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PD-1-TALEN activity screen in murine thymoma EL4 cells.PD-1 gene inactivation in pmel-1 CD8+T cells and functional analysis. PD-1-TALEN activity in TRL from MCA205. Effector/memory phenotype of TRL from MCA205.

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Despite the promising efficacy of adoptive cell therapies (ACT) in melanoma, complete response rates remain relatively low and outcomes in other cancers are less impressive. The immunosuppressive nature of the tumor microenvironment and the expression of immune-inhibitory ligands, such as PD-L1/CD274 by the tumor and stroma are considered key factors limiting efficacy. The addition of checkpoint inhibitors (CPI) to ACT protocols bypasses some mechanisms of immunosuppression, but associated toxicities remain a significant concern. To overcome PD-L1–mediated immunosuppression and reduce CPI-associated toxicities, we used TALEN technology to render tumor-reactive T cells resistant to PD-1 signaling. Here, we demonstrate that inactivation of the PD-1 gene in melanoma-reactive CD8+ T cells and in fibrosarcoma-reactive polyclonal T cells enhanced the persistence of PD-1 gene-modified T cells at the tumor site and increased tumor control. These results illustrate the feasibility and potency of approaches incorporating advanced gene-editing technologies into ACT protocols to silence immune checkpoints as a strategy to overcome locally active immune escape pathways. Cancer Res; 76(8); 2087–93. ©2016 AACR.

Relapse following CD19-directed chimeric antigen receptor T-cells (CAR-T) for relapsed/refractory large B-cell lymphoma (r/r LBCL) is commonly ascribed to antigen loss or CAR-T exhaustion. Multi-antigen targeting and PD-1 blockade are rational approaches to prevent relapse. Here, we test CD19/22 dual-targeting CAR-T (AUTO3) plus pembrolizumab in r/r LBCL as inpatient or outpatient therapy (NCT03289455, https://clinicaltrials.gov/ct2/show/NCT03289455). Endpoints include toxicity (primary) and response rates (secondary). AUTO3 was manufactured for 62 patients using autologous leukapheresis, modified with a bicistronic transgene. 52 patients received AUTO3 (7/52,50x106; 45/52,150-450x106) and 48/52 received pembrolizumab. Median age was 59 years (range,27-83) and 46/52 had stage III/IV disease. Median follow-up was 21.6 months (range,15.1-51.3) at last data cut (Feb 28, 2022). AUTO3 was safe: grade 1-2 and grade 3 CRS affected 18/52 (34.6%) and 1/52 (1.9%) patients, neurotoxicity arose in 4 patients (2/4, grade 3-4), HLH affected 2 patients, and no Pembrolizumab-associated autoimmune sequalae were observed. On this basis, outpatient administration was tested in 20 patients, saving a median of 14 hospital days/patient. AUTO3 was effective: overall response rates were 66% (48.9%, CR; 17%, PR). For patients with CR, median DOR was not reached, with 54.4% (CI: 32.8, 71.7) projected to remain progression-free beyond 12 months after onset of remission. DOR for all responding patients was 8.3 months (95% CI: 3.0, NE) with 42.6% projected to remain progression-free beyond 12 months after onset of remission. Overall, AUTO3 +/- pembrolizumab for r/r LBCL was safe, lending itself to outpatient administration, and delivered durable remissions in 54.4% of complete responders, associated with robust CAR-T expansion. Neither dual-targeting CAR-T nor pembrolizumab prevented relapse in a significant proportion of patients, and future developments include next-generation-AUTO3, engineered for superior expansion/persistence in vivo, and selection of CAR binders active at low antigen densities.

Chimeric antigen receptor (CAR) T cells targeting CD19 or CD22 have shown remarkable activity in B cell acute lymphoblastic leukemia (B-ALL). The major cause of treatment failure is antigen downregulation or loss. Dual antigen targeting could potentially prevent this, but the clinical safety and efficacy of CAR T cells targeting both CD19 and CD22 remain unclear. We conducted a phase 1 trial in pediatric and young adult patients with relapsed or refractory B-ALL (n = 15) to test AUTO3, autologous transduced T cells expressing both anti-CD19 and anti-CD22 CARs (AMELIA trial, EUDRA CT 2016-004680-39). The primary endpoints were the incidence of grade 3–5 toxicity in the dose-limiting toxicity period and the frequency of dose-limiting toxicities. Secondary endpoints included the rate of morphological remission (complete response or complete response with incomplete bone marrow recovery) with minimal residual disease-negative response, as well as the frequency and severity of adverse events, expansion and persistence of AUTO3, duration of B cell aplasia, and overall and event-free survival. The study endpoints were met. AUTO3 showed a favorable safety profile, with no dose-limiting toxicities or cases of AUTO3-related severe cytokine release syndrome or neurotoxicity reported. At 1 month after treatment the remission rate (that is, complete response or complete response with incomplete bone marrow recovery) was 86% (13 of 15 patients). The 1 year overall and event-free survival rates were 60% and 32%, respectively. Relapses were probably due to limited long-term AUTO3 persistence. Strategies to improve CAR T cell persistence are needed to fully realize the potential of dual targeting CAR T cell therapy in B-ALL., Bicistronic CAR T cells targeting CD19 and CD22 exhibit clinical activity and low toxicity in pediatric and young adult patients with B cell acute lymphoblastic leukemia, with relapses associated with limited CAR T cell persistence.

Intratumoral regulatory T (Treg) cell abundance associates with diminished antitumor immunity and poor prognosis in human cancers. Recent work demonstrates that CD25, the high-affinity receptor subunit for interleukin (IL)-2, is a selective target for Treg depletion in mouse and human malignancies; however, anti-human CD25 antibodies have failed to deliver clinical responses against solid tumors due to bystander IL-2 receptor signaling blockade on effector T cells, which limits their antitumor activity. Here we demonstrate potent single-agent activity of anti-CD25 antibodies optimized to deplete Treg cells, while preserving IL-2-STAT5 signaling on effector T cells and show synergy with immune checkpoint blockade in vivo. Pre-clinical evaluation of an anti-human CD25 (RG6292) antibody with equivalent features demonstrates, in both nonhuman primates and humanized mouse models, efficient Treg cell depletion with no overt immune-related toxicities. Our data support the clinical development of RG6292 and evaluation of new combination therapies incorporating non-IL-2-blocking anti-CD25 antibodies in clinical studies. Quezada and colleagues develop improved anti-CD25 antibodies that preserve IL-2 signaling and enhance single-agent antitumor immunity and immunotherapy through specific and efficient Treg cell depletion.

Intratumoral regulatory T cell (Treg) abundance associates with diminished anti-tumor immunity and poor prognosis in human cancers. Recent work demonstrates that CD25, the high affinity receptor subunit for IL-2, is a selective target for Treg depletion in mouse and human malignancies; however, anti-human CD25 antibodies have failed to deliver clinical responses against solid tumors due to bystander IL-2 receptor signaling blockade on effector T cells, which limits their anti-tumor activity. Here we demonstrate potent single-agent activity of anti-CD25 antibodies optimized to deplete Tregs whilst preserving IL-2-STAT5 signaling on effector T cells, and demonstrate synergy with immune checkpoint blockade in vivo. Pre-clinical evaluation of an anti-human CD25 (RG6292) antibody with equivalent features demonstrates, in both non-human primates and humanized mouse models, efficient Treg depletion with no overt immune-related toxicities. Our data supports the clinical development of RG6292 and evaluation of novel combination therapies incorporating non-IL-2 blocking anti-CD25 antibodies in clinical studies.

Background Regulatory T cell (Treg) depletion with antibodies against CD25 is effective in tumor models but response rates are low in poorly infiltrated B16 melanomas. Combination with a tumor vaccine enhances efficacy, but relapse usually occurs following partial control, similar to what is seen clinically. How resistance develops is unknown. Materials and Methods C57BL/6 mice were injected subcutaneously with B16 cells. Treatments included a depleting mouse IgG2a αCD25 antibody and/or a genetically modified, granulocyte-macrophage colony-stimulating factor (GM-CSF) secreting whole B16 tumor vaccine (Gvax). Changes in the immune landscape were assessed with high dimensional flow cytometry. Results Compared to monotherapies, combined Gvax/αCD25 significantly delayed tumour growth and prolonged survival, in association with enhanced infiltration of T cells with an activated phenotype. Approximately 50% of mice achieved partial response with relapse at day 35–45 post tumor injection. To characterize immune evolution prior to relapse, we analysed stable, partially responding tumors and paired draining lymph nodes (DLNs). Over time, activated PD-1+ICOS+TCF7- T cells with an effector memory (CD44+CD62L-) phenotype fell from 30% to 10% whilst resting, TCF7+ early differentiated cells rose in abundance towards levels seen in untreated tumors. Abundance of Ki67-, resting Tregs also recovered. Similar results were obtained in analysis of DLNs. Conclusions Combined Treg depletion/whole tumor vaccination therapy is effective in a poorly infiltrated B16 melanoma model. Combined treatment promotes T cell infiltration and activation. In mice achieving a partial response, treatment effects on the immune landscape were observed to decay over time suggesting a return to immune equilibrium. Further studies to explore the mechanistic basis of this observation are underway. Disclosure Information C. Qing: None. E. Ghorani: None. I. Solomon: None. F. Galvez-Cancino: None. F. Vargas: None. K. Peggs: None. S. Quezada: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; F. HOFFMANN-LA ROCHE LTD.

INTRODUCTION We have previously described AUTO1, a CD19 CAR with a fast off-rate binding domain, designed to reduce CAR T-cell immune toxicity and improve engraftment. Clinical testing in two academic studies in relapsed/refractory (r/r) paediatric [NCT02443831; CARPALL] and adult B-ALL, B-NHL and B-CLL [NCT02935257; ALLCAR19] confirmed the intended function of the receptor, with low levels of cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) and long-term engraftment of CAR T-cell 1,2. Based on data in adult B-ALL, we initiated a phase Ib/II registration study in r/r adult B-ALL [NCT04404660; FELIX]. To facilitate this study and future commercialization, industrialization of the manufacturing process was required. METHODS & RESULTS The original process in the academic studies was based on the Miltenyi CliniMACS Prodigy T cell transduction process. Leukapheresis was performed at the same site as manufacture. T cells were isolated from pheresis by CD4/CD8 positive selection and seeded onto the Prodigy to be activated using the Miltenyi CD3/CD28 targeting activating reagent, TransAct. The following day, transduction was carried out using a lentiviral vector. Cells were cryopreserved after an expansion phase of up to day 10 of the process. To facilitate industrialization of the AUTO1 manufacture in the multi-center, multi-regional FELIX study, we first explored the use of cryopreserved pheresis (81.3% median viability pre-selection (range 71.9 - 94.3), 1.0 days median doubling time (range 0.9 - 1.5) and 47.6% median CD19 CAR expression (range 19.1-62.1)). We concluded that optimal manufacture includes the use of fresh pheresis and the initiation of manufacture within 72 hours (99.0% median viability pre-selection (range 92.5 - 99.7), 1.3 days median doubling time (range 1.1 - 2.1) and 69.3% median CD19 CAR expression (range 22.9-86.2)). To further simplify the process, we explored removal of the pre-selection step. Full-scale runs using starting material from 4 healthy donors were conducted to compare CD4/CD8 selected with unselected cells. On the day following activation, selected cells displayed a higher percentage of viable cells, defined as cPARP-FVS780- (median: 76.1%, range: 84.5-66.4) as compared to unselected cells (median: 52.2%, range: 43.6-59.0). In addition, selected cells demonstrated a median of 23-fold expansion (range: 20.0 - 29.1) compared to a 13.3-fold expansion for unselected cells (range 6.1-17.4). Median transduction efficiencies of viable CAR+ T-cells were 53.9% (range: 43.2-56.9) and 78.0% (range: 64.5-81.1) in selected and unselected cells, respectively. CD4/8 pre-selection was determined to be a critical part of the process. A comparison of phenotype between 18 batches manufactured using the academic process and 5 batches produced from fresh material using the industrial process was carried out. No significant differences, as determined by 2-way ANOVA, were observed between the percentage of CAR+ CD3+ cells, the memory phenotype (% TSC/naive, % TCM, % TEM and % TEMRA) and the percentage expression of PD1 (figure 1). The CD4/CD8 ratio was also comparable between products of the two processes. Data from the initial 6 fresh in patients show that engraftment in the FELIX study is consistent with ALLCAR19 engraftment results. Additional patients, updated clinical data and longer follow-up will be presented at the conference. CONCLUSION Industrialization of an autologous Miltenyi CAR T process is feasible, leading to a comparable product to that manufactured in an academic setting. We have now opened the pivotal multi-center phase II part of the FELIX study in r/r adult B-ALL patients. REFERENCES Ghorashian S et al. (2019) Enhanced CAR T cell expansion and prolonged persistence in pediatric patients with ALL treated with a low-affinity CD19 CAR. Nat Med, 25(9):1408-1414.Roddie C et al. (2021) Durable responses and low toxicity after fast off-rate CD19 CAR-T therapy in adults with relapsed/ refractory B-ALL. J Clin Oncol, in press Figure 1. Comparison of phenotype between 18 CAR T cell batches manufactured using the academic process and 5 batches produced using the industrial process. Boxes represent median, 25th and 75th percentiles and whiskers represent minimum and maximum. Figure 1 Figure 1. Disclosures Culshaw: Autolus Ltd.: Current Employment. Arce Vargas: Autolus Ltd.: Current Employment. Santiago Toledo: Autolus Ltd.: Current Employment. Roddie: Novartis: Consultancy; Celgene: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau. Shaughnessy: BMS: Honoraria, Speakers Bureau; Sanofi: Honoraria, Speakers Bureau; Kite: Honoraria, Speakers Bureau. Cerec: Autolus Ltd.: Current Employment. Duffy: Autolus Ltd.: Current Employment. Perna: Autolus Ltd.: Current Employment. Brugger: Autolus Ltd.: Current Employment. Merges: Autolus Ltd.: Current Employment. Pule: Autolus Ltd: Current Employment.

Immune-checkpoint blockade therapies has had impressive results in the clinic and are approved in multiple cancer indications. However, meaningful responses remain elusive in several solid tumor types and durable responses in some of these indications, or subtypes, are rarely observed. It has been postulated that this is in part due to elaborate poorly understood immunosuppressive mechanisms that the therapeutic approach must overcome to elicit the desired durable responses. Chimeric Antigen Receptor T (CAR-T) cell therapies had been therapeutically highly successful in haematological malignancies but such successes have not been replicated yet in solid tumors, primarily due to immune suppressive mechanisms in the tumor microenvironment (TME) of these settings. To address these issues in developing our CAR-T therapeutics, we deployed tissue cytometry to conduct a multiplex analysis of 30 markers on the same tissue sections from each patient from a set of patients from various solid tumor indications expressing the antigen GD2 (e.g. SCLC, Melanoma, Neuroblastoma), a disialoganglioside to which we have developed a selective CAR. The panel aimed to characterize and phenotype key players within the TME (i.e. tumour cells, T cells, DCs, macrophages, MDSCs, CAFs, as well as endothelial cells). We then deployed machine learning analytics to assist in inferring about potential interactions the different cell types have in the 3D context of the tumor tissue which allowed the identification of therapeutically relevant elements in the TMEs of these indications. Next, we generated CAR-T cells from healthy donors targeting GD2, an antigen we validated its expression in multiple solid tumor types. A first generation GD2 CAR is currently being developed in the clinic for the treatment of Neuroblastoma. We proceeded to incorporate a CCR module (IL7R chimeric protein designed to improve cell persistence), a dnTGFβ (dominant negative TGF-βRII protein designed to block inhibitor signals from TGFβ), and other function enhancing modules into the GD2 CAR. The multi module CAR-T cells showed potent in vitro activity against human patient derived cancer cell lines as measured by both tumor lysis and cytokine secretion. These CAR-T cells also demonstrated enhanced tumor infiltration and anti-tumor activity in vivo in xenografts mouse models. Our findings suggest that GD2 targeted CAR-T cell therapy is a viable novel approach for the treatment of multiple solid tumors indications. Citation Format: Margarida Neves, Lukas Stanczuk, Alireza Alavi, Meggan Czapiga, Rebecca Moore, Frederick Vargas, Muhammad A. Al-Hajj. AUTO6NG overcomes immune suppressive mechanisms in the TME and demonstrate preclinical anti-tumor activity in GD2-expressing solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2661.

Summary CD25 is expressed at high levels on regulatory T (Treg) cells and was initially proposed as a target for cancer immunotherapy. However, anti-CD25 antibodies have displayed limited activity against established tumors. We demonstrated that CD25 expression is largely restricted to tumor-infiltrating Treg cells in mice and humans. While existing anti-CD25 antibodies were observed to deplete Treg cells in the periphery, upregulation of the inhibitory Fc gamma receptor (FcγR) IIb at the tumor site prevented intra-tumoral Treg cell depletion, which may underlie the lack of anti-tumor activity previously observed in pre-clinical models. Use of an anti-CD25 antibody with enhanced binding to activating FcγRs led to effective depletion of tumor-infiltrating Treg cells, increased effector to Treg cell ratios, and improved control of established tumors. Combination with anti-programmed cell death protein-1 antibodies promoted complete tumor rejection, demonstrating the relevance of CD25 as a therapeutic target and promising substrate for future combination approaches in immune-oncology., Graphical Abstract, Highlights • CD25 expression is largely restricted to Treg cells in mice and humans • FcγRIIb inhibits anti-CD25-mediated depletion of intra-tumoral Treg cells • Fc-optimized anti-CD25 efficiently depletes intra-tumoral Treg cells • Anti-CD25 synergizes with PD-1 blockade to reject established tumors, Anti-CD25 antibodies have displayed only modest therapeutic activity against established tumors. Arce Vargas et al. demonstrate that existing anti-CD25 antibodies fail to deplete intra-tumoral Treg cells due to upregulation of FcγRIIb within tumors. Fc-optimized anti-CD25 mediates effective depletion of tumor-infiltrating Treg cells and synergizes with PD-1 blockade to promote tumor eradication.

Immunomodulatory antibodies influence the direction and magnitude of immune responses against cancer. Significant efficacy has been demonstrated across multiple solid tumour types within clinical trials. Recent preclinical studies indicate that successful outcome relies upon mechanistic activity extending beyond simple receptor stimulation or blockade. In addition to blocking co-inhibitory signals in secondary lymphoid organs, cytotoxic T-lymphocyte antigen (CTLA)-4 antibodies mediate depletion of tumour-infiltrating regulatory T cells by antibody-dependent cellular cytotoxicity (ADCC). This mechanism appears to be common to other immunomodulatory antibodies including those targeting OX40 and glucocorticoid-induced TNFR-related protein (GITR). If verified in the human setting, these findings have significant implications for antibody design, biomarker discovery, and the development of synergistic combinatorial therapies.

The accumulation of regulatory T-cells (Tregs) in the tumor hampers effector antitumor activity and correlates with a poor prognosis in several human cancers. Increasing the effector T-cell (Teff) to regulatory T-cell (Treg) ratio is known to result in improved control of established tumors. Studies demonstrating high levels of CD25 expression on Tregs and only low levels on Teff in human tumors have underscored its relevance as a target for Treg depletion. This supported the development of anti-CD25 depleting monoclonal antibodies (mAbs) as a promising monotherapy as well as combination partner in cancer immunotherapies. To date, anti-CD25 antibodies for clinical use have been designed to block IL-2 binding and signaling through the IL-2 receptor complex and also depletes CD25 positive cells. Since IL-2 is a critical cytokine involved in T-cell activation and proliferation, we hypothesized that a depleting antibody targeting CD25 but unable to block IL-2 signaling would promote a more potent anti-tumor response by preferentially depleting Tregs while still allowing IL-2 to stimulate effector T-cells expressing low levels of CD25 on their surface. We therefore compared the functional activity of anti-mouse CD25 depleting mAbs with and without IL-2 blocking activity. After having confirmed their differential impact on IL-2 signaling in vitro and in vivo, we evaluated the therapeutic activity of these mAbs in various syngeneic tumor models. While both the IL-2 blocking and non-IL-2 blocking mAbs showed equivalent Treg-depleting activity, the antibody sparing IL-2 signaling (αCD25NIB) promoted stronger antitumor effect than the IL-2 blocking mAb, with complete tumor regression observed in 70-100% of the mice after a single administration of the antibody. We demonstrated that the αCD25NIB would be an ideal partner for combination against several tumor types including “immune cold” tumors and αPD-L1 resistant tumors. Our data demonstrate that targeting CD25 with ADCC enabled antibodies preserving IL-2 signaling is a novel and powerful strategy for rejection of established tumors via depletion of Treg cells and enhanced, cell intrinsic, IL-2-driven effector T-cell activation. Citation Format: Anne Goubier, Isabelle Solomon, Frederick Arce Vargas, Dimitrios Zervas, Chen Qing, Josephine Salimu, Mark Brown, Pascal Merchiers, Karl S. Peggs, Sergio A. Quezada. Non-IL-2 blocking anti-CD25-targeting antibodies: depletion of regulatory T-cells driving optimal effector response for rejection of established tumors [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A192.

Summary Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known for specifically killing cancer cells, whereas in resistant cancers, TRAIL/TRAIL-R can promote metastasis via Rac1 and PI3K. It remains unknown, however, whether and to what extent TRAIL/TRAIL-R signaling in cancer cells can affect the immune microenvironment. Here we show that TRAIL-triggered cytokine secretion from TRAIL-resistant cancer cells is FADD dependent and identify the TRAIL-induced secretome to drive monocyte polarization to myeloid-derived suppressor cells (MDSCs) and M2-like macrophages. TRAIL-R suppression in tumor cells impaired CCL2 production and diminished both lung MDSC presence and tumor growth. In accordance, the receptor of CCL2, CCR2, is required to facilitate increased MDSC presence and tumor growth. Finally, TRAIL and CCL2 are co-regulated with MDSC/M2 markers in lung adenocarcinoma patients. Collectively, endogenous TRAIL/TRAIL-R-mediated CCL2 secretion promotes accumulation of tumor-supportive immune cells in the cancer microenvironment, thereby revealing a tumor-supportive immune-modulatory role of the TRAIL/TRAIL-R system in cancer biology., Graphical Abstract, Highlights • TRAIL induces a cytokine cancer secretome in a FADD- and caspase-8-dependent manner • FADD promotes tumor growth along with accumulation of M2-like immune cells in vivo • The TRAIL-induced secretome recruits M2-like immune cells to tumors via CCR2 • TRAIL/CCL2 correlate with a tumor-supportive immune profile in lung cancer patients, Hartwig et al. show that endogenous TRAIL signaling in cancer cells induces a FADD-dependent secretome that promotes the accumulation of M2-like immune cells and tumor growth via host CCR2.

Regulatory T cells (Treg) are key players of the suppressive tumour microenvironment (TME). Their presence is correlated with a bad prognosis in multiple cancers, while a greater ratio of effector T cells (Teff) to Treg is associated with improved outcome. Studies demonstrating high expression of CD25 on Tregs but not Teff in human tumors have underscored its relevance as a target for Treg depletion. However, clinical trials conducted to target Treg in cancer patients with existing anti-CD25 (aCD25) antibodies have shown contradicting results, and no aCD25 antibody is in clinical development for this application. Importantly, aCD25 antibodies tested to date block IL2 signalling via CD25. We demonstrated (Solomon et al. AACR2018) that such blocking drastically reduces the therapeutic activity of aCD25 antibodies and that a single administration of a depleting antibody targeting CD25 but not blocking the IL2 signalling effectively depletes Treg, increases Teff activity and promotes eradication of established tumors in several mouse models of cancer. With this rationale in mind, we have generated a panel of fully human aCD25 IgG1 antibodies, which we characterized for their binding to human and cynomolgus monkey CD25. The antibodies were then screened for their impact on IL2 binding, using a sandwich binding assay on Octet, and on IL2 signalling, using a STAT5 phosphorylation assay. Antibodies were also tested for their ability to deplete CD25 positive cell lines and in vitro-derived human Treg in ADCC and ADCP assays. Finally, the impact of selected antibodies on Treg within the TME and on Teff responses was evaluated in human samples. Among the antibodies binding to human and cynomolgus CD25, we have selected a panel interfering with neither IL2 binding to CD25 nor IL2 signalling. Interestingly, epitope binning assays demonstrated that none of the selected antibodies bind to epitopes overlapping with those of the existing clinical antibodies, Daclizumab and Basiliximab. Among the non-IL2 blocking antibodies, those showing the maximum target cell lysis via ADCC and highest phagocytosis via ADCP represent potential lead clinical candidates. Most interestingly, contrary to the existing clinical aCD25 antibodies, which block IL2 signalling, our candidates do not inhibit Teff proliferation and function, confirming the specificity of action toward Treg and the importance of preserving IL2 signalling. Finally, we have shown that our clinical candidates efficiently deplete Treg in the TME. We present the first anti-human CD25 antibodies selected for their capacity to deplete human Treg while preserving IL2 signalling and activity of Teff. These antibodies provide a novel therapeutic approach to alleviate immune suppression in the TME, which would provide an ideal combination partner for existing standard of care and IO treatments, and could potentially be used as a monotherapy. Citation Format: Josephine C. Salimu, Mark Brown, Pascal Merchiers, Beatriz Goyenechea, Kevin Moulder, Robert Dejonge, Aghiles Boughetane, Isabelle Solomon, Frederick Arce Vargas, Karl S. Peggs, Anne Goubier, Sergio A. Quezada. Generation of first-in-class anti-CD25 antibodies depleting Treg without interfering with IL2 signalling for cancer therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2787.

International audience; In November 2015, the CALYM Carnot Institute held a 2-d workshop to discuss the current and future development of immunomodulatory antibodies for the treatment of lymphoma. Highlights from the workshop are presented in this article. © 2016 Taylor & Francis Group, LLC.

Despite the promising efficacy of adoptive cell therapies (ACT) in melanoma, complete response rates remain relatively low and outcomes in other cancers are less impressive. The immunosuppressive nature of the tumor microenvironment and the expression of immune-inhibitory ligands, such as PD-L1/CD274 by the tumor and stroma are considered key factors limiting efficacy. The addition of checkpoint inhibitors (CPI) to ACT protocols bypasses some mechanisms of immunosuppression, but associated toxicities remain a significant concern. To overcome PD-L1–mediated immunosuppression and reduce CPI-associated toxicities, we used TALEN technology to render tumor-reactive T cells resistant to PD-1 signaling. Here, we demonstrate that inactivation of the PD-1 gene in melanoma-reactive CD8+ T cells and in fibrosarcoma-reactive polyclonal T cells enhanced the persistence of PD-1 gene-modified T cells at the tumor site and increased tumor control. These results illustrate the feasibility and potency of approaches incorporating advanced gene-editing technologies into ACT protocols to silence immune checkpoints as a strategy to overcome locally active immune escape pathways. Cancer Res; 76(8); 2087–93. ©2016 AACR.

Cytomegalovirus (CMV) remains a significant cause of morbidity after allogeneic hematopoietic stem cell transplantation (HSCT). Clinical risk varies according to a number of factors, including recipient/donor CMV serostatus. Current dogma suggests risk is greatest in seropositive recipient (R+)/seronegative donor (D-) transplants and is exacerbated by T-cell depletion. We hypothesized that in the setting of reduced-intensity T-cell-depleted conditioning, recipient-derived CMV-specific T cells escaping deletion may contribute significantly to CMV-specific immunity and might therefore also influence chimerism status. We evaluated 105 recipients of alemtuzumab-based reduced-intensity HSCT and collated details on CMV infection episodes and T-cell chimerism. We used CMV-specific HLA multimers to enumerate CMV-specific T-cell numbers and select cells to assess chimerism status in a subset of R+/D- and R+/seropositive donor patients. We show that in R+/D- patients, CMV-specific T cells are exclusively of recipient origin, can protect against recurrent CMV infections, and significantly influence the chimerism status toward recipients. The major findings were replicated in a separate validation cohort. T-cell depletion in the R+/D- setting may actually, therefore, foster more rapid reconstitution of protective antiviral immunity by reducing graft-vs-host directed alloreactivity and the associated elimination of the recipient T-cell compartment. Finally, conversion to donor chimerism after donor lymphocytes is associated with clinically occult transition to donor-derived immunity.

Acute inflammation is traditionally characterized by polymorphonuclear leukocytes (PMN) influx followed by phagocytosing macrophage (Mφs) that clear injurious stimuli leading to resolution and tissue homeostasis. However, using the peritoneal cavity, we found that although innate immune-mediated responses to low-dose zymosan or bacteria resolve within days, these stimuli, but not hyperinflammatory stimuli, trigger a previously overlooked second wave of leukocyte influx into tissues that persists for weeks. These cells comprise distinct populations of tissue-resident Mφs (resMφs), Ly6c(hi) monocyte-derived Mφs (moMφs), monocyte-derived dendritic cells (moDCs), and myeloid-derived suppressor cells (MDSCs). Postresolution mononuclear phagocytes were observed alongside lymph node expansion and increased numbers of blood and peritoneal memory T and B lymphocytes. The resMφs and moMφs triggered FoxP3 expression within CD4 cells, whereas moDCs drive T-cell proliferation. The resMφs preferentially clear apoptotic PMNs and migrate to lymph nodes to bring about their contraction in an inducible nitric oxide synthase-dependent manner. Finally, moMφs remain in tissues for months postresolution, alongside altered numbers of T cells collectively dictating the magnitude of subsequent acute inflammatory reactions. These data challenge the prevailing idea that resolution leads back to homeostasis and asserts that resolution acts as a bridge between innate and adaptive immunity, as well as tissue reprogramming.

Anti-tumor monoclonal antibodies eliminate tumor cells through different mechanisms including antibody-mediated cell cytotoxicity and generation of long-term anti-tumor T cell responses. In a recent publication, DiLillo and Ravetch demonstrate how this vaccinal effect is mediated by engagement of Fcγ receptors expressed on antigen-presenting cells.

Different components of the tumor microenvironment hamper the immune response against cancer cells and are a critical impediment for the success of cancer immunotherapy. Among these, the accumulation of regulatory T cells (Treg) and the reduction of the effector T cells (Teff)/Treg ratio favors tumor progression and is associated with worse prognosis in several human cancers. Treg elimination in the tumor microenvironment can be achieved through antibodies that target molecules expressed preferentially in Treg and that induce antibody-mediated cell cytotoxicity (ADCC). CTLA-4 is highly expressed on tumor-infiltrating Treg and therapy with anti-CTLA-4 has been effective for cancer treatment in murine models and in humans. In mice, we and others have shown that anti-CTLA-4 depletes Treg in the tumor but not in peripheral lymphoid organs. This site specificity in mice is given by the expression of the Fcγ-receptor IV (FcγRIV) in myeloid cells present in the tumor microenvironment but not in the periphery. However, the role of FcγRs in the effectiveness of anti-CTLA-4 therapy or therapies with other immunomodulatory antibodies in humans is not known. We therefore investigated the density of targets of clinically relevant immunomodulatory antibodies in different subpopulations of tumor-infiltrating immune cells as well as the expression of FcγR subtypes in murine tumor models. Because of the inter-species differences in the repertoire and the cellular distribution of FcγRs, we also studied this in humanized-FcγR (huFcγR) mice, in which there is no expression of murine FcγRs but of their human homologues. We compared this with samples of human melanoma and observed surprisingly similar expression profiles of both FcγRs and targets of immunomodulatory antibodies, including CTLA-4. We then asked whether anti-CTLA-4 would also lead to selective Treg depletion in the tumor microenvironment in huFcγR mice. Using an antibody targeting mCTLA-4 with a human IgG1 isotype, we observed that was the case. Furthermore, mutation of the Fc region of the antibody to enhance its affinity to activating huFcγRs resulted in more effective selective Treg depletion in the tumor. These findings are not only relevant for CTLA-4 but also for other targets expressed preferentially on Treg instead of Teff and must be taken into account when designing immunomodulatory antibodies for clinical use. Citation Format: Frederick Arce Vargas, Andrew J. Furness, Marta H. Lesko, Martin Pule, Jeffrey V. Ravetch, Karl S. Peggs, Sergio A. Quezada. Enhancing intratumoural Treg depletion through antibody engineering and analysis of the checkpoint landscape of mouse and human cancers. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B179.

A number of factors influence the incidence and degree of T-cell mixed chimerism following allogeneic stem cell transplantation (HSCT). The most well-recognized of these include donor source, GvHD and T-cell depletion of the graft. We hypothesized that in the ‘pro-tolerogenic’ environment following T-cell-depleted reduced intensity HSCT, viral infection could drive differential expansion of donor or recipient memory T cell (Tmem) populations, contributing to differences in the degree of mixed chimerism. Viral infection, more specifically cytomegalovirus (CMV) infection, has been shown to drive pauci-clonal T cell expansions in the early post-HSCT period, skewing T-cell repertoire reconstitution. Since CMV infection is associated with maintenance of relatively large memory T cell pools, and viral ‘reactivation’ can be associated with very large expansions of virus-specific T cells, we predicted that recipient T-cell chimerism would be greater following recipient seropositive/ donor seronegative (R+/D-) versus recipient seronegative/ donor seropositive (R-/D+) HSCT due to differential expansion of virus-specific Tmem. We evaluated the outcomes of 46 consecutive reduced intensity transplants incorporating in vivo alemtuzumab, in which these CMV serostatus combinations were present. Recipient chimerism was markedly greater in the R+/D- cohort (median 50% recipient, range 0-95% versus median 4%, range 0-95% respectively, P = 0.0044), and the correlation was greater when analysis was restricted to those with sibling donors (n=34, median 68% (2-95%) recipient versus 4% (0-95%) respectively, P = 0.0018), or to patients without grade II-IV acute or extensive chronic GvHD (n=33, median 63% (2-95%) recipient versus 2% (0-70%) respectively, P = 0.0019). At 6+ months post transplant, CMV-specific T cells accounted for 0.4-24% of the CD8+ T cell compartment. All such virus-specific T cells in R+/D- patients were recipient-derived, as demonstrated by chimerism analyses of CMV pp65-, pp50- or IE-1-specific HLA-streptamer-selected CD8+ T cells. Furthermore, chimerism was consistently more skewed towards recipient in the CD8+ as opposed to the CD4+ T cell compartment, in keeping with larger clonal burst sizes of CD8+ versus CD4+ virus-specific T cells. Since the majority of recipient-derived T cells were not CMV-specific, we hypothesize that events early post-transplant might have a role in ‘imprinting’ the level of recipient chimerism, which thereafter remains stable in the absence of GvHD. Interestingly, these recipient-derived CMV-specific T cells conferred apparent protection against multiple CMV reactivation episodes. The number of reactivation episodes and number of days on treatment within the R+/D- cohort were significantly higher for those with 10% (n=16) recipient chimerism (median 3 (range 1-10) versus 1 (1-3), P = 0.0044, and median 88 days (19-412) versus 21 days (17-63), P = 0.001, respectively). R+/D- transplants were not, therefore, generally associated with recurrent problematic CMV infection. Indeed, this scenario was largely confined to the group of patients developing GvHD, who suffered both from reduced absolute lymphocyte counts secondary to corticosteroids, and eradication of the recipient-derived T cell compartment including the CMV-specific Tmem pool. Paradoxically, this suggests that T-cell depletion may actually reduce the incidence of problematic CMV infection in this ‘high-risk’ group by facilitating persistence of recipient CMV-specific T cells. Twelve R+/D- patients received donor-lymphocytes at later time points for persistent recipient chimerism (median 78% recipient (5-95%)). All converted to Disclosures: No relevant conflicts of interest to declare.

Summary In addition to helper and regulatory potential, CD4+ T cells also acquire cytotoxic activity marked by granzyme B (GzmB) expression and the ability to promote rejection of established tumors. Here, we examined the molecular and cellular mechanisms underpinning the differentiation of cytotoxic CD4+ T cells following immunotherapy. CD4+ transfer into lymphodepleted animals or regulatory T (Treg) cell depletion promoted GzmB expression by tumor-infiltrating CD4+, and this was prevented by interleukin-2 (IL-2) neutralization. Transcriptional analysis revealed a polyfunctional helper and cytotoxic phenotype characterized by the expression of the transcription factors T-bet and Blimp-1. While T-bet ablation restricted interferon-γ (IFN-γ) production, loss of Blimp-1 prevented GzmB expression in response to IL-2, suggesting two independent programs required for polyfunctionality of tumor-reactive CD4+ T cells. Our findings underscore the role of Treg cells, IL-2, and Blimp-1 in controlling the differentiation of cytotoxic CD4+ T cells and offer a pathway to enhancement of anti-tumor activity through their manipulation., Graphical Abstract, Highlights • Tumor-infiltrating CD4+ T cells exhibit T helper and cytotoxic features • Treg cells limit IL-2 availability vital for the acquisition of cytotoxic features • T-bet is required for IFN-γ, but not GzmB, expression in CD4+ TILs • Blimp-1 is required for GzmB expression in response to IL-2 in CD4+ TILs, Śledzińska et al. show that upon depletion of regulatory T cells, a surplus of interleukin-2 in the tumor microenvironment supports acquisition of cytotoxic activity by T helper cells orchestrated by the transcription factor Blimp-1. These polyfunctional CD4+ T cells exhibit potent anti-tumor activity in an adoptive transfer setting with therapeutic implications.