Regulatory T Cells Contribute to Sexual Dimorphism in Neonatal Hypoxic-Ischemic Brain Injury

Background and Purpose: Neonatal encephalopathy caused by hypoxia-ischemia (HI) is a major cause of death and disability in newborns. Clinical and experimental studies suggest a sexual dimorphism in HI-induced brain injury and therapy responses. A major hallmark of HI pathophysiology is the infiltration of peripheral immune cells into the injured brain. However, the specific role of regulatory T cells (Tregs) in neonatal HI is still unknown. Methods: Nine-day-old mice were exposed to HI by ligation of the right common carotid artery followed by 1 hour hypoxia (10% oxygen). Using immunohistochemistry, flow cytometry, and microarray analyses, Tregs were investigated in the brain, spleen, and blood 24 hours post HI. The functional role of Tregs was evaluated by acute Treg depletion in depletion of regulatory T cells transgenic mice. Brain injury, neuroinflammatory responses, and vascular injury were analyzed via immunohistochemistry and Western blot 48 hours and 7 days after HI. Functional outcome was assessed 3 days and 5 weeks after HI. Results: Female mice revealed an increased cerebral Treg infiltration, coinciding with elevated chemokine receptor expression. Treg depletion in females aggravated HI-induced brain tissue injury, short-term motor deficits, and long-term deficits in exploratory activity, paralleled by an increased microglia and endothelial activation and leukocyte infiltration. Treg depletion in male mice reduced HI-induced brain injury, short-term motor, and long-term cognitive deficits, associated with reduced vascular injury. Ex vivo isolated female Tregs displayed an increased immunosuppressive activity on effector T cell proliferation and an increased gene enrichment in pathways related to enhanced Treg activity. Conclusions: Tregs from neonatal female mice provide endogenous neuroprotection, whereas Tregs from male mice increase secondary neurodegeneration. As potential mechanisms, we identified intrinsic transcriptional differences associated with enhanced anti-inflammatory activity of female Tregs. Our study emphasizes the urgent need for sex-stratified clinical and preclinical analyses.

231 Molecular insights on safety and anti-tumor activity of a non-irAE-inducing anti-CTLA-4 monoclonal antibody ONC-392

BackgroundAnti-CTLA-4 antibodies have brought about limited clinical benefit because severe toxicity limits dosing levels and/or duration. We used CTLA-4 knockin mice to screen for antibodies with higher anti-tumor activity but lower autoimmunity. We have revealed that the key for better safety and preclinical efficacy is preservation of CTLA-4 for immune tolerance and intratumorial Treg depletion. Our work established that, independent of blocking activities, mAbs that preserve CTLA-4 recycling maintain the physiological immune tolerance checkpoint function while allowing more efficient and selective elimination of tumor-infiltrating regulatory T cells, resulting in highest efficacy and lowest toxicity and was thus developed for clinical testing of all antibodies tested.1–6 The antibody with best safety and efficacy profile, ONC-392 was developed for clinical testing. The first-in human studies showed that ONC-392 is safe and well tolerated. Remarkably, no irAE has been reported among patients who has received repeated dosing of 3.0 mg/kg and 10.0 mg/kg of ONC-392. The molecular and cellular characterization of ONC-392 will be presented.MethodsIn vitro binding and disassociation assay were determined between pH 4.0–7.0. The intracellular traffic of both antibodies and CTLA-4 molecules were visualized by confocal microscopy. The binding to human and mouse FcgRI, IIA, IIB, and III (A), FcRn as well as mouse FcgRIV were evaluated by surface plasmon resonance (SPR). Depletion of regulatory T cells in tumor and lymphoid tissues were determined by flow cytometry.ResultsONC-392 is a pH-sensitive antibody that preserves CTLA-4 recycling. By preserving cell surface CTLA-4, Onco-392 preserves immune tolerance. Preserving CTLA-4 on tumor-infiltrating Treg contribute to more effective immunotherapy. In addition to its unique feature of pH sensitive binding, OncoC4 also have several important features in Fc. ONC-392 shown comparable binding to human FcgRI and IIIA as wild-type IgG1s. As expected from the mutations introduced, ONC-392 show about 6 fold higher affinity for FcRn than wild-type IgG1. Interestingly, ONC-392 has shown 7–10-fold reduction to FcgRIIB, which is generally considered to be a negative signaling FcR. ONC-392 binding to mouse FcgRI-IV was lower that WT IgG1.ConclusionsUnlike other clinical anti-CTLA-4 antibodies, ONC-392 preserves CTLA-4 recycling and thus Treg function in the peripheral tissues. The higher cell surface CTLA-4 allows more efficient Treg depletion in the tumor microenvironment. In addition, despite reduced binding to mouse activating Fc?RI, III/IV, ONC-392 was more effective in intratumor Treg depletion in the mice. Therefore, lacking negative signaling from Fc?RIIB may also contribute to its anti-tumor activity.Trial RegistrationNCT04140526ReferencesDu X, et al. Uncoupling therapeutic from immunotherapy-related adverse effects for safer andeffective anti-CTLA-4 antibodies in CTLA4 humanized mice. Cell Res 2018;28:433–447.Du X, et al. A reappraisal of CTLA-4 checkpoint blockade in cancer immunotherapy. Cell Res 2018;28:416–432.Liu Y, Zheng P. How does an anti-CTLA-4 antibody promote cancer immunity? Trends Immunol 2018;39:953–956.Zhang Y, et al. Hijacking antibody-induced CTLA-4 lysosomal degradation for safer and more effective cancer immunotherapy. Cell Res 2019;29:609–627.Liu Y, Zheng P. Preserving the CTLA-4 checkpoint for safer and more effective cancer immunotherapy. Trends Pharmacol Sci 2020;41(1):4–12.Zhang P, et al. Mechanism- and immune landscape-based ranking of therapeutic responsiveness of 22 major human cancers to next generation anti-CTLA-4 antibodies. Cancers 2020;12:284.

857 Selective Treg depletion in solid tumors with ALD2510, a novel humanized CD25-specific, IL-2 sparing monoclonal antibody

BackgroundRegulatory T cells (Tregs) inhibit immune responses in solid cancers using cell-cell contacts and anti-inflammatory cytokine release. Also, due to high and constitutive levels of IL2Ralpha chain (CD25) expression, Tumor infiltrating (TIL)-Tregs cells preferably consume local Interleukin-2 (IL2), thus depriving conventional T cells from IL2-induced activation and proliferation. Therefore, the selective depletion of TIL-Tregs using therapeutic antibodies targeting CD25 represents a promising strategy to unleash tumor-specific immune responses in solid cancers.MethodsCD25 expression was evaluated by flow and mass cytometry on T -cell subsets from tumor biopsies collected in patients with various solid cancers (Breast, Endometrial and Cervix). ALD2510 potency was demonstrated in vitro and in vivo in human CD25 Knock-In huGEMM (huCD25-KI) MC38-bearing mice and in CD34+ humanized NSG mice grafted with human cancer cell lines (MDA-MB-231 and HT29).ResultsIn tumor biopsies, CD25 is highly and homogeneously expressed by TIL-Tregs, while being much less expressed by only a fraction of conventional CD4+ T cells and barely expressed by TIL-CD8+ cells. This confirms CD25 as the most selective marker to target TIL-Tregs in cancer patients.In vitro, ALD2510 shows potent ADCC and ADCP as well as strong Treg depletion capacity. Importantly, CD8+ and CD4+ conventional T cells are not impacted by ALD2510 even after activation confirming ALD2510 ability to selectively deplete Tregs. Accordingly, ALD2510 neither blocks IL-2 binding to CD25 nor inhibits IL-2 induced proliferation of activated T cells. In CD34+-humanized mice, ALD2510 efficiently depletes human Tregs but spares conventional T cells. Also, in the MC38 model in huCD25-KI mice, ALD2510 shows a strong anti-tumor activity as a single agent with 60% overall tumor growth inhibition together with massive Treg depletion 7 days after a single administration. In addition, combination of ALD2510 with anti-PD1 leads to complete tumor regression and strong activation of conventional T cells. Importantly, Basiliximab, a CD25-specific IL-2 blocking antibody, although efficient at depleting Treg cells, did not impact tumor growth, thus demonstrating that the IL-2 sparing feature of ALD2510 is critical to elicit anti-tumour response in vivo.ConclusionsThis preclinical data package supports CD25 as a potent and selective Treg marker allowing Tregs depletion while sparing conventional T cells. In this context, ALD2510, a novel humanized CD25-specific and IL-2 sparing antibody presents all the required attributes for selective and efficient TIL-Tregs depletion, making it a promising drug candidate to treat a broad range of solid tumor patients.Ethics ApprovalThe studies involving human material were approved by the ethical committee “Comité de Protection des Personnes Sud Méditerranée » under approval numbers 1362 and 1048. All participants gave informed consent before taking part.

746 Vectorized Treg-depleting anti-CTLA-4 elicits antigen cross-presentation and CD8+ T cell immunity to reject “cold” tumors

BackgroundImmune checkpoint blockade (ICB) is a clinically proven concept to treat cancer. Still, a majority of cancer patients including those with poorly immune infiltrated “cold” tumors are resistant to currently available ICB therapies. CTLA-4 is one of few clinically validated targets for ICB, but toxicities linked to efficacy in approved anti-CTLA-4 regimens have restricted their use and precluded full therapeutic dosing. At a mechanistic level, accumulating preclinical and clinical data indicate dual mechanisms for anti-CTLA-4; immune checkpoint blockade and Treg depletion are both thought to contribute efficacy and toxicity in available, systemic, anti-CTLA-4 regimens. Accordingly, strategies to deliver highly effective, yet safe, anti-CTLA-4 therapies have been lacking. Here, BioInvent and Transgene present and preclinically characterize a highly efficacious and potentially safe strategy to target CTLA-4 in the context of oncolytic virotherapy.MethodsA novel human IgG1 CTLA-4 antibody (4-E03) was identified using function-first screening for mAbs and targets associated with superior Treg depleting activity. A tumor-selective oncolytic Vaccinia vector was then engineered to encode this novel, strongly Treg-depleting, checkpoint-blocking, anti-CTLA-4 antibody and GM-CSF (VVGM-ahCTLA4, BT-001). Viruses encoding a matching Treg-depleting mouse surrogate antibody were additionally generated, enabling proof-of-concept studies in syngeneic immune competent mouse tumor models.ResultsOur studies demonstrate that intratumoral (i.t.) administration of VVGM-aCTLA4 achieved tumor-restricted CTLA-4 receptor saturation and Treg-depletion, which elicited antigen cross-presentation and stronger systemic expansion of tumor-specific CD8+ T cells and antitumor immunity compared with systemic anti-CTLA-4 antibody therapy. Efficacy correlated with FcgR-mediated intratumoral Treg-depletion and the reduction of exhausted CD8+ T cells. Remarkably, in a clinically relevant mouse model resistant to systemic immune checkpoint blockade, i.t. VVGM-aCTLA4 synergized with anti-PD-1 to reject “cold” tumors.ConclusionsOur findings demonstrate in vivo proof-of-concept for spatial restriction of strongly Treg-depleting, immune checkpoint blocking, vectorized anti-CTLA-4 as a highly effective and safe strategy to target CTLA-4 which is able to overcome current limitations of approved anti-CTLA-4 regimens. A clinical trial evaluating i.t. VVGM-ahCTLA4 (BT-001) alone and in combination with anti-PD-1 in metastatic or advanced solid tumors has commenced.Ethics ApprovalAll mouse experiments were approved by the local ethical committee for experimental animals (Malmö/Lunds djurförsöksetiska nämnd); at BioInvent under permit numbers 17196/2018 or 2934/2020; or at Transgene APAFIS Nr21622 project 2019072414343465 and performed in accordance with local ethical guidelines.

664 Pulmonary priming of tumor-reactive CD8+ T cells by DC1 is impaired by regulatory T cells

BackgroundAlthough failure to respond to checkpoint blockade immunotherapies (CBT) is frequently associated with a lack of T cell infiltration into the tumor, emerging clinical data suggests that specifically in patients with lung cancer, T cell-inflamed tumors can also be resistant to therapy.1 Recent work by our group identified that immunotherapy resistance in a T cell-inflamed pre-clinical mouse model of lung cancer is driven by a lung cancer-specific CD8+ T cell dysfunctional program (TLdys), characterized by blunted production of IFNg and reduced cytolytic capacity. Intriguingly, this TLdysprogram is established during priming in the tumor-draining mediastinal lymph nodes (mLN). Understanding the lung-specific mechanisms blunting the activation of anti-tumor T cell responses could enable development of novel therapies needed to improve outcomes of patients with CBT-resistant T cell-inflamed lung cancer.MethodsTo study anti-tumor immune responses against lung tumors, a syngeneic lung cancer cell line (KP) was implanted orthotopically or subcutaneously into C57BL/6 mice. KP cells were engineered to express SIINFEKL and ZsGreen to enable studies of tumor-reactive T cells and antigen uptake by dendritic cells (DC).ResultsLung KP tumors led to the induction of tumor-reactive TLdys CD8+ T cells lacking CD25 and GzmB in the mLN, in contrast to subcutaneous KP tumors, which induced CD25high GzmBhigh tumor-reactive CD8+ T cells in the inguinal LN (iLN). Mouse models lacking DC1 revealed that DC1 are necessary to prime tumor-reactive CD8+ T cells in both LNs. Flow cytometry characterization of DC1 from LNs revealed equivalent levels of antigen load, but reduced levels of costimulatory molecules CD80, CD86 and the cytokine IL-12 in the mLN compared to iLN, suggesting a blunted stimulatory capacity in the lung setting. Regulatory T cell (Treg) depletion using FoxP3DTR mice rescued expression of effector T cell priming in tumor-draining mLN, suggesting that TLdys induction requires the presence of local Treg. Ex vivo co-cultures of antigen-specific CD8+ T cells with DC1 and Treg sorted from the mLN fully recapitulated the in vivo observation, suggesting that both DC1 and Treg are required and sufficient for TLdys induction. Blockade of the MHCII-dependent DC1:Treg interaction restored an effector-like profile of tumor-reactive CD8+ T cells.ConclusionsTreg restrain DC1 stimulatory function in the tumor-draining mLN, leading to the induction of lung cancer-specific dysfunction in tumor-reactive CD8+ T cells and thus rendering the T cell response refractory to CBT-mediated reinvigoration. Blockade of Treg:DC1 interactions can restore priming of lung cancer-reactive effector T cell responses.AcknowledgementsPew-Stewart Scholarship, Training grantReferenceHerbst RS, et al. Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients. Nature 2014;515:563–567.Ethics ApprovalAll mouse experiments in this study were approved by MIT's Committee on Animal Care (CAC) - DHHS Animal Welfare Assurance # D16-00078

Transient regulatory T-cell targeting triggers immune control of multiple myeloma and prevents disease progression

Multiple myeloma remains a largely incurable disease of clonally expanding malignant plasma cells. The bone marrow microenvironment harbors treatment-resistant myeloma cells, which eventually lead to disease relapse in patients. In the bone marrow, CD4+FoxP3+ regulatory T cells (Tregs) are highly abundant amongst CD4+ T cells providing an immune protective niche for different long-living cell populations, e.g., hematopoietic stem cells. Here, we addressed the functional role of Tregs in multiple myeloma dissemination to bone marrow compartments and disease progression. To investigate the immune regulation of multiple myeloma, we utilized syngeneic immunocompetent murine multiple myeloma models in two different genetic backgrounds. Analyzing the spatial immune architecture of multiple myeloma revealed that the bone marrow Tregs accumulated in the vicinity of malignant plasma cells and displayed an activated phenotype. In vivo Treg depletion prevented multiple myeloma dissemination in both models. Importantly, short-term in vivo depletion of Tregs in mice with established multiple myeloma evoked a potent CD8 T cell- and NK cell-mediated immune response resulting in complete and stable remission. Conclusively, this preclinical in-vivo study suggests that Tregs are an attractive target for the treatment of multiple myeloma.

Global anti-tumor immunity after localized, bioengineered Treg depletion

Over 90% of deaths from cancer occur due to solid tumors, occurring at a rate of ∼1,500 deaths per day in the US, highlighting a profound and unmet need for new therapies. Solid tumors evade clearance by T cells due to a variety of immunosuppressive properties of the tumor microenvironment. However, this immunosuppression cannot be easily blocked on a global level because systemic activation of the immune system elicits a host of complications. An ideal therapy for solid tumors would act locally to activate the immune response without evoking global adverse effects. Here we present a biodegradable, macroporous scaffold that is implanted adjacent to the tumor and suppresses the main obstacle to cancer immunosurveillance: intratumoral regulatory T cells. The scaffold also promotes the recruitment and activation of T cell effectors into the tumor, resulting in clearance of otherwise aggressive and fatal tumors in mice. Unexpectedly, the local depletion of Tregs results in an “immunological abscopal effect” acting on distant tumors. We demonstrate that this versatile platform can also deliver tumor-antigen-specific T cells directly to the peri-tumoral environment, bypassing difficulties in intravenous delivery including the environmental barriers imposed by the tumor’s vasculature. By orchestrating multiple local immunomodulatory treatments, this scaffold offers a general approach to engineer T-cell responses to solid tumors without systemic toxicities.

Lack of blocking activity in anti-CTLA-4 antibodies reduces toxicity, but not anti-tumor efficacy

Anti-CTLA-4 antibodies such as ipilimumab were among the first immune-oncology agents to show significantly improved outcomes for patients. However, existing anti-CTLA-4 therapies fail to induce a response in a majority of patients and can induce severe, immune-related adverse events. It has been assumed that checkpoint inhibition, i.e., blocking the interaction between CTLA-4 and its ligands, is the primary mechanism of action for ipilimumab. In this study we present evidence that checkpoint inhibition is not a primary mechanism of action for efficacy of anti-CTLA-4 antibodies. Instead, the primary mechanism for efficacy is FcR-mediated Treg depletion in the tumor microenvironment. First, we identified a monoclonal antibody (mAb) that binds to CTLA-4 at an epitope that differs from ipilimumab's by only a few amino acids, yet has limited checkpoint inhibitor activity. Surprisingly, the weak checkpoint inhibitor has superior anti-tumor activity compared to ipilimumab in a murine model. The weak checkpoint inhibitor also induces less Treg proliferation and has increased ability to induce in vitro FcR signaling and in vivo depletion of intratumoral Tregs. Further experiments showed that the enhanced FcR activity of the weak checkpoint inhibitor likely contributes to its enhanced anti-tumor activity. Importantly, we also showed that weak checkpoint inhibition was associated with lower toxicity in murine models. Our work suggests that new anti-CTLA-4 drugs should be optimized for Treg depletion rather than checkpoint inhibition.

KY1044 to target the ICOS pathways inducing intratumoral Treg depletion and agonism of effector T cells: Preliminary pharmacodynamic markers from a phase 1/2 multicenter trial.

2626 Background: Inducible T-cell co-stimulator (ICOS) is an important co-stimulatory receptor on effector T cells (Teffs) that also promotes tumor growth due to its high expression on regulatory T cells (Tregs). KY1044 is a fully human IgG1 that targets ICOS, acting via a dual mode of action (MoA) by depleting ICOShigh Tregs and stimulating ICOSLow Teffs. A Phase 1/2 clinical trial (NCT03829501) is currently assessing the safety and preliminary efficacy of KY1044, as a single agent and in combination with atezolizumab, in subjects with advanced relapsed/refractory malignancies. Using longitudinal blood samples and tumor biopsies, we aim to correlate KY1044 target engagement levels with pharmacodynamic (PD) properties (e.g. dual MoA) in the tumor microenvironment (TME) and the circulation. Methods: Phase 1 subjects were enrolled in dose escalation and enrichment cohorts to evaluate the effect of KY1044 as monotherapy (0.8 – 240 mg) Q3W and in combination (0.8 – 80 mg) with atezolizumab (1200 mg) Q3W. PBMCs, plasma and tumor biopsies were collected over the first 3 cycles to confirm target engagement and KY1044 MoA. The sample analysis included: immunohistochemistry (IHC) of tumor samples (ICOS, FOXP3 and CD8); circulating T cell immunoprofiling and receptor occupancy by chip-cytometry; PBMC and tumor sample pre- and post-treatment transcriptomic analysis; and the assessment of circulating cytokines (e.g. GM-CSF). Results: As assessed in PBMCs, full/prolonged ICOS target engagement on T cells was confirmed in subjects receiving a flat dose of 8 to 240 mg, while partial/transient saturation was observed at lower doses (0.8-2.4 mg). The target engagement was not affected by atezolizumab. The immune cell profiling showed changes in some populations, but there was no significant depletion of peripheral ICOS+ cells. In contrast, pre- and post-treatment IHC analysis of ICOS+/FOXP3+ cells in tumor biopsies confirmed a KY1044-dose dependent reduction of ICOS+ Tregs and maintenance of CD8+ T cells in the TME. Together, this resulted in an increased intratumoral CD8+/ICOS+ Treg ratio at all doses, plateauing from subjects receiving a flat KY1044 dose of 8 mg. KY1044-dependent agonism was indirectly assessed by measuring circulating cytokine levels. A post-dosing transient induction of GM-CSF was evident in subjects dosed with KY1044 at the 0.8 and 2.4 mg dose, whereas minimal induction was observed at dose of 8 mg and higher. Conclusions: LongitudinalPDdata confirmed the expected KY1044 MoA, namely ICOS Treg depletion and increased CD8/ICOS Treg ratio in the TME as well as T cell co-stimulation. The observed PD responses are currently being further explored in a more homogenous patient population.