlyn Lucas, Becca Weekley, Samantha Youngblood, Cathy Langford, Guoling Chen, Louis B. Nabors. Maintenance-phase Temozolomide as a lymphodepletion platform for intracranialadoptiveγδ T cell-basedtherapy in primary high-grade gliomas [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 1173.

Phase I study of drug-resistant immunotherapy (DRI) with gene-modified autologous γδ T cells in patients with newly diagnosed glioblastoma multiforme (GBM) receiving maintenance temozolomide (TMZ).

2044 Background: γδ T cells, MHC unrestricted immune cells, target NKG2D ligands differentially expressed on tumor cells. DeltEx drug resistant immunotherapy (DRI), a novel ex vivo expanded, activated γδ T cell expresses MGMT, conveying TMZ resistance. NCT04165941 , a phase 1 trial assessing the safety of single and multiple infusions of autologous DeltEx DRI cells presents updated safety and efficacy data. Methods: Adult newly diagnosed GBM patients with adequate organ function and KPS≥70% are enrolled. Cells engineered from apheresis after tumor resection were infused through a Rickham catheter placed during surgery. Cohort (C) 1, 2 and 3 receive 1, 3 and 6 doses of cells respectively on day (D) 1 of each 28-day maintenance cycle. Patients receive 1 x 107 γδ T cells intratumorally on D1 with 150 mg/m2 of TMZ intravenously with the Stupp regimen. Primary endpoint is safety; secondary endpoints include progression free and overall survival. Immunologic and genomic correlative analyses are being conducted. Dose limiting toxicities (DLTs) are defined as treatment related ≥ grade 3 cardiopulmonary or hepatic toxicity, grade 4 toxicity exceeding 72 hours or neurologic deterioration that exceeds 2 weeks. Results: 12 patients (58% male; median age 66.5 (range: 21-76); 66.7% IDH-WT, 66.7% MGMT unmethylated) were enrolled with 6 dosed (3 in C1, 3 in C2). No patients had DLTs, cytokine release syndrome (CRS), or neurotoxicity. The most common adverse events (AEs) were Grade 1/2 events including fever, leukopenia, nausea, and vomiting attributable to TMZ or radiotherapy. One subject had Grade 3 treatment related AEs of UTI, dehydration, and thrombocytopenia. Three evaluable C1 patients have PFS of 8.3, 11.9, 7.4 months and OS of 15.6, 17.7 and 9.6 months respectively. In C2, three patients have been dosed, with one patient with stable disease at 8.2 months after receiving all three doses and no DLTs. Patient recruitment continues with anticipated completion in 2022. Conclusions: Data demonstrates that single, repeat doses of DRI T cells have manageable toxicity with encouraging trend in PFS.

23P Engineered chemotherapy resistant γδ T-cells with combinatorial chemotherapies can enhance tumor cell immunogenicity and killing

Cell Immunotherapies have had limited efficacy in solid tumors due to challenges trafficking to the tumor and efficiently targeting tumor heterogeneity. Gamma-delta (γδ) T cells can directly recognize and kill malignant tissue through recognition of upregulated Natural Killer Group D Ligands (NKG2DL). We engineered γδ T-cells using a non-signaling chimeric antigen receptor (CAR) that is resistant to chemotherapies such as temozolomide (TMZ), a construct we call drug resistant immunotherapy (DRI).

A combined treatment regimen of MGMT-modified \u03b3\u03b4 T cells and temozolomide chemotherapy is effective against primary high grade gliomas

Chemotherapeutic drugs such as the alkylating agent Temozolomide (TMZ), in addition to reducing tumor mass, can also sensitize tumors to immune recognition by transient upregulation of multiple stress induced NKG2D ligands (NKG2DL). However, the potential for an effective response by innate lymphocyte effectors such as NK and γδ T cells that recognize NKG2DL is limited by the drug’s concomitant lymphodepleting effects. We have previously shown that modification of γδ T cells with a methylguanine DNA methyltransferase (MGMT) transgene confers TMZ resistance via production of O6-alkylguanine DNA alkyltransferase (AGT) thereby enabling γδ T cell function in therapeutic concentrations of TMZ. In this study, we tested this strategy which we have termed Drug Resistant Immunotherapy (DRI) to examine whether combination therapy of TMZ and MGMT-modified γδ T cells could improve survival outcomes in four human/mouse xenograft models of primary and refractory GBM. Our results confirm that DRI leverages the innate response of γδ T cells to chemotherapy-induced stress associated antigen expression and achieves synergies that are significantly greater than either individual approach.

Abstract 1490: Dual chlorotoxin and methylguanine methyltransferase γδ-T cells for drug resistant immunotherapy of glioblastoma multiforme

Abstract While recent advances in immunotherapies have shown promise in extracranial tumors, Glioblastoma Multiforme (GBM) has remained impervious to these advances with a consistent median survival of 15 months. We have developed a novel approach to the treatment of primary GBM by combining simultaneous intracranial administration of gene-modified γδ T cells and standard temozolomide (TMZ) maintenance chemotherapy. These γδ T cells are transduced with O-6-Methylguanine-DNA Methyltransferase (MGMT), conveying resistance to alkylating chemotherapies, thereby allowing effector function at therapeutic concentrations of TMZ when tumor NKG2DL expression is significantly elevated. We modified γδ T cells with a lentivector construct expressing a chimeric antigen receptor (CAR) with a chlorotoxin binding domain (CLTX-CAR) to improve GBM targeting. CLTX is a small natural peptide derived from scorpion venom that specifically targets altered expression of matrix metalloproteinase 2 (MMP2) and chloride channel CLCN3 on GBM. MGMTp140k was co-expressed within the same CLTX-CAR vector to confer TMZ resistance to the CAR-T cells. The CLTX-CAR vectors contain a CD8α signal peptide, a mono or dual CLTX binding domain, a Myc-Tag peptide, a CD8α hinge domain, a CD28 transmembrane domain, and a costimulatory domain followed by a CD3ζ activation domain. We used P2A peptide to co-express MGMTp140k with the CAR. Initially, we demonstrated efficient transduction of the CLTX-CAR with a dual-CLTX construct as the binding domain (dCLTX-CAR) in Jurkat T cells. Cell surface localization of the dCLTX-CAR was verified by flow cytometry. When compared with the mono-CLTX-CARs (mCLTX-CARs) transduced Jurkat cells, the dCLTX-CARs demonstrated increased CD69 activation (MFI= 4873 vs 1078) and greater cytotoxicity against tumor cells in vitro. The modified T cells also demonstrated enhanced cytotoxicity against tumor cells under TMZ exposure in vitro. Since γδ T cells recognize and kill tumors through NKG2DL stress antigen recognition, we hypothesized that a CLTX-CAR without an activation signal may be sufficient to enhance recognition and cytotoxicity of γδ T cells to tumor cells and mitigate CAR-T activation-induced cell death. We then developed dCLTX-CAR constructs without the CD3ζ domain (dCLTX- Δz-CARs). As expected, the dCLTX-Δz-CAR lentivirus transduced Jurkat cells demonstrated enhanced cell-cell binding compared to the full dCLTX-CAR but no CD69 expression when co-cultured with GBM cells. Overall, we were able to generate dCLTX-CAR T cells with resistance to TMZ and showed improved activation and cytotoxicity against GBM cells under TMZ exposure. Our approach of combining the dCLTX-CAR and TMZ resistance will be further validated in animal model experiments and could be a potential candidate for clinical development for GBM. Citation Format: Lei Ding, Lawrence S. Lamb. Dual chlorotoxin and methylguanine methyltransferase γδ-T cells for drug resistant immunotherapy of glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1490.

Phase 1 trial of drug resistant immunotherapy: A first-in-class combination of MGMT-modified γδ t cells and temozolomide chemotherapy in newly diagnosed glioblastoma.

2057 Background: Temozolomide (TMZ) transiently upregulates NKG2D ligands targeted by innate immune effector cells. Lymphodepletion impairs this immune response, however, genetic modification of ex vivo expanded γδ T cells with an MGMT-expressing lentivector confers resistance to TMZ, allowing concurrent chemotherapy and γδ T cell infusion, thereby targeting the tumor when NKG2DL are maximally expressed. This Drug Resistant Immunotherapy (DRI) is currently being evaluated in a Phase 1 first in human study (NCT04165941) and interim safety and biologic correlative analysis are detailed here for the first dosing cohort. Methods: Adults with newly diagnosed, untreated glioblastoma (GBM), adequate organ function, and a KPS≥70% will be enrolled. Subjects undergo subtotal resection and placement of a Rickham reservoir followed 3-4 weeks by apheresis from which γδ T cells are expanded, transduced with an MGMT-expressing lentivector, harvested, and cryopreserved. Standard of care induction TMZ/radiation therapy is initiated followed by 6 cycles of maintenance TMZ. Intravenous TMZ (150mg/m2) and intracranial dosing of 1 x 107 γδ T cells occur on day 1 of each maintenance cycle. Daily oral TMZ 150mg/m2 follows for Days 2-5. Dose level 1 (DL1) subjects receive 1 fixed dose of γδ T cells and DL2 receive 3 doses administered on Day 1 of each of first 3 cycles of TMZ dependent on absence of dose limiting toxicity. Primary endpoint is safety; secondary endpoints include progression free and overall survival. Immunologic and genomic correlative analyses are being conducted at specific time points from peripheral blood and cerebral spinal fluid collected from the Rickham. Results: Six subjects (4 females, 2 males) have been enrolled in DL1. All subjects were IDH1-WT with 5 subjects MGMT unmethylated and 1 methylated. Of these, 1 generated inadequate gd T cells and 2 withdrew consent prior to DRI treatment. For the 3 that received DRI, treatment-related adverse events with maximum CTCAE Grade 3 occurred in 1 subject; UTI, dehydration, and thrombocytopenia. The most common Grade 1/2 events included: fever, leukopenia, nausea, and vomiting which were attributable to TMZ or radiotherapy. Circulating T cells remained below normal range throughout maintenance phase in 2/3 subjects. NK and gd T cell numbers remained within low normal range for 3/3 and 2/3 subjects, respectively. Serum Th1 (IFNg, IL-2, TNFa) and Th2 (IL4, IL5, IL-10) cytokines were within clinical range although TNFa remained elevated from the gdT cell infusion through day +30 in 2 subjects. Conclusions: Administration of MGMT-gene modified gdT cells and TMZ as DRI is feasible in lymphodepleted subjects during TMZ maintenance phase and sufficiently safe to warrant further investigation at additional doses. Clinical trial information: NCT04165941.

Phase I trial of drug resistant immunotherapy: A first-in-class combination of MGMT-modified γδ t cells and temozolomide chemotherapy in newly diagnosed glioblastoma multiforme.

TPS3150 Background: Temozolomide (TMZ) transiently upregulates GBM-specific stress-induced NKG2D ligands that are targeted by innate immune effector cells. Leveraging this effect is problematic, however, due to the lymphodepleting effects of TMZ.Genetic modification of ex vivo expanded and activated with an MGMT-expressing lentivector allows simultaneous chemotherapy and γδ T cell therapy that targets the tumor when NKG2DL are maximally expressed. We have termed this Drug Resistant Immunotherapy (DRI). Patient-derived xenograft mouse models of both primary and recurrent GBM treated with DRI have shown a significant survival advantage that were otherwise impervious to either cell therapy or TMZ. These preclinical findings and associated safety data provide the rationale to initiate a Phase I trial of DRI in primary GBM. Methods: This first in human study will evaluate the safety and optimal dosing frequency of the DRI with TMZ (NCT04165941).Eligibility criteria include the following: GBM eligible for resection, ≥18y, adequate organ and marrow function, and KPS≥70. Six to 12patients with newly diagnosed GBM are being enrolled in a 3 + 3 design into 1 of 2 fixed dose levels (DL) of DRI. Following tumor resection and immediately prior to induction chemo/radiotherapy, an apheresis product is collected and γδ T cells expanded in Zoledronic Acid (Novartis) and rhIL-12 (Miltenyi) and transduced with a P140K-MGMT lentivector (Miltenyi Lentigen, Gaithersburg, MD), harvested, and cryopreserved. At initiation of maintenance phase TMZ therapy, patients receive 150mg/m2 intravenous TMZ concurrently with intracranial injection of 1 x 107 γδ T cells (DL1) delivered through a Rickham reservoir previously inserted into the tumor cavity at resection. The patient then receives 4 daily doses of oral TMZ followed by 24d rest. Treatment cycles escalate from 1 to 3 (DL2) DRI doses following a safety observation period and absence of dose limiting toxicity. Maintenance TMZ treatment will continue for 6 cycles. Safety evaluations consist of routine laboratory analyses, clinical measurements (physical exams, vital signs), neurological function and evidence DRI γδ T cell related toxicity. Peripheral blood will be obtained for comprehensive immuno-phenotyping and T cell function analysis. Clinical benefit of DRI will be characterized by evaluating responses (CR, PR, SD and PD) and determining progression-free, median, and overall survival. As of February 2020, enrollment into DL 1 is ongoing. Clinical trial information: NCT04165941 .

ATIM-22. PHASE I TRIAL OF DRUG RESISTANT IMMUNOTHERAPY: A FIRST-IN-CLASS COMBINATION OF MGMT-MODIFIEDγδ T CELLS AND TEMOZOLOMIDE CHEMOTHERAPY IN NEWLY DIAGNOSED GLIOBLASTOMA MULTIFORME

Abstract INTRODUCTION Alkylating agents such as Temozolomide (TMZ) significantly upregulate stress-induced NKG2D ligands targeted by innate immune effector cells. Genetic modification of γδT cells with an MGMT-expressing lentivector abrogates TMZ-induced lymphodepletion allowing simultaneous chemotherapy and γδT cell therapy in a process we have termed Drug Resistant Immunotherapy (DRI) resulting in a significant improvement in long-term survival in patient-derived xenograft (PDXT) models of primary and recurrent GBM. We have now initiated a Phase I clinical trial of DRI in primary GBM (BB-IND-17928) to determine the safety of this approach. METHODS Effector γδT cells are manufactured from a fractional apheresis product obtained immediately prior to post-resection chemo/radiotherapy. Cultures are propagated in a Miltenyi Prodigy® bioreactor using media containing Zoledronic Acid and rhIL-12, transduced with a P140K-MGMT lentivector, maintained for up to 14 days, harvested, and cryopreserved. At the initiation of maintenance TMZ therapy, patients receive 150mg/m2 intravenous TMZ and 1 x 107 γδT cells delivered through a Rickham reservoir inserted into the tumor cavity at primary resection. Treatment frequency escalates from one to three 28-day cycles in a standard 3 x 3 design. RESULTS Closed system manufacturing in the Prodigy bioreactor produced 4.5 – 11.9 x 108 γδT cells (44–103 fold expansion) from the apheresis starting material (0.6–1.9% γδT cells) with vector copy numbers of 0.9 - 2.1 copies/cell and in vitro cytotoxicity against K562 44.3% - 77.9% at E:T 40:1. Products were successfully cryopreserved and retained in vivo potency against GBM PDXT in mice over single-agent TMZ (p= 0.0286). All cell products met sterility and identity criteria. CONCLUSIONS These findings show that genetically modified γδT cells can be manufactured to clinical scale under GMP compliance in an automated bioreactor and cryopreserved to produce multiple doses of functionally-competent γδT cells. Patient accrual will initiate in 2019.

γδT cells and the future of drug resistant immunotherapy

γδT cells and the future of drug resistant immunotherapy

Abstract 2262: Chemotherapy, checkpoint inhibition, and MGMT-modified adoptive gamma-delta (γδ) T cell-based therapy to treat post-resection, primary glioblastomas

Abstract Introduction: Recent evidence supports the crucial contribution of innate immunity to chemotherapy-based cancer treatments. Our previous in vitro modeling shows improved killing of glioblastoma (GBM) cell lines when γδ T cells are combined with alkylating agents such as Temozolomide (TMZ), which cause DNA damage but are also lymphodepleting. We now report findings from combined TMZ therapy with MGMT-modified γδ T cells (Drug Resistant Immunotherapy - DRI) in a xenograft (PDXT) mouse model of primary and recurrent GBM. Methods: Effectorγδ T cells were manufactured from apheresis product in media containing 5mM Zoledronic Acid and 50U/mL rhIL-12. Cultures were transduced with a P140K-MGMT lentivector, maintained for up to 14 days, harvested, and evaluated in vitro for PDXT lysis +PD-1, CTLA-4 and PD-L1 by flow cytometry. Primary (P) and recurrent TMZ-resistant (T) GBM were modeled orthotopically using classical JX12P and mesenchymal JX59P intracranial (IC) xenografts versus JX12T, JX22T and JX59T. After placement of IC xenografts, mice received concurrent intraperitoneal TMZ 60mg/kg and 1 x 106IC DRI γδ T cells on post-tumor days 6, 8,13 and 15. Control mice received either γδ T cells, TMZ, or no therapy. Survival was assessed by Kaplan-Meier analysis.NKG2DL and PD-L1 expression was assessed on FFPE sections of tumor during therapy and at termination. Results: DRI-treated JX12P mice showed significant improvement in median survival (MS) over TMZ alone (p=0.0001) with 80% tumor-free survivors. DRI also improved survival for JX59P-bearing mice with 60% survivors as compared to 30% for mice receiving TMZ alone (p=0.044). TMZ reduced tumor growth in primary but not TMZ-resistant PDXT-bearing mice while γδ T cells alone had no survival benefit over untreated controls for any model. For JX12T, DRI improved MS from 27 to 38 days (+41%) over TMZ alone (p=0.017). JX22T and JX59T tumors were resistant to DRI (p=0.0966 and 0.1390). DRI γδ T cells upregulated PD-1 and CTLA-4 over baseline and showed improved function against TMZ-resistant xenolines after blockade of PD-1 and/or CTLA-4 by as much as 200%. TMZ induced upregulation of NKG2DL (MIC-A/B, ULBP-4), and PD-L1 on both primary and TMZ-resistant PDXT following TMZ injection and for several days thereafter. Conclusions: Primary GBM PDXT are significantly more sensitive to DRI than TMZ-resistant GBM suggesting that DRI would be more effective if administered as adjuvant therapy in the TMZ-maintenance phase following primary resection and chemo/radiotherapy. Additionally, TMZ increases GBM immunogenicity by upregulating NKG2DL expression but also PD-L1. Checkpoint blockade improves γδ T cell function. These findings show the potential to augment the effect of DRI and improve immune effectiveness against more resistant tumors. Citation Format: Sailesh Pillai, George Y. Gillespie, Louis B. Nabors, Samantha Langford, Catherine P. Langford, Lawrence S. Lamb. Chemotherapy, checkpoint inhibition, and MGMT-modified adoptive gamma-delta (γδ) T cell-based therapy to treat post-resection, primary glioblastomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2262.

Abstract 1638: Improved outcomes with drug-resistant immunotherapy in a human xenograft model of glioblastoma multiforme

Abstract INTRODUCTION: Conventional treatment strategies for high-grade gliomas have been uniformly dismal. We have previously shown that both primary and Temozolomide (TMZ)-resistant glioblastoma (GBM) cell lines upregulate stress-associated NKG2D ligands (NKG2DL) during the first several hours following exposure to TMZ, thereby creating an opportunity for NKG2DL-directed cell therapy, particularly γδ T cells that directly recognize these stress-associated antigens. Using a human/mouse patient-derived tumor xenograft (PDTX) model, we combined TMZ chemotherapy and TMZ-resistant ex vivo expanded/activated γδ T cells as Drug Resistant Immunotherapy (DRI). Drug resistance in this example is conferred by O-6-methylguanine-DNA-methyltransferase (MGMT) gene transfer, thereby enabling cytotoxic lymphocyte function in a chemotherapy-rich environment when the tumor is maximally stressed. METHODS: A total of five GBM PDTX were examined, three parent (X12P, X22P, X59P) and two TMZ-resistant (X12T, X22T). Tumor NKG2DL expression and cytotoxicity of DRI were assessed using flow cytometry with cultured human astrocytes as controls. Intracranial (IC) glioma xenografts were established using either an unmodified (P) or a TMZ resistant clone (T) of human GBM explants passaged exclusively in immunodeficient mice. Tumor-bearing mice received intraperitoneal 60mg/kg TMZ on days 6, 8,13, and 15 and received IC injection of 1.5 x 106 DRI 4 hours following TMZ injection. Control mice received DRI T cells alone, TMZ alone or no therapy. Survival was assessed using Kaplan-Meier analysis. RESULTS: All xenografts were found to constitutionally express NKG2DL which were upregulated upon exposure to TMZ. DRI T cells were cytotoxic to all tumors in vitro, showing 65%-80% specific lysis at an E:T ratio of 20:1 with no evidence of toxicity against cultured human astrocytes. Median survival (MS) for all groups of untreated mice was approximately 25 days, and γδ T cell therapy alone did not improve survival in the absence of TMZ. For the parent tumors, TMZ therapy significantly improved MS over untreated controls for both X12P and X59P (29 vs. 59 days and 20 vs 51 days respectively, p =0.0001) and eliminated tumors in X22P. DRI γδ T cells + TMZ significantly increased median survival additionally over TMZ alone with 80% of animals in both X12P and X59P surviving long-term (p=0.0001 and 0.05 respectively). DRI had no clear effect over TMZ for X22T (p=0.46), however, for X12T, DRI + TMZ significantly increased median survival from 22 to 38 days (72.7%) over TMZ alone (p = 0.04). CONCLUSIONS: Combined TMZ chemotherapy and drug-resistance modified γδ T cell therapy can produce a significant increase in time to progression and improvement in median and overall survival for both primary and TMZ-resistant GBM using a strategy is readily adaptable to the clinical setting. Citation Format: Samantha B. Langford, Harold T. Spencer, Anindya Dasgupta, George Y. Gillespie, Kathryn Sutton, Larisa Pereboeva, Lawrence S. Lamb. Improved outcomes with drug-resistant immunotherapy in a human xenograft model of glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1638. doi:10.1158/1538-7445.AM2017-1638

287. Improved Outcomes Following Drug-Resistant Immunotherapy in a Human Xenograft Model of Temozolomide-Resistant Glioblastoma Multiforme

INTRODUCTION: Conventional treatment strategies for Temozolomide (TMZ)-resistant high-grade gliomas have been uniformly dismal. We have shown that TMZ-resistant tumors up-regulate stress-associated NKG2D ligands (NKG2DL) during the first hours after exposure to TMZ, thereby creating an opportunity for NKG2DL-directed cell therapies. We report improved survival using a combination of TMZ chemotherapy and gene modified TMZ-resistant γΔ T cells which we term Drug Resistant Immunotherapy (DRI). TMZ resistance is conferred by lentivector transfer of methylguanine methyltransferase (MGMT) enabling cytotoxic lymphocyte function during high-dose chemotherapy.METHODS: Tumor NKG2DL expression and DRI cytotoxicity were assessed by flow cytometry. Intracranial (IC) glioma xenografts were established on Day 0 with either unmodified (P) or a TMZ resistant clone (T) of human primary GBM-X12 passaged exclusively in immunodeficient mice. Tumor-bearing mice were treated with intraperitoneal TMZ 60mg/kg on days 6, 8,13, and 15 followed by IC injection of 1.5 × 106 DRI 4h after each TMZ dose × 2 weeks. Control mice received non-modified cells, TMZ, or no therapy. Survival was assessed using Kaplan-Meier analysis.RESULTS: Both GBM-X12P and X12T express NKG2DL MIC-A, MIC-B, and ULBP-4. DRI cells killed both tumors in vitro at approximately 80% at an effector: target ratio of 20:1 with no toxicity against cultured human astrocytes. Cell therapy alone did not improve survival beyond that of untreated mice. For the parent tumor X12P (Figure 1), TMZ therapy significantly improved median survival over untreated controls (29 vs. 59 days, p < 0.0001), and addition of DRI increased median survival to 120 days with 80% long term survivors. For the TMZ-resistant X12T (Figure 2), TMZ alone did not improve survival over untreated controls (22 vs. 27 days p = 0.4827). Addition of DRI significantly increased median survival from 22 days to 38 days (p = 0.0004) with 10% of animals surviving to day +119. View Large Image | Download PowerPoint Slide View Large Image | Download PowerPoint SlideCONCLUSIONS: This is the first report of combined TMZ chemotherapy and drug-resistance modified γΔ T cell therapy resulting in a significant increase in time to progression and improvement in median and overall survival in immunodeficient mice bearing otherwise impervious human primary xenograft tumors using a strategy is readily adaptable to the clinical setting

Improved Outcomes Following Drug-Resistant Immunotherapy in a Hunan Xenograft Model of Temozolomide-Resistant Glioblastoma Multiforme

Improved Outcomes Following Drug-Resistant Immunotherapy in a Hunan Xenograft Model of Temozolomide-Resistant Glioblastoma Multiforme

Treatment of a Solid Tumor Using Engineered Drug-Resistant Immunocompetent Cells and Cytotoxic Chemotherapy

Multimodal therapy approaches, such as combining chemotherapy agents with cellular immunotherapy, suffers from potential drug-mediated toxicity to immune effector cells. Overcoming such toxic effects of anticancer cellular products is a potential critical barrier to the development of combined therapeutic approaches. We are evaluating an anticancer strategy that focuses on overcoming such a barrier by genetically engineering drug-resistant variants of immunocompetent cells, thereby allowing for the coadministration of cellular therapy with cytotoxic chemotherapy, a method we refer to as drug-resistant immunotherapy (DRI). The strategy relies on the use of cDNA sequences that confer drug resistance and recombinant lentiviral vectors to transfer nucleic acid sequences into immunocompetent cells. In the present study, we evaluated a DRI-based strategy that incorporates the immunocompetent cell line NK-92, which has intrinsic antitumor properties, genetically engineered to be resistant to both temozolomide and trimetrexate. These immune effector cells efficiently lysed neuroblastoma cell lines, which we show are also sensitive to both chemotherapy agents. The antitumor efficacy of the DRI strategy was demonstrated in vivo, whereby neuroblastoma-bearing NOD/SCID/γ-chain knockout (NSG) mice treated with dual drug-resistant NK-92 cell therapy followed by dual cytotoxic chemotherapy showed tumor regression and significantly enhanced survival compared with animals receiving either nonengineered cell-based therapy and chemotherapy, immunotherapy alone, or chemotherapy alone. These data show there is a benefit to using drug-resistant cellular therapy when combined with cytotoxic chemotherapy approaches.

549. A Drug Resistant Immunotherapy Strategy Allowing for the Combined Administration of Chemo- and Immunotherapy

549. A Drug Resistant Immunotherapy Strategy Allowing for the Combined Administration of Chemo- and Immunotherapy

253. Regression of Large Solid Tumors Using Engineered Drug Resistant Immunocompetent Cells and Selective Chemotherapy

Chemotherapy remains a central approach for the treatment of cancer but is limited by severe toxic side effects, particularly on the hematopoietic system. Various immunotherapy strategies are less toxic, such as tumor vaccines and cytokine therapy, and are emerging areas of cancer treatment. Although promising, these methods have limited effectiveness on established tumors. Combining chemotherapy agents with immunotherapy has proven difficult because development of an immune response can be significantly impaired in a chemotherapy-induced immunosuppressive environment. Therefore, we are investigating strategies that will allow cytotoxic chemotherapy and immunotherapy treatments to be used concurrently. Our aim is to generate drug resistant immunocompetent T-lymphocytes using retroviral gene transfer of cDNA sequences that confer drug resistance. Our drug resistant immunotherapy approach entails generating tumor-specific genetically engineered immunocompetent cells, such as T and NK cells, by transducing hematopoietic stem cells (HSC) with a retroviral vector encoding the L22Y variant of dihydrofolate reductase (L22YDHFR). This construct confers >10-fold resistance to the cytotoxic agent trimetrexate (TMTX) compared to non-modified hematopoietic cells. Drug resistant lymphocytes are generated by transplanting drug resistant bone marrow into irradiated recipient mice. Upon engraftment immunocompetent cells encoding the drug resistant marker are generated. To examine the effectiveness of drug resistance immunotherapy, C3H mice reconstituted with drug resistant bone marrow were inoculated with AG104 sarcoma cells and treated with either an agonistic anti-4-1BB antibody alone, TMTX alone, or the combination. Approximately 60% of animals transplanted with gene-modified cells and treated with anti-4-1BB, which induces CD8+ and NK anti-tumor responses, achieved complete tumor regression within 30 days. Although TMTX treatment alone regressed tumor size transiently, there was no effect on tumor clearance. However, animals treated with the combination of TMTX and anti-4-1BB antibody completely eliminated tumors in 100% of mice. Also important, compared to animals transplanted with non-modified marrow, hematopoiesis of animals transplanted with drug resistant marrow is protected against chemotherapy-induced toxicities. These results show that drug resistant immunocompetent cells can be conserved after a chemotherapy challenge and appear to retain their anti-tumor activity. This study supports our hypothesis that, in the context of drug resistant immunocompentent cells, chemotherapy can be used to control tumor growth and provide the critical time needed for the establishment of an effective anti-tumor immune response.