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Background: PD-1 blockade (αPD-1) has shown limited efficacy in breast & gynecologic cancers. In other types of solid tumor, regulatory T cells (Tregs) harbouring markers of highly suppressive effector Tregs (eTregs) correlates with poor responses to PD-1 blockade, prompting combination therapy based on eTregs depletion. Yet, subsets of Tregs remain to be determined in breast & gynecologic cancers, in order to: (i) evaluate the role of eTregs in resistances to PD-1 blockade, (ii) identify the most selective target for eTregs depletion tailored to tumors context and combination strategy. Methods: We collected public single-cell RNA/TCR-sequencing data from primary tumors and metastases of Triple-Negative Breast Cancer patients (TNBC, N = 28) biopsied before and after αPD-1 (pembrolizumab). Microarray data from primary TNBC (N = 124) biopsied before and after αPD-1 were quired for association with clinical responses. Deep phenotyping of Tregs from normal breast tissues (N = 4) and primary breast tumors (N = 8) or gynecologic tumors (N = 17) was performed by mass cytometry. ALD2510 (αCD25NIB, Alderaan Biotechnology) is a novel non-IL-2 blocking Fc-optimized anti-CD25 mAb (1). αPD-1 and αCD25NIB combination was evaluated using: (i) a humanized αCD25NIB in CD34+ humanized NSG mice grafted with human TNBC cell lines, (ii) a murine surrogate of αCD25NIB in a syngeneic TNBC mouse model. Results: CD25high Tregs were accumulating in breast & gynecologic tumor tissues. In TNBC patients treated with αPD-1, fractions of CD25high Tregs were further increased. CD25high Tregs highly expressed eTregs-related molecules in primary tumors, invaded tumor-draining lymph nodes and distant metastases. Amongst the potential therapeutic targets for Treg depletion, only CD25, 4-1BB and CCR8 were largely restricted to intratumoral CD25high eTregs. Yet, CD25 was the best candidate for CD25high eTregs depletion with limited on-target off-Treg effects, as: (i) CCR8 marked a small fractions of CD25high eTregs in primary tumors and was not detected in metastasis, (ii) 4-1BB marked antigen-experienced αβCD8 T cell revigorated by PD-1 blockade, (iii) only CD25high eTregs expressed 4-1BB and CCR8, yet TCR clonotype analysis revealed that CD25high eTregs originate in part from activated CD25+ Tregs, suggesting that anti-CCR8/-4-1BB mAbs may be ineffective due to CD25high eTregs replenishment in tumors. In murine models resistant to αPD-1 alone, αCD25NIB effectively depleted intratumoral CD25high eTregs, with limited effects on peripheral Tregs, and synergized with αPD-1 by restoring a positive effector αβCD8 T cells/Tregs ratio, leading to tumor clearance without adverse effects. Conclusions: This study supports clinical evaluation of CD25high effector Tregs depletion by ALD2510 in patients with breast or gynecologic cancers resistant to PD-1 blockade. Citation Format: Stéphane FATTORI, Aude Le Roy, Jemila Houacine, Lucie Robert, Riad Abes, Laurent Gorvel, Samuel Granjeaud, Marie-Sarah Rouvière, Amira Ben Amara, Nicolas Boucherit, Carole Tarpin, Jihane Pakradouni, Emmanuelle Charafe-Jauffret, Julien Barrou, Gilles Houvenaeghel, Eric Lambaudie, François Bertucci, Philippe Rochigneux, Anthony Gonçalves, Arnaud Foussat, Anne-Sophie Chrétien, Daniel Olive. Selective depletion of regulatory T cells by ALD2510, a novel IL-2-sparing anti-CD25 antibody, synergizes with PD-1 blockade in breast and gynecologic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3239.

Within the tumor microenvironment (TME), TREGS have been associated with worse prognosis and/or resistance to checkpoint inhibitor (CPI) therapy in various cancer diseases, making these cells a promising target for the development of new immunotherapies. Here, we present recent preclinical development data about ALD2510, a next-gen low-fucose IL-2/TCONV-sparing anti-CD25 antibody designed for the selective depletion of CD25high TREGS and the boost of host anti-tumor immunity. Ultra-humanization of parental anti-CD25 antibody was achieved through phage display and, in parallel, all putative liability sequences were replaced for maximizing developability while minimizing off-target and immunogenicity risks. ALD2510 sequences were re-formatted and used for further CHO cell line development, using the GlymaxX™ technology which boosts Fc effector functions. Early tox ALD2510 material was produced following 10L fed-batch USP/DSP and analytical characterization, including a 6-month early stability study. Preliminary safety and pharmacokinetic/pharmacodynamic (PKPD) profile of ALD2510 were then determined through a non-GLP MTD/DRF study in cynomolgus monkey. ALD2510 demonstrated excellent potency (TREG depletion, ADCC, ADCP), manufacturability and stability together with very low immunogenicity potential. CHO productivity reached ~4g/L in 10L fed-batch bioreactor and characterization revealed excellent purity and activity in line with its low fucose content, together with very low levels of process- or cell-related impurities. This material showed a good behavior during the 6-month stability study and was therefore administered in cynomolgus monkeys during a non-GLP exploratory MTD/DRF study. Increasing doses of ALD2510 (from 0.3 to 100mg/kg) were given to four animals during MTD and same animals received three additional doses at 100mg/kg during DRF. Very good tolerability and safety were reported in animals. ALD2510 half-life and exposure were found consistent with that of humanized IgG1 in cynomolgus. Strong TREG depletion was monitored in the blood in all animals with, importantly, no drop was observed in CD4+ or CD8+ T-cells, confirming ALD2510 capacity to selectively target TREGS while sparing TCONV. Overall, ALD2510 demonstrated excellent potency, manufacturability and stability during the 1st phase of preclinical development. Very good tolerability and safety profile together with satisfying PKPD data were reported during exploratory MTD/DRF study in cynomolgus monkeys. Noteworthy, strong TREG depletion was confirmed while CD4+ or CD8+ T-cells were not impacted, confirming ALD2510 selectivity for TREGS while sparing TCONV. This makes ALD2510 a promising candidate for the treatment of solid tumors, in particular those where the presence of TREGS in the TME is associated with worse prognosis and/or resistance to CPI. Citation Format: Jemila Houacine, Riad Abes, Anne Marie-Cardine, Aude Le Roy, Bettina Serbin, Lucie Robert, Jérôme Giustiniani, Anne-Sophie Chrétien, Stéphane Fattori, Laurent Gorvel, Armand Bensussan, Daniel Olive, Arnaud Foussat. Preclinical development of ALD2510, a Next-Gen Fc-enhanced, TREG-selective and IL-2-sparing anti-CD25 antibody for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 710.

Regulatory T cells (Treg) are an immunosuppressive subtype of CD4+ T cells essential for maintaining self-tolerance in physiological settings. Tregs also abundantly infiltrate inflamed tumor tissues, impeding the host's antitumor immune response and contributing to tumor growth and metastasis. In breast cancers, subsets of Tregs express highly immunosuppressive effector phenotypes that favor tumorigenesis, progression, and resistance to immune-checkpoint inhibitor therapies. Tregs share phenotypic features with cytotoxic lymphocytes, rendering them difficult to inhibit without compromising productive antitumor immunity. In addition, systemic targeting of Tregs causes serious autoimmune adverse events in patients with cancer. Hence, the identification of candidate targets or methodologies allowing the specific elimination of tumor antigen-specific Tregs, including tumor-infiltrating Tregs, is a prerequisite for developing efficient and safe combinatorial immunotherapeutic strategies in breast cancers. To date, numerous preclinical studies have demonstrated that specific targeting of breast tumor–infiltrating Tregs restores a competent antitumor immune response and improves responses to immune-checkpoint inhibitors such as PD-1/PD-L1 blockade. Herein, we discuss major candidate molecules for Treg-targeted therapeutic strategies in breast cancers, detailing the pros and cons of various approaches, including mAb-mediated depletion, homeostasis destabilization, and functional blockade.

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.

Pathological amino acid substitutions in the amyloid precursor protein (APP) and chemical ? secretase modulators affect the processing of APP by the ? secretase complex and the production of the amyloid beta peptide Aß42 the accumulation of which is considered causative of Alzheimer's disease. Here we demonstrate that mutations in the transmembrane domain of APP causing aggressive early onset familial Alzheimer's disease affect both ? and e cleavage sites by raising the Aß42/40 ratio and inhibiting the production of AICD50 99 one of the two physiological APP intracellular domains (ICDs). This is in sharp contrast to ? secretase modulators which shift Aß42 production towards the shorter Aß38 but unequivocally spare the e site and APP and Notch ICDs production. Molecular simulations suggest that familial Alzheimer's disease mutations modulate the flexibility of the APP transmembrane domain and the presentation of its ? site modifying at the same time the solvation of the e site. © 2009 2012 IEEE.