Long-term Antitumor Activity Research Articles

Exploring the multifaceted antitumor activity of axitinib in lung carcinoids.

Lung carcinoids (LCs) are a type of neuroendocrine tumor (NET) that originate in the bronchopulmonary tract. LCs account for 20-25% of all NETs and approximately 1-2% of lung cancers. Given the highly vascularized nature of NETs and their tendency to overexpress vascular growth factor receptors (VEGFR), inhibiting angiogenesis appears as a potential therapeutic target in slowing down tumor growth and spread. This study evaluated the long-term antitumor activity and related mechanisms of axitinib (AXI), a VEGFR-targeting drug, in LC cell lines. Three LC cell lines (NCI-H727, UMC-11 and NCI-H835) were incubated with their respective EC50 AXI concentrations for 6 days. At the end of the incubation, FACS experiments and Western blot analyses were performed to examine changes in the cell cycle and the activation of apoptosis. Microscopy analyses were added to describe the mechanisms of senescence and mitotic catastrophe when present. The primary effect of AXI on LC cell lines is to arrest tumor growth through an indirect DNA damage. Notably, AXI triggers this response in diverse manners among the cell lines, such as inducing senescence or mitotic catastrophe. The drug seems to lose its efficacy when the DNA damage is mitigated, as observed in NCI-H835 cells. The ability of AXI to affect cell viability and proliferation in LC tumor cells highlights its potential as a therapeutic agent. The role of DNA damage and the consequent activation of senescence seem to be a prerequisite for AXI to exert its function.

T lymphocytes expressing the switchable chimeric Fc receptor CD64 exhibit augmented persistence and antitumor activity

Chimeric antigen receptor (CAR) Tcell therapy lacks persistent efficacy with "on-target, off-tumor" toxicities for treating solid tumors. Thus, an antibody-guided switchable CAR vector, the chimeric Fc receptor CD64 (CFR64), composed of a CD64 extracellular domain, is designed. Tcells expressing CFR64 exert more robust cytotoxicity against cancer cells than CFR Tcells with high-affinity CD16 variant (CD16v) or CD32A as their extracellular domains. CFR64 Tcells also exhibit better long-term cytotoxicity and resistance to Tcell exhaustion compared with conventional CAR Tcells. With trastuzumab, the immunological synapse (IS) established by CFR64 is more stable with lower intensity induction of downstream signaling than anti-HER2 CAR Tcells. Moreover, CFR64 Tcells exhibit fused mitochondria in response to stimulation, while CARH2 Tcells contain predominantly punctate mitochondria. These results show that CFR64 Tcells may serve as a controllable engineered Tcell therapy with prolonged persistence and long-term antitumor activity.

TGF-β blocking combined with photothermal therapy promote tumor targeted migration and long-term antitumor activity of CAR-T cells.

TGF-β is widely existed in tumor microenvironment, taking part in tumorigenesis process including angiogenesis, cancer associated fibroblast (CAF) proliferation, and immunosuppression. It inhibited the activation, proliferation, migration and differentiation of T cells, in which way caused a limited therapeutic effects of chimeric antigen receptor T (CAR-T) towards solid tumor such as lymphoma. To targeted block TGF-β at tumor site, we take advantages of nano-techniques to deliver TGF-β inhibitors LY2157299 (LY) towards the tumor sites, in order to help achieve a improved and long-term functions of CAR-T towards lymphoma. Based on amphipathic hydroxyethyl starch-polycaprolactone (HES-PCL), LY and photosensitizer indocyanine green (ICG) were co-loaded in HES-PCL to achieve LY/ICG@HES-PCL nanoparticle. The enhanced function of CAR-T benefited from LY/ICG@HES-PCL were verified through lymphoma Raji cells in vitro and Nod scid gamma mice engrafted with the Raji cells in vivo. LY was targeted transported to tumor site and accelerated release by mild ICG photothermal. Chemokines CXCL9/10/11​at the tumor site relevant to CAR-T migration and chemokines receptor CXCR3 of CAR-T could be up-regulated by LY, thus facilitated the enhanced accumulation of CAR-T at lymphoma site. T effector memory cells differentiation could also be accelerated by LY/ICG@HES-PCL. Combined therapy of LY/ICG@HES-PCL and CAR-T achieved 2.4 times higher antitumor activity and 2.7 times higher relapse inhibiting rates than CAR-T alone within 15 days and 11 days, respectively. The results suggested that LY/ICG@HES-PCL facilitated the enhanced therapeutic index of CAR-T cells towards lymphoma simply and safely, it may be further potentiated applied for other solid tumors.

Overcoming Heterogeneity in AML with a Novel or-Gated Hitcar-1XX Platform

Target heterogeneity limits therapeutic success of conventional CAR therapies in B cell and other malignancies. Therefore, novel concepts for efficient combinatorial targeting are urgently needed. Several OR-gated targeting approaches based on CARs have been described and include dual CARs (T cells co-expressing two CARs), tandem CARs (T cells expressing a single bi-specific CAR) and pooled single CAR-T cells (T cells expressing a single CAR). Clinical efficacy of such OR-gated CAR therapies is still under ongoing investigation, and emerging pre-clinical data suggests that optimization of OR-gated approaches requires fine-tuning of combinatorial CAR signaling to balance effector and memory functions, avoiding premature differentiation and eventual exhaustion. Here, we present a novel OR-gated platform based on co-expression of a CAR and a HIT (HLA-Independent TCR), which we previously described to afford T cells with increased antigen sensitivity (Mansilla-Soto et al., Nature Medicine 2022). Since HIT receptors do not incorporate engineered co-stimulatory domains like conventional CARs, we hypothesized that T cells co-expressing CAR and HIT display limited differentiation in comparison to dual CAR T cells, thus resulting in improved long-term anti-tumor activity. We termed this novel OR-gated therapy HITCAR and evaluated it in the setting of AML which stands out with a high degree of inter- and intra-individual phenotypic heterogeneity. We selected two target antigens which we had previously demonstrated to have a favorable and non-overlapping expression profile in normal hematopoietic cells and other tissues (Perna et al., Cancer Cell 2017). We selected a lower-density target for the HIT, using its increased sensitivity to avoid antigen-low escape, and selected a higher-density target for the CAR. We utilized an NSG xenograft model for target heterogeneity, based on co-engraftment of engineered MOLM13 AML cell line populations endogenously expressing either HIT target alone, CAR target alone, or both. To identify an optimal HITCAR design, we treated target-heterogeneous MOLM13 AML-bearing mice with HITCAR T cells containing different CAR architectures and found that only the 28z1XX CAR allowed for complete AML remission and long-term survival in conjunction with the HIT. All other CAR architectures failed to control AML long-term, highlighting the importance for fine-tuned CAR signaling in combination with HIT. We then selected the superior HITCAR-1XX and compared it to dual CARs containing BBz and 28z1XX architectures. In our NSG AML xenograft heterogeneity model, the HITCAR-1XX induced complete AML remission while the alternative dual CARs failed to control AML. T cell phenotyping demonstrated a less differentiated T cell phenotype over time with HITCAR-1XX as compared to the alternative dual CARs, supporting our hypothesis. Further validation of our HITCAR-1XX platform in clinically relevant AML PDX models is ongoing. Overall, we established a novel OR-gated HITCAR T cell platform based on combinatorial expression of HIT and CAR which can be applied to overcome target heterogeneity in various malignancies. In the setting of AML, we compared different HITCAR configurations and identified HITCAR-1XX as most promising. Furthermore, we demonstrated superiority of HITCAR-1XX over alternative dual CAR designs, highlighting its high potential for clinical translation.

Abstract P1-17-11: Updated data from AMEERA-1: Phase 1/2 study of amcenestrant (SAR439859), an oral selective estrogen receptor (ER) degrader (SERD), combined with palbociclib in postmenopausal women with ER+/HER2- advanced breast cancer

Abstract Background: In Arm 2 of the ongoing AMEERA-1 trial (NCT03284957), amcenestrant, an optimized oral SERD combined with the CDK4/6 inhibitor (CDK4/6i) palbociclib demonstrated favorable safety and encouraging antitumor activity among patients with endocrine-resistant ER+/HER2− advanced breast cancer in dose escalation (Part C) and dose expansion (Part D) (Chandarlapaty et al., ASCO 2021; abstract 1058). Here we report an update of safety, antitumor activity data, and progression-free survival (PFS), of amcenestrant 200 mg in combination with palbociclib. Analysis of genomic data, including modulation over time and correlation with clinical outcome, will also be presented. Methods: The trial enrolled postmenopausal women with ER+/HER2- locally-advanced or metastatic breast cancer with disease progression while on ≥ 6 months of prior endocrine therapy (ET) in the advanced setting, or who relapsed on adjuvant ET after the first 2 years of treatment or within 12 months of completing adjuvant ET. Prior chemotherapy (≤ 1) was allowed as well as prior CDK4/6i-based therapy (≤ 1, in Part C only). In this pooled analysis (N = 39), patients in Parts C + D received amcenestrant 200 mg once daily + palbociclib 125 mg (21 days on/7 days off), administered in 28-day cycles. Safety in the pooled analysis was reported using methods previously described (Chandarlapaty et al., ASCO 2021; abstract 1058). Data from investigator-assessed, response-evaluable patients in the pooled analysis without prior exposure to targeted therapies (N = 34) were used to evaluate antitumor activity per RECIST v1.1, including the objective response rate (ORR), clinical benefit rate (CBR), and PFS. Results: At a data cutoff of May 30, 2021, in the pooled analysis (N = 39), the median (range) duration of treatment exposure was 44.3 weeks (1-80). Of 39 patients, 24 (61.5%) had initiated at least 10 cycles (40 weeks) of treatment, with 20/39 (51.3%) still receiving ongoing treatment. Among the 34/39 (87.2%) patients in the response-evaluable population, median follow-up was 48.3 weeks with a PFS probability of being event free at 24 weeks of 78.2% (95% CI: 59.6%; 89.0%). Median PFS is not yet mature, with 14/34 (41.2%) patients having had a PFS event (all were progression events and no deaths occurred). The ORR was 11/34 (32.4%; all partial responses). Clinical benefit at 24 weeks was seen in 25/34 (CBR = 73.5%) patients. Median (range) time to first response was 16.3 weeks (8-32). Amcenestrant treatment-related adverse events (TRAEs) and palbociclib TRAEs, respectively, occurred in 27/39 (69.2%) and 35/39 (89.7%) patients for all grade events and in 5/39 (12.8%) and 18/39 (46.2%) patients for Grade ≥ 3 events. Non-hematological amcenestrant and palbociclib TRAEs are reported in Table 1. Neutrophil count decrease based on hematological laboratory abnormalities was observed in the majority of patients (94.9%; with Grade ≥ 3 in 56.4%). Conclusions: Among postmenopausal women with endocrine-resistant ER+/HER2- advanced breast cancer, amcenestrant 200 mg in combination with the approved dose of palbociclib continues to demonstrate encouraging long-term antitumor activity, sustained clinical benefit, and a favorable safety profile consistent with previous results. Funding: Sanofi. Table 1.Non-hematological amcenestrant and palbociclib TRAEs occurring in > 10% of patientsPooled Analysis. Amcenestrant 200 mg + Palbociclib. (Parts C + D; N = 39)Amcenestrant Non-hematological TRAEs, n (%)All GradesGrade ≥ 3–Fatigue7 (17.9)0–Nausea7 (17.9)0–Arthralgia4 (10.3)0–Asthenia4 (10.3)0–Hot flush4 (10.3)0Palbociclib Non-hematological TRAEs, n (%)All GradesGrade ≥ 3–Fatigue12 (30.8)0–Nausea10 (25.6)0–Asthenia4 (10.3)0–Dysgeusia4 (10.3)0–Stomatitis4 (10.3)0 Citation Format: Sarat Chandarlapaty, Hannah M Linden, Patrick Neven, Katarina Petrakova, Aditya Bardia, Peter Kabos, Sofia Braga, Valentina Boni, Alice Gosselin, Marina Celanovic, Patrick Cohen, Gautier Paux, Vasiliki Pelekanou, Nils Ternès, Joon Sang Lee, Mario Campone. Updated data from AMEERA-1: Phase 1/2 study of amcenestrant (SAR439859), an oral selective estrogen receptor (ER) degrader (SERD), combined with palbociclib in postmenopausal women with ER+/HER2- advanced breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-17-11.

Results from the Phase I Clinical Studies Evaluating Cyad-01, a First-Generation NKG2D CAR T-Cell Product in Relapsed or Refractory Acute Myeloid Leukemia and Myelodysplastic Syndrome Patients

Background CYAD-01 is a T-cell product engineered to express a chimeric antigen receptor (CAR) based on the NKG2D receptor (NKG2D CAR) which binds 8 ligands (MICA/B, ULBP1-6) over-expressed by a large variety of malignancies, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). The phase I THINK study (NCT03018405) evaluated the safety and clinical activity of multiple injections of CYAD-01 infused every 2 weeks, without preconditioning chemotherapy, in 13 relapsed/refractory (r/r) AML and MDS patients. While an encouraging objective response rate according to ELN2017 (AML) or revised IPSS (MDS) and reduction in bone marrow blasts were seen with good safety profile, the responses were short-lived (≤ 3 months - see ASH 2019, poster 3826). To enhance CAR T-cell persistence, we evaluated a weekly dose schedule without preconditioning (THINK study) or the addition of cyclophosphamide and fludarabine (CyFlu) as a preconditioning regimen prior to CAR T-cell infusion (phase I DEPLETHINK study, NCT03466320). Aim To further increase persistence and potency of the T-cell product, optimization of the previously used mAb manufacturing process was performed by shortening the duration of production along with modification of PI3K inhibitor. This optimized manufacturing process (termed "OptimAb") aimed to generate CYAD-01 cells with a higher frequency of early memory T-cells with high cytokine secretion upon activation, as compared to the original "mAb" process. Results As compared to the previous mAb manufacturing process, the OptimAb manufacturing process generates a product that secretes higher levels of IFN-γ upon co-culture with tumor cells and contains a higher frequency of CD62L+ T-cells in vitro, characteristic of an early memory phenotype. In an in vivo aggressive AML (THP-1) model, CYAD-01 OptimAb displayed a strong improvement in long-term anti-tumor activity as compared to the CYAD-01 mAb at the same dose chosen to have a minimal anti-tumor activity (stress-test dose, see figure). Based on these results, both THINK and DEPLETHINK clinical studies were amended to evaluate the OptimAb process. As of August 2020, 5 patients have been treated with multiple infusions of the OptimAb CYAD-01 as standalone treatment at the dose of 3x108 cells/infusion in the small expansion segment of the THINK study. 7 patients were treated with a single infusion of OptimAb CYAD-01 administered after a CyFlu preconditioning in the dose-escalation segment at the doses of 3x108 cells/infusion or 1x109 cells/infusion in the DEPLETHINK study. To date, the results demonstrate the safety and tolerability for CYAD-01 OptimAb with or without a prior lymphodepletion in patients with r/r AML and MDS. Preliminary data of the clinical and pharmacokinetics evaluation of CYAD-01 manufactured with the improved OptimAb process, as compared with the mAb process at the same dose, in two Phase I studies will be provided at the time of presentation. Conclusion/summary The autologous CYAD-01, a first generation NKG2D CAR T-cell product is currently investigated in r/r AML/MDS patients, a difficult to target disease due in part to the absence of truly AML-specific surface antigens, its rapid clinical progression and the absence of disease control by the CyFLu preconditioning. CYAD-01 manufactured using an optimized process, OptimAb, aims to improve CAR T-cell persistence and clinical responses. The data analysis of the same CAR-T product with different manufacturing processes, with or without preconditioning chemotherapy, will provide the medical community with clinical and scientific insights to guide the future of this therapeutic modality. Figure Disclosures Sallman: Agios, Bristol Myers Squibb, Celyad Oncology, Incyte, Intellia Therapeutics, Kite Pharma, Novartis, Syndax: Consultancy; Celgene, Jazz Pharma: Research Funding. Al-Homsi:Celyad: Membership on an entity's Board of Directors or advisory committees. Pollyea:Janssen: Consultancy; 47: Consultancy, Research Funding; Amgen: Consultancy; Genentech: Consultancy; Novartis: Consultancy; Karyopharm: Consultancy; Syndax: Consultancy; Syros: Consultancy; Abbvie: Consultancy, Research Funding; Daiichi Sankyo: Consultancy; Takeda: Consultancy; Pfizer: Consultancy; Celgene/BMS: Consultancy; Agios: Consultancy; Glycomimetics: Other. Wang:Abbvie: Consultancy; Pfizer: Speakers Bureau; Genentech: Consultancy; Stemline: Speakers Bureau; PTC Therapeutics: Consultancy; Macrogenics: Consultancy; Astellas: Consultancy; Bristol Meyers Squibb (Celgene): Consultancy; Jazz Pharmaceuticals: Consultancy. Demoulin:Celyad Oncology: Current Employment. Sotiropoulou:Celyad Oncology: Current Employment. Alcantar-Orozco:Celyad Oncology: Current Employment. Breman:Celyad Oncology: Current Employment. Dheur:Celyad Oncology: Current Employment. Braun:Celyad Oncology: Current Employment. Lonez:Celyad Oncology: Current Employment. Gilham:Celyad Oncology: Current Employment. Flament:Celyad Oncology: Current Employment. Lehmann:Celyad Oncology: Current Employment.

Preclinical Evaluation of ALLO-605, an Allogeneic BCMA Turbocar TTM Cell Therapy for the Treatment of Multiple Myeloma

Chimeric antigen receptor (CAR) T cells have demonstrated unprecedented efficacy in heavily pretreated relapsed and/or refractory multiple myeloma (MM) patients but may require further engineering to achieve their greatest potential. Enhancing cell expansion and persistence by providing cytokine support has been shown to improve the long-term antitumor activity of adoptively transferred CAR T cells in preclinical models. Combining CAR T cells with systemically-administered cytokines or cytokine mimetics can however result in toxicities and adverse events. Alternatively, cytokine signaling can be provided in a constitutive, CAR T cell-intrinsic fashion, by exogenous expression of a Constitutively Active Chimeric Cytokine Receptor (CACCR). CACCRs can be engineered by combining a membrane-tethered dimerization and JAK-binding domain derived from the thrombopoietin receptor (TpoR) fused to an intracellular signaling domain derived from a cytokine receptor. We investigated the impact of CACCR expression on the phenotype, functionality, persistence, and safety profile of allogeneic CAR T cells targeting BCMA to produce a second generation allogeneic BCMA "TurboCAR T™" candidate (ALLO-605). BCMA CAR T cells were generated from healthy donor T cells by lentiviral transduction with a CAR construct followed by genetic inactivation of the TRAC and CD52 loci using TALEN® gene editing. Allogeneic BCMA TurboCAR™ T cells, engineered for stoichiometric expression of the CAR and a CACCR via a self-cleaving peptide, were produced similarly. Constitutive expression of the CACCR during manufacturing had no negative effects on CAR T cell phenotype or yield and resulted in a product with over 60% stem cell memory/central memory T cells. In vitro, BCMA TurboCAR T™ cells showed enhanced cytokine secretion, polyfunctionality and improved serial killing activity. In a disseminated mouse model of multiple myeloma, BCMA TurboCAR T™ cells exhibited at least a 2-fold increase in peak expansion and enhanced survival and persistence compared to BCMA CAR T cells, resulting in prolonged antitumor responses and delaying relapses. Despite this enhanced persistence, we found that exposure to target cells was absolutely required for the expansion and long-term activity of BCMA TurboCAR T™ cells and no evidence of target- and cytokine-independent proliferation was observed. Since adoptive cell therapies have the potential to elicit toxicities in some patients, two alternative approaches to modulate BCMA TurboCAR T™ cell activity were investigated. First, we tested the ability of a CD20-based off-switch incorporated within the CAR to sensitize cells to rituximab and found effective depletion of BCMA TurboCAR T™ cells by complement or effector cells in vitro and in vivo in the presence of the antibody. In addition, we confirmed rapid inhibition of BCMA TurboCAR T™ cells by the protein tyrosine kinase inhibitor dasatinib, which has been shown to interfere with LCK activity. The prolonged persistence and antitumor responses seen in preclinical models along with a favorable safety profile of BCMA TurboCAR™ T cells support clinical investigation of ALLO-605 in relapsed or refractory multiple myeloma. Disclosures Sommer: Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Lin:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Sutton:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Bentley:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Nguyen:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Yoon:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Au:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Vargas-Inchaustegui:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Cheng:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Van Blarcom:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Panowski:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Sasu:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company.

Allogeneic CAR T-cells resistant to both T- and NK-cell cytotoxicity

Background & Aim The last years have seen the adoptive transfer of engineered autologous T-cells making great strides in the development of new treatments against cancer. The use of third-party donor-derived T-cells represents an attractive alternative to generate CAR T-cells readily accessible to patients. These off-the-shelf cells derived from a possibly non-HLA-matched donor nevertheless carry the risk of graft-versus-host-disease (GvHD) through the expression of their endogenous T-cell receptors (TCRs). The recent advances in precise genome editing using designer nucleases allowed to mitigate this issue as demonstrated by the molecular remissions observed in patients after infusion of universal TALEN® multiplex gene-edited CAR T-cells. In particular, these universal CAR T-cells were engineered to reduce the risk of graft versus host disease (GvHD) by TALEN® inactivation of TCRαβ. The clinical outcome of CAR T-cell therapies is intimately linked to the ability of effector cells to engraft and proliferate in order to eradicate tumor cells within patients. Although the transient activity of off-the-shelf CAR T-cells represents an important safety feature, the possibility to extend their therapeutic window may be desirable in particular disease indications and after lymphodepleting regimens where the patient's immune system has been restored. Thus, we are developing novel approaches to render these cells less visible to both host T- and NK-cells. Methods, Results & Conclusion The single genetic disruption of the beta-2 microglobulin (B2M) gene, a required component of all MHC class I molecules, promotes resistance to host CD8+ T-cell attack but may trigger NK-cell activation, leading to the rejection of the edited T-cells lacking MHC-I. We therefore further engineered these B2M negative cells via a TALEN®-mediated and B2M gene-specific targeted integration matrix to express inhibitors of NK cytotoxicity. We have identified an NK-cell inhibitor that successfully blocks the so-called NK-cell “missing self-response” in vitro and in vivo. Altogether, the precise TALEN®-mediated inactivation of B2M coupled with insertion of an NK-cell inhibitor enabled the generation of allogeneic CAR T-cells, resistant to host T- and NK-cells, with improved persistence and long-term anti-tumor activity.

Abstract 1547: DNA-encoded bispecific T-cell engagers and antibodies present long-term antitumor activity

Abstract Specific antibody therapy, including monoclonal antibodies and bispecific T cell engagers (BiTEs), are important new tools for cancer immunotherapy. However, these approaches are slow to develop and may be limited in their production thus restricting the patients who can access these treatments. BiTEs exhibit a particularly short half-life and difficult production. The development of an approach allowing simplified development, delivery and in vivo production would be an important advance. Here we describe development of a designed synthetic DNA plasmid, which we optimized to permit high expression of an anti-HER2 antibody (HER2DMAb) and delivered it into animals through adaptive electroporation. HER2DMAb was efficiently expressed in vitro and in vivo, reaching levels of 50ug/ml in mouse sera. Mechanistically, HER2DMAb blocked HER2 signaling and induced antibody-dependent cytotoxicity. HER2DMAb delayed tumor progression for HER2-expressing ovarian and breast cancer models. We next used the HER2DMAb scFv portion to engineer a DNA-encoded BiTE. This HER2DBiTE was expressed in vivo for approximately 4 months after a single administration. The HER2DBiTE was highly cytolytic and delayed cancer progression in mice. These studies illustrate a novel approach to generate DBiTEs in vivo which represent promising immunotherapies for HER2+ tumors including ovarian and potentially other cancers. Citation Format: Alfredo Perales-Puchalt, Elizabeth K. Duperret, Kar Muthumani, David B. Weiner, Xue Yang, Xizhou Zhu, Krzysztof Wojtak, Edgar Tello-Ruiz, Megan C. Wise. DNA-encoded bispecific T-cell engagers and antibodies present long-term antitumor activity [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 1547.

DNA-encoded bispecific T cell engagers and antibodies present long-term antitumor activity.

Specific antibody therapy, including mAbs and bispecific T cell engagers (BiTEs), are important new tools for cancer immunotherapy. However, these approaches are slow to develop and may be limited in their production, thus restricting the patients who can access these treatments. BiTEs exhibit a particularly short half-life and difficult production. The development of an approach allowing simplified development, delivery, and in vivo production would be an important advance. Here we describe the development of a designed synthetic DNA plasmid, which we optimized to permit high expression of an anti-HER2 antibody (HER2dMAb) and delivered it into animals through adaptive electroporation. HER2dMAb was efficiently expressed in vitro and in vivo, reaching levels of 50 μg/ml in mouse sera. Mechanistically, HER2dMAb blocked HER2 signaling and induced antibody-dependent cytotoxicity. HER2dMAb delayed tumor progression for HER2-expressing ovarian and breast cancer models. We next used the HER2dMAb single-chain variable fragment portion to engineer a DNA-encoded BiTE (DBiTE). This HER2DBiTE was expressed in vivo for approximately 4 months after a single administration. The HER2DBiTE was highly cytolytic and delayed cancer progression in mice. These studies illustrate an approach to generate DBiTEs in vivo, which represent promising immunotherapies for HER2+ tumors, including ovarian and potentially other cancers.

Remissions in Relapse/Refractory Acute Myeloid Leukemia Patients Following Treatment with NKG2D CAR-T Therapy without a Prior Preconditioning Chemotherapy

▪Introduction:CYAD-01 is a chimeric antigen receptor T-cell (CAR-T) product based on the receptor NKG2D with specificity for a broad range of ligands (MICA, MICB and ULBP1-6) expressed on most tumors. In vivo preclinical studies showed long-term anti-tumor activity of CYAD-01, whilst not only targeting tumor cells but also cells from the tumor neo-vasculature and immunosuppressive environment in the absence of pre-conditioning therapy.Methods:Exploiting this unique mode of action of CYAD-01, the THINK trial (NCT03018405) is an open-label Phase I study assessing the safety and clinical activity of multiple CYAD-01 administrations without prior preconditioning in 2 parallel cohorts: one in patients (pt) with metastatic solid tumors and the other one in hematological malignancies, including relapsing/refractory (r/r) acute myeloid leukemia (AML), multiple myeloma (MM) and myelodysplastic syndrome (MDS). The dose escalation segment of the study evaluates 3 dose levels (DL; 3x108, 1x109 and 3x109 cells per injection) of one cycle of 3 CYAD-01 administration with 2-weeks intervals. The study has been amended at the stage of DL-2 to authorize a second cycle of 3 CYAD-01 administrations in case of no progressive disease after 2 months.Results:As of July 31, 2018, 12 pts in the hematological cohort (8 AML, 3 MM and 1 MDS) have been enrolled at the 3 DLs (6 pts in DL-1, 3 in DL-2 and 3 in DL-3) without prior preconditioning. Median age was 64 (range 29-83) and median number of prior therapies was 3. DL-3 (3 pts) has been fully accrued as of data cutoff. Over 34 injections, 5 pts experienced grade (G) 3/4 treatment-related AEs: in DL-1, one pt experienced G3 lymphopenia and a second pt experienced G4 lymphopenia and G4 pneumonitis in DL-2, one pt experienced G3 lymphopenia and G3 thrombocytopenia and two other pts experienced G3 cytokine release syndrome (CRS). Treatment related AEs occurring in ≥ 1pt include pyrexia, CRS, hypoxia, lymphopenia, fatigue and nausea. CRS occurred in 5 pts, three G1/2 and two G3 AEs, with rapid resolution to appropriate treatment including tocilizumab. No neurotoxicity AEs have been observed to date.Out of the 8 r/r AML pts enrolled, 7 were response evaluable (2 at DL-1, 3 at DL-2 and 2 at DL-3). The third DL-3 pt has just initiated the first cycle of CYAD-01 treatment. Overall response rate in r/r AML pts was 42% (3/7 patients) with 1 complete remission with partial hematologic recovery (CRh) in DL-1 and 2 CR with incomplete marrow recovery (CRi; 1 in DL-1 and 1 in DL-3). All responding pts achieved response by day 29 (i.e. after 2 CYAD-01 administrations). The AML pt with CRh in DL-1 was bridged to allogeneic hematopoietic stem cell transplantation (allo-HSCT) on day +97 post CYAD-01 and is in durable complete molecular remission (CRMRD-) for more than 1 year (ongoing). The two other responding pts had CRi for 1 month. Two other AML patients at DL-2 had clinical benefit/disease stabilization with hematologic improvement and bone marrow blasts decrease: one pt for 3 months and with a decrease from 24% to 10% and the second pt for at least 4 months (ongoing) and with a decrease from 9.8% to 5.5%. The first patient in CRh had a relative increase in the systemic levels of SDF-1, RANTES and MCP-1 which correlated with the timing of injections. CYAD-01 engraftment kinetics, NKG2D ligand expression (including blasts and soluble ligand expression), and the kinetics of cytokine induction will be correlated with patient's responses. The 3 MM and the MDS pt, all in DL-1, did not present any sign of clinical activity.Conclusions:We have demonstrated the feasibility and safety of multiple injections of CYAD-01 without preconditioning chemotherapy. We evidenced promising anti-leukemic activity with 42% ORR in r/r AML with 5/7 pts having clinical benefit. Rates of G3/4 CRS were low and manageable. Updated safety, activity and correlative science data of the complete dose-escalation segment will be presented. DisclosuresSallman:Celgene: Research Funding, Speakers Bureau. Kerre:Celyad: Consultancy; BMS: Consultancy; Celgene: Consultancy, Research Funding. Davila:Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees. Wang:Jazz: Speakers Bureau; Jazz: Speakers Bureau; Amgen: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Amgen: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Dekker:Celyad: Employment. Snykers:Celyad: Employment. Sotiropoulou:Celyad: Employment. Breman:Celyad: Employment. Braun:Celyad: Employment. Lonez:Celyad: Employment. Verma:Celyad: Employment. Lehmann:GSK: Patents & Royalties; Celyad: Employment, Honoraria, Patents & Royalties.

Long-Term Antitumor Activity and Safety of Enzalutamide Monotherapy in Hormone Naïve Prostate Cancer: 3-Year Open Label Followup Results

A phase 2 study of enzalutamide monotherapy in patients with hormone naïve prostate cancer demonstrated high prostate specific antigen response rates at 25 weeks, 1 year and 2 years with minimal effects on total body bone mineral density and favorable safety. In this followup analysis we evaluated enzalutamide antitumor activity and safety at 3 years. In a single arm analysis 67 patients with hormone naïve prostate cancer and noncastrate testosterone (230 ng/dl or greater) received enzalutamide 160 mg per day orally until disease progression or unacceptable toxicity. The primary end point was the prostate specific antigen response (80% or greater decline from baseline). No patients discontinued treatment during year 3. Of 42 patients with prostate specific antigen assessments at 3 years 38 (90.5%, 95% CI 77.4-97.3) maintained a prostate specific antigen response. Of 26 patients with metastases at baseline 17 (65.4%) had a complete or partial response as the best overall response during 3 years. In patients who completed the 3-year visit minimal mean changes from baseline were observed in total body bone mineral density or bone mineral density of the femoral neck, trochanter, spine L1-L4 or forearm (range -2.7% to -0.1%). At 3 years total body fat had increased a mean of 16.5%, total lean body mass had decreased a mean of -6.5% and global health status had minimally decreased from baseline. Common adverse events were gynecomastia, fatigue, hot flush and nipple pain. Enzalutamide antitumor activity was maintained in patients with hormone naïve prostate cancer at 3 years. Overall bone mineral density, global health status and safety results were similar to those at 2 years.

A NKG2D-based CAR-T therapy in a multinational phase I dose escalation and expansion study targeting multiple solid and hematologic tumor types.

TPS3093 Background: Chimeric Antigen Receptor (CAR)-T therapy has potentially serious limitations related to target antigen loss, toxicity due to pre-conditioning regimen, and lack of activity in many tumor types. To overcome these limitations, we have developed a novel CAR-T, called NKR-2, incorporating the full-length human natural killer receptor NKG2D fused with the human CD3 zeta signaling domain. When expressed in T-cells, the naturally-expressed DAP10 provides co-stimulatory signals to NKR-2 to produce cytokines and selectively target tumor cells upon recognition of up to 8 different stress-induced NKG2D ligands expressed in many solid and hematologic malignancies. In preclinical studies, NKR-2 demonstrated long-term anti-tumor activity towards a breadth of tumor indications, in the absence of pre-conditioning, whilst simultaneously targeting tumor cells and cells from the local tumor neo-vasculature and suppressive immune environment. In our recently completed First-in-Human Phase 1 study (NCT02203825) in hematologic cancers, a single administration of autologous NKR-2 was safe with initial signs of clinical benefit. Methods: Exploiting the multiple ligand targeting capability and unique mode of action of NKR-2, the THINK trial (THerapeutic Immunotherapy with NKR-2) is an open-label Phase I study that will assess the safety and clinical activity of multiple infusion NKR-2 treatment (every 2 weeks x 3 infusions) in relapse/refractory patients with metastatic or locally advanced CRC, urothelial carcinoma, TNBC, pancreatic cancer, recurrent epithelial ovarian and fallopian tube carcinoma, AML/MDS and MM, post standard treatment. The study contains two consecutive segments. The dose escalation segment will enroll 18 patients in two separate hematologic and solid malignancy arms, and will evaluate 3 dose levels of NKR-2 (3x108, 1x109 and 3x109 per injection) following a 3+3 design. The expansion segment will then enroll 96 additional patients in 7 separate cohorts for each indication with 3 steps of statistical analysis (overall futility, cohort futility and final evaluation). The study is open for recruitment in both EU and US. Clinical trial information: NCT03018405.

X-Ray Psoralen Activated Cancer Therapy (X-PACT).

This work investigates X-PACT (X-ray Psoralen Activated Cancer Therapy): a new approach for the treatment of solid cancer. X-PACT utilizes psoralen, a potent anti-cancer therapeutic with current application to proliferative disease and extracorporeal photopheresis (ECP) of cutaneous T Cell Lymphoma. An immunogenic role for light-activated psoralen has been reported, contributing to long-term clinical responses. Psoralen therapies have to-date been limited to superficial or extracorporeal scenarios due to the requirement for psoralen activation by UVA light, which has limited penetration in tissue. X-PACT solves this challenge by activating psoralen with UV light emitted from novel non-tethered phosphors (co-incubated with psoralen) that absorb x-rays and re-radiate (phosphoresce) at UV wavelengths. The efficacy of X-PACT was evaluated in both in-vitro and in-vivo settings. In-vitro studies utilized breast (4T1), glioma (CT2A) and sarcoma (KP-B) cell lines. Cells were exposed to X-PACT treatments where the concentrations of drug (psoralen and phosphor) and radiation parameters (energy, dose, and dose rate) were varied. Efficacy was evaluated primarily using flow cell cytometry in combination with complimentary assays, and the in-vivo mouse study. In an in-vitro study, we show that X-PACT induces significant tumor cell apoptosis and cytotoxicity, unlike psoralen or phosphor alone (p<0.0001). We also show that apoptosis increases as doses of phosphor, psoralen, or radiation increase. Finally, in an in-vivo pilot study of BALBc mice with syngeneic 4T1 tumors, we show that the rate of tumor growth is slower with X-PACT than with saline or AMT + X-ray (p<0.0001). Overall these studies demonstrate a potential therapeutic effect for X-PACT, and provide a foundation and rationale for future studies. In summary, X-PACT represents a novel treatment approach in which well-tolerated low doses of x-ray radiation are delivered to a specific tumor site to generate UVA light which in-turn unleashes both short- and potentially long-term antitumor activity of photo-active therapeutics like psoralen.

Adoptively transferred natural killer cells maintain long-term antitumor activity by epigenetic imprinting and CD4+ T cell help

ABSTRACTNatural killer (NK) cell infusions can induce remissions in subsets of patients with different types of cancer. The optimal strategies for NK cell activation prior to infusion are still under debate. There is recent evidence that NK cells can acquire long-term functional competence by preactivation with the cytokines IL-12/15/18. The mechanisms supporting the maintenance of long-term NK cell antitumor activity are incompletely under-stood. Here, we show that NK cells preactivated in vitro with IL-12/15/18, but not with IL-15 alone, maintained high antitumor activity even 1 mo after transfer into lymphopenic RAG-2−/−γc−/− mice. The NK cell intrinsic ability for IFNγ production coincided with demethylation of the conserved non-coding sequence (CNS) 1 in the Ifng locus, previously shown to enhance transcription of Ifng. In a xenograft melanoma mouse model, human IL-12/15/18-preactivated NK cells rejected tumors more efficiently. In RAG-2−/−γc−/− mice, co-transfer of CD4+ T cells further improved the long-term competence of NK cells for IFNγ production that was dependent on IL-2. CD4+ T cell activation during homeostatic proliferation required macrophages and further promoted the long-term NK cell antitumor activity. Thus, NK cells can “remember” a previous exposure to cytokines by epigenetic imprinting resulting in a remarkable stability of the IFNγ-producing phenotype after adoptive transfer. In addition, our results support combination of cytokine-preactivated NK cells with CD4+ T cell activation upon lymphopenic conditioning to achieve long-term NK cell effector function for cancer immunotherapy.

Abstract B63: Improving CAR T cell function by reversing the immunosuppressive tumor environment of pancreatic cancer

Abstract Background: Adoptive transfer of T cells redirected to tumor-associated antigens (TAAs) by expression of chimeric antigen receptors (CARs) can produce tumor responses, even in patients with resistant malignancies. Although these CAR-T cells have potent anti-tumor activity in vitro and in vivo, pancreatic tumors employ immune evasion mechanisms, such as the production of inhibitory cytokines, which limit in vivo CAR-T cell persistence and effector function. Methods: To target pancreatic ductal adenocarcinoma (PDAC), we generated T cells expressing a CAR directed to the TAA prostate stem cell antigen (PSCA). We also engineered a chimeric cytokine receptor in which the IL4 receptor exodomain was fused to the IL7 receptor endodomain (IL4/7 ChR). Expansion and selection profiles and short- and long-term anti-tumor activity of these transgenic T cells were assessed. Results: T cells expressing CAR-PSCA kill PSCA(+) tumor cell lines CAPAN1 and K562-PSCA but not PSCA(-) targets, such as 293T (74±4%, 73±6% and 9±3% specific lysis, respectively, 10:1 E:T, n=3). Although these CAR-T cells had potent anti-tumor activity in vitro and in vivo, pancreatic tumors employ immune evasion mechanisms, such as the production of inhibitory cytokines, which limit in vivo CAR-T cell persistence and effector function. Indeed, when the serum of patients with pancreatic cancer (n=8) was examined, we found the levels of the immunosuppressive cytokine IL4 to be elevated relative to patients with benign pancreatic disorders or normal healthy controls (14.25±19.48 pg/mL vs 7.28±9.03 vs 1.13±1.42 pg/mL). Thus, to protect the CAR-PSCA T cells from the negative influences of IL-4, we generated a chimeric cytokine receptor in which the IL4 receptor exodomain was fused to the IL7 receptor endodomain (IL4/7 ChR). Transgenic expression of this molecule in CAR-PSCA T cells should invert the inhibitory effects of tumor-derived IL4 and instead promote the proliferation of the effector CAR T cells. In preliminary experiments we successfully co-expressed both CAR-PSCA and IL4/7 ChR (47.5±12.3% double-positive cells, n=4) on primary T cells. These T cells retained their tumor-specific activity (80±8% specific lysis against CAPAN1, 10:1 E:T, n=3) and when cultured in conditions that mimic the tumor milieu (IL4 12.5 ng/ml), CAR-PSCA 4/7R ChR-modified T cells continued to expand unlike unmodified CAR-PSCA T cells (from 2x106 cells on day 0 to 5.53x1010±8.46x1010 cells (CAR-PSCA 4/7R ChR) on day 28, in comparison to CAR-PSCA T cells that reached only 3.84x108±5.43x108 cells, n=4). Indeed, in the presence of IL4, transgenic cells had a selective advantage (comprising 44.8±11.0% of the population on day 0 and 87.6±10.0% on day 28, n=4), but even after prolonged cytokine exposure these T cells remained both antigen- and cytokine-dependent. Conclusions: CAR-PSCA 4/7 ChR-modified tumor-specific T cells can effectively target pancreatic cancer cells and should be equipped to expand, persist, and retain their cytotoxic function even in the presence of high levels of IL4 in the tumor microenvironment. Citation Format: Somala Mohammed, Sujita Sukumaran, Usanarat Anurathapan, Pradip Bajgain, Helen E. Heslop, Cliona M. Rooney, Malcolm K. Brenner, Ann M. Leen, Juan F. Vera. Improving CAR T cell function by reversing the immunosuppressive tumor environment of pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B63.

Sustained Functional T Cell Persistence and B Cell Aplasia Following CD19-Targeting Adoptive T Cell Immunotherapy for Relapsed, Refractory CD19+ Malignacy

Abstract 756 BackgroundAdvances in ex vivo T cell engineering have facilitated clinical trials to evaluate the potential for adoptive T cell transfer to target malignancy. Gene-modified T cells have the potential to expand, functionally persist and mediate long-term anti-tumor activity. Most clinical studies have shown only limited persistence of infused cells, with modest and often temporary anti-tumor activity. We reported initial clinical data on CAR T cells targeting CD19 expressed on normal and malignant B cells (CART19 cells) (Porter, et al NEJM 2011; Kalos et al Sci Trans Med 2011). The CAR included signaling domains from CD3zeta and CD137 (4-1BB) that mediated effector and proliferation/persistence signals. Here we report functional persistence of CART19 cells from the initial cohort at approximately 2 years post-infusion, and data from a more recently treated cohort of CLL patients and a pediatric patient with advanced, treatment refractory ALL. MethodsPersistence of gene-modified T cells was assessed by quantitative ABI-Taqman based PCR and a qualified assay that detects the CD137-TcR-zeta junction in the anti-CD19 CAR. CAR19 surface expression was detected by flow cytometry with an anti-CAR-19 idiotype specific antibody. B cell aplasia was evaluated by multiparametric flow cytometry. Multiplex cytokine analyses were performed using Luminex™assays. Results10 patients with relapsed, refractory disease have been treated to date: 9 adults w/CLL and one child w/pre- B cell ALL. Each had been extensively pre-treated and had active disease at the time of CART19 infusion. All CLL patients received lymphodepleting chemotherapy 4–6 days before infusions, while the ALL patient did not get further lymphodepletion. Patients were infused with an average total of 1.7–50 × 108 total T cells which corresponded to 0.14–5.9 x108CART-19 cells.9/10 treated patients were evaluable on 8.14.12. Detailed clinical outcomes will be reported separately at this meeting (Porter, D.L., Grupp S. et al). 4 pts (3 CLL, 1 ALL) had achieved CR at the primary endpoint (30 days post infusion) which is sustained and ongoing in all patients (range 1–24 months). Two CLL patients had a partial response (PR) lasting 3 and 5 months, while 3 patients did not respond (NR). In all patients with CR, robust in vivo expansion of CART19 cells was observed. By molecular analysis, CART19 cells demonstrated in vivo expansion, followed by contraction and an ongoing stable persistence at all evaluated timepoints. Expansion kinetics were unique for each patient; in all cases maximal expansion was observed by day +30 post CART-19 infusion. In patients with CR, observed peak marking for CART-19 ranged from 1 × 102-1 × 103 CART-19 cells/uL blood. Patients with PR demonstrated less robust in vivo expansion, with peak observed marking ∼1 × 101 CART-19 cells/uL blood. In NR patients, peak marking was <1 × 101CART-19 cells/uL blood. Long term peripheral blood persistence of CART19 cells and CAR19 surface expression was observed in all patients with CR in both CD3+/CD8+ and CD3+/CD4+ subsets. In patients with CR, elimination of peripheral B cells was observed at the time of CART19 in vivo expansion. Ongoing B cell aplasia has been documented in each CR patient in both peripheral blood and marrow by flow cytometry. Patients with PR showed transient elimination of malignant and normal B cells.Multiplex-cytokine analysis of serum samples from CR patients revealed a broad pro-inflammatory signature with significant elevation in a subset of soluble immune modulators including IL-6, IL-8, IFN-g, MIP1b, and IL2ra. In contrast, NR patients did not have elevated serum cytokines. In CR patients, elevation of cytokines tracked with expansion of CART19 cells and elimination of B cells, suggesting the potential for a cytokine-based diagnostic signature to monitor CART19 treatment efficacy. ConclusionsAdoptive transfer of CART19 cells engineered to express CD137 and TCR-zeta signaling domains can result in in vivo expansion, homing to disease sites, and long-term functional persistence of CART19 cells, accompanied by ongoing complete clinical responses and long-term B cell aplasia in a substantial fraction of patients with advanced, refractory and high risk CLL and relapsed refractory ALL. A detailed cytokine profile and persistent B cell aplasia has been identified that may correlate with treatment efficacy. Disclosures:Kalos:University of Pennsylvania: Patents & Royalties. Levine:TxCell: Consultancy, Membership on an entity’s Board of Directors or advisory committees; University of Pennsylvania: financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight, financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight Patents & Royalties. June:Novartis: Research Funding, institution owned patents have been licensed by Novartis, institution owned patents have been licensed by Novartis Patents & Royalties.

Potent Tumor-Specific Protection Ignited by Adoptively Transferred CD4+ T Cells

Administration of anti-CD25 mAb before an aggressive murine breast tumor inoculation provoked effective antitumor immunity. Compared with CD4(+) T cells purified from anti-CD25 mAb-pretreated mice that did not reject tumor, CD4(+) T cells purified from anti-CD25 mAb-pretreated mice that rejected tumor stimulated by dendritic cells (DCs) produced more IFN-gamma and IL-2, and less IL-17 in vitro, and ignited protective antitumor immunity in vivo in an adoptive transfer model. Tumor Ag-loaded DCs activated naive CD8(+) T cells in the presence of these CD4(+) T cells in vitro. Tumor Ag and adoptively transferred CD4(+) T cells were both required for inducing a long-term tumor-specific IFN-gamma-producing cellular response and potent protective antitumor activity. Although adoptively transferred CD4(+) T cells ignited effective tumor-specific antitumor immunity in wild-type mice, they failed to do so in endogenous NK cell-depleted, Gr-1(+) cell-depleted, CD40(-/-), CD11c(+) DC-depleted, B cell(-/-), CD8(+) T cell-depleted, or IFN-gamma(-/-) mice. Collectively, the data suggest that adoptively transferred CD4(+) T cells orchestrate both endogenous innate and adaptive immunity to generate effective tumor-specific long-term protective antitumor immunity. The data also demonstrate the pivotal role of endogenous DCs in the tumor-specific protection ignited by adoptively transferred CD4(+) T cells. Thus, these findings highlight the importance of adoptively transferred CD4(+) T cells, as well as host immune components, in generating effective tumor-specific long-term antitumor activity.

DNA aneuploidy in ulcerative colitis

Administration of anti-CD25 mAb before an aggressive murine breast tumor inoculation provoked effective antitumor immunity. Compared with CD4⁺ T cells purified from anti-CD25 mAb-pretreated mice that did not reject tumor, CD4⁺ T cells purified from anti-CD25 mAb-pretreated mice that rejected tumor stimulated by dendritic cells (DCs) produced more IFN-γ and IL-2, and less IL-17 in vitro, and ignited protective antitumor immunity in vivo in an adoptive transfer model. Tumor Ag-loaded DCs activated naive CD8⁺ T cells in the presence of these CD4⁺ T cells in vitro. Tumor Ag and adoptively transferred CD4⁺ T cells were both required for inducing a long-term tumor-specific IFN-γ-producing cellular response and potent protective antitumor activity. Although adoptively transferred CD4⁺ T cells ignited effective tumor-specific antitumor immunity in wild-type mice, they failed to do so in endogenous NK cell-depleted, Gr-1⁺ cell-depleted, CD40^−/−, CD11c⁺ DC-depleted, B cell^−/−, CD8⁺ T cell-depleted, or IFN-γ^−/− mice. Collectively, the data suggest that adoptively transferred CD4⁺ T cells orchestrate both endogenous innate and adaptive immunity to generate effective tumor-specific long-term protective antitumor immunity. The data also demonstrate the pivotal role of endogenous DCs in the tumor-specific protection ignited by adoptively transferred CD4⁺ T cells. Thus, these findings highlight the importance of adoptively transferred CD4⁺ T cells, as well as host immune components, in generating effective tumor-specific long-term antitumor activity.