anti-CD4 mAb Research Articles

Short-term cultured tumor fragments to study immunotherapy combinations based on CD137 (4-1BB) agonism

ABSTRACT Biomarkers for cancer immunotherapy are an unmet medical need. The group of Daniela Thommen at the NKI recently reported on novel methodologies based on short-term cultures of patient-derived tumor fragments whose cytokine concentrations in the supernatants and activation markers on infiltrating T cells were associated with clinical response to PD-1 blockade. We set up a similar culture technology with tumor-derived fragments using mouse tumors transplanted into syngeneic immunocompetent mice to test an agonist anti-CD137 mAb and its combinations with anti-PD-1 and/or anti-TGF-β. Increases in IFNγ concentrations in the tissue culture supernatants were detected upon in-culture activation with the anti-CD137 and anti-PD-1 mAb combinations or concanavalin A as a positive control. No other cytokine from a wide array was informative of stimulation with these mAbs. Interestingly, increases in Ki67 and other activation markers were substantiated in lymphocytes from cell suspensions gathered at the end of 72 h cultures. In mice bearing bilateral tumors in which one was excised prior to in vivo anti-CD137 + anti-PD-1 treatment to perform the fragment culture evaluation, no association was found between IFNγ production from the fragments and the in vivo therapeutic outcome in the non-resected contralateral tumors. The experimental system permitted freezing and thawing of the fragments with similar functional outcomes. Using a series of patient-derived tumor fragments from excised solid malignancies, we showed IFNγ production in a fraction of the studied cases, that was conserved in frozen/thawed fragments. The small tumor fragment culture technique seems suitable to preclinically explore immunotherapy combinations.

Inflammatory Profiles Induced by Intranasal Immunization with Ricin Toxin-immune Complexes.

The underlying contribution of immune complexes in modulating adaptive immunity in mucosal tissues remains poorly understood. In this report, we examined, in mice, the proinflammatory response elicited by intranasal delivery of the biothreat agent ricin toxin (RT) in association with two toxin-neutralizing mAbs, SylH3 and PB10. We previously demonstrated that ricin-immune complexes (RICs) induce the rapid onset of high-titer toxin-neutralizing Abs that persist for months. We now demonstrate that such responses are dependent on CD4+ T cell help, because treatment of mice with an anti-CD4 mAb abrogated the onset of RT-specific Abs following intranasal RICs exposure. To define the inflammatory environment associated with RIC exposure, we collected bronchoalveolar lavage fluid (BALF) and sera from mice 6, 12, and 18 h after they had received RT or RICs by the intranasal route. A 32-plex cytometric bead array revealed an inflammatory profile elicited by RT that was dominated by IL-6 (>1500-fold increase in BALF) and secondarily by KC (CXCL1), G-CSF, GM-CSF, and MCP-1. RICs induced inflammatory profiles in both BALF and serum response that were similar to RT, albeit at markedly reduced levels. These results demonstrate that RICs retain the capacity to induce local and systemic inflammatory cytokines/chemokines that, in turn, may influence Ag sampling and presentation in the lung mucosa and draining lymph nodes. A better understanding of the fate of immune complexes following intranasal delivery has implications for the development of mucosal vaccines for biothreats and emerging infectious diseases.

Non-allergic eosinophilic inflammation and airway hyperresponsiveness induced by diesel engine exhaust through activating ILCs

Diesel engine exhaust (DEE) is associated with the development and exacerbation of asthma. Studies have shown that DEE can aggravate allergen-induced eosinophilic inflammation in lung. However, it remains not clear that whether DEE alone could initiate non-allergic eosinophilic inflammation and airway hyperresponsiveness (AHR) through innate lymphoid cells (ILCs) pathway. This study aims to investigate the airway inflammation and hyperresponsiveness and its relationship with ILC after DEE exposure. Non-sensitized BALB/c mice were exposed in the chamber of diesel exhaust or filtered air for 2, 4, and 6 weeks (4 h/day, 6 days/week). Anti-CD4 mAb or anti-Thy1.2 mAb was administered by intraperitoneal injection to inhibit CD4+T or ILCs respectively. AHR、airway inflammation and ILCs were assessed. DEE exposure induced significantly elevated level of neutrophils, eosinophils, collagen content at 4, 6 weeks. Importantly, the airway AHR was only significant in the 4weeks-DEE exposure group. No difference of the functional proportions of Th2 cells was found between exposure group and control group. The proportions of IL-5+ILC2, IL-17+ILC significantly increased in 2, 4weeks-DEE exposure group. After depletion of CD4+T cells, both the proportion of IL-5+ILC2 and IL-17A ILCs was higher in the 4weeks-DEE exposure group which induced AHR, neutrophilic and eosinophilic inflammation accompanied by the IL-5, IL-17A levels. Diesel engine exhaust alone can imitate asthmatic characteristics in mice model. Lung-resident ILCs are one of the major effectors cells responsible for a mixed Th2/Th17 response and AHR.

Experimental evidence that carbon-ion radiotherapy utilizes cytotoxic T lymphocyte-mediated anti-tumor immunity for shrinking tumors compared to X-ray therapy

The therapeutic efficacy of radiotherapy (RT) is primarily driven by two factors: biophysical DNA damage in cancer cells and radiation-induced anti-tumor immunity. However, Anti-tumor immune responses between X-ray RT (XRT) and carbon-ion RT (CIRT) remain unclear. In this study, we, employed mouse models to assess the immunological contribution, especially cytotoxic T-lymphocyte (CTL)-mediated immunity, to the therapeutic effectiveness of XRT and CIRT in shrinking tumors. We irradiated mouse intradermal tumors of B16F10-ovalbumin (OVA) mouse melanoma cells and 3LL-OVA mouse lung cancer cells with carbon-ion beams or X-rays in the presence or absence of CTLs. CTL removal was performed by administration of anti-CD8 monoclonal antibody (mAb) in mice. Based on tumor growth delay, we determined the tumor growth and regression curves. The enhancement ratio (ER) of the slope of regression lines in the presence of CTLs, relative to the absence of CTLs, indicates the dependency of RT on CTLs for shrinking mouse tumors, and the biological effectiveness (RBE) of CIRT relative to XRT were calculated. Tumor growth curves revealed that the elimination of CD8+ CTLs by administrating anti-CD8 mAb accelerated tumor growth compared to the presence of CTLs in both RTs. The ERs were larger in CIRT compared to XRT in the B16F10-OVA tumor models, but not in the 3LL-OVA models, suggesting a greater contribution of CTL-mediated anti-tumor immunity to tumor reduction in CIRT compared to XRT in the B16F10-OVA tumor model. In addition, the RBE values for both models were larger in the presence of CTLs compared to models without CTLs, suggesting that CIRT may utilize CTL-mediated anti-tumor immunity more than X-ray. The findings from this study suggest that although immunological contribution to therapeutic efficacy may vary depending on the type of tumor cell, CIRT utilizes CTL-mediated immunity to a greater extent compared to XRT.

Peripherally activated brain CD8+ T cells promote AD in 5xFAD mice

Abstract The role of the adaptive immunity in Alzheimer’s Disease (AD) remains poorly understood. Recent findings from our group, which link B cells to manifestation AD 1, and others, which show increase of CD8+ T cells in the AD brain 2, suggest that the adaptive immunity is an important but overlooked player in this progressive neurodegenerative disease. Here, our aim was to determine the role of CD8+ T cells in the brain in transgenic 5xFAD mice, which develop human AD-like symptoms. We manipulated brain CD8+ T cells in AD mice by depleting circulating CD8+ T cells with anti-CD8 mAb or expanding antigen-specific CD8+ T cells with Aβ-CoreS (encodes Ab1-11) RNA immunization. We found that while the CD8+ T cell depletion decreases Aβ plaques and disease-associated microglia in the hippocampus, and improves cognition, Aβ-CoreS exacerbates AD symptoms via increasing brain CD8+ T cells with cytotoxic and exhausted phenotype. Overall, our results indicate the AD manifestation requires brain antigen-specific CD8+ T cells, which upon peripheral induction/expansion infiltrate the brain and exacerbate AD symptoms. We propose that CD8+ T cells may similarly exacerbate AD in humans, as we detected their significant increase in the brain of deceased people with AD. 1. Kim et al. Nat Commun. 2021;12(1):2185. 2. Gate et al. (2020). Nature 577, 399-404. This research was supported by the Intramural Research Program of the National Institute on Aging, NIH

Abstract CT249: Results from KEYMAKER-U01 substudy 3: a phase 2 umbrella study of pembrolizumab plus investigational agents in patients with non-small-cell lung cancer (NSCLC) previously treated with anti-PD-(L)1 therapy

Abstract Background: The anti-PD-1 monoclonal antibody (mAB) pembrolizumab with or without chemotherapy is a standard of care for first-line advanced or metastatic NSCLC and as monotherapy for NSCLC with PD-L1 TPS ≥1% previously treated with chemotherapy. However, there are limited treatment options for patients whose disease progresses after anti-PD-(L)1 therapy. We present results from the KEYMAKER-U01 study (NCT04165798) substudy 3, which evaluated pembrolizumab in combination with investigational mAbs for patients with NSCLC previously treated with anti-PD-(L)1 therapy. Methods: This randomized, phase 2, rolling-arm, multicenter, open-label, adaptive-design study enrolled patients aged ≥18 y with ECOG PS of 0 or 1 and confirmed stage IV squamous or nonsquamous NSCLC (per current AJCC) who progressed on or after prior anti-PD-(L)1 therapy and platinum-based chemotherapy given sequentially or concomitantly. Patients were randomized to receive up to 35 cycles (~2 y) of pembrolizumab 200 mg Q3W plus either the anti-CD27 mAb boserolimab 30 mg Q3W (arm A), the anti-ILT4 mAb MK-4830 800 mg Q3W (arm B), or the anti-ILT3 mAb MK-0482 750 mg Q3W (arm C). The primary endpoint was ORR per RECIST version 1.1 by investigator review. Secondary endpoints were PFS per RECIST version 1.1 by investigator review and safety (AEs and treatment discontinuation due to AEs). Results: Overall, 127 patients received ≥1 dose of study treatment in arms A (n = 37), B (n = 45), and C (n = 45). At data cutoff (Nov 10, 2023), median study follow-up was 35 (range, 31-45) mo, 33 (range, 30-36) mo, and 23 (range, 18-25) mo, respectively. ORR (95% CI) was 8% (2%-22%; 1 CR, 2 PR) in arm A, 11% (4%-24%; 1 CR, 4 PR) in arm B, and 4% (1%-15%; 0 CR, 2 PR) in arm C. Median (95% CI) PFS was 2.4 (1.4-2.8) mo, 2.4 (1.5-2.7) mo, and 2.6 (1.4-4.7) mo, respectively. AEs of any cause occurred in 37 patients (100%) in arm A, 42 (93%) in arm B, and 41 (91%) in arm C. These were grade 3-5 in 21 (57%), 20 (44%), and 17 patients (38%), respectively. Treatment-related AEs occurred in 27 patients (73%; grade 3/4, 16%) in arm A, 25 patients (56%; grade 3/4, 9%) in arm B, and 23 patients (51%; grade 3/4, 11%) in arm C. Six (16%), 8 (18%), and 3 patients (7%) discontinued treatment due to any AE. Grade 5 AEs occurred in 2 (5%), 4 (9%), and 2 patients (4%), respectively; none were deemed to be treatment related. Conclusions: In the KEYMAKER-U01 substudy 3 evaluating novel treatment combinations of pembrolizumab plus boserolimab, MK-4830, or MK-0482 in patients with NSCLC that progressed on/after platinum-based chemotherapy and anti-PD-(L)1 therapy, modest antitumor activity was observed with all 3 treatment combinations. No new safety signals were observed for the treatment combinations investigated. Citation Format: Charu Aggarwal, James Stevenson, Dariusz M. Kowalski, Tibor Csőszi, Nir Peled, Byoung Chul Cho, Belén Rubio-Viqueira, Jiaxin Niu, David P. Carbone, Susanne M. Arnold, Mark M. Awad, Justin F. Gainor, Suman Rao, Anne S. Tsao, Heng Zhou, Omobolaji O. Akala, Elliot Chartash, Ernest Nadal. Results from KEYMAKER-U01 substudy 3: a phase 2 umbrella study of pembrolizumab plus investigational agents in patients with non-small-cell lung cancer (NSCLC) previously treated with anti-PD-(L)1 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT249.

Differential induction of donor-reactive Foxp3+ regulatory T cell via blockade of CD154 vs CD40

Recently published studies in both murine models and a meta-analysis of non-human primate renal transplant studies showed that anti-CD154 reagents conferred a significant survival advantage over CD40 blockers in both animal models and across multiple organs. Here we sought to compare the induction of donor-reactive forkhead box P3+-induced regulatory T cells (Foxp3+ iTreg) in mice treated with anti-CD154 versus anti-CD40 monoclonal antibodies (mAbs). Results indicated that while treatment with anti-CD154 mAb resulted in a significant increase in the frequency of donor-reactive CD4+ Foxp3+ iTreg following transplantation, treatment with anti-CD40 or Cd40 deficiency failed to recapitulate this result. Because we recently identified CD11b as an alternate receptor for CD154 during alloimmunity, we interrogated the role of CD154:CD11b interactions in the generation of Foxp3+ iTreg and found that blockade of CD11b in Cd40−/− recipients resulted in increased donor-reactive Foxp3+ iTreg as compared with CD40 deficiency alone. Mechanistically, CD154:CD11b inhibition decreased interleukin (IL)-1β from CD11b+ and CD11c+ dendritic cells, and blockade of IL-1β synergized with CD40 deficiency to promote Foxp3+ iTreg induction and prolong allograft survival. Taken together, these data provide a mechanistic basis for the observed inferiority of anti-CD40 blockers as compared with anti-CD154 mAb and illuminate an IL-1β-dependent mechanism by which CD154:CD11b interactions prevent the generation of donor-reactive Foxp3+ iTreg during transplantation.

Antibody-Suppressor CXCR5+CD8+ T Cells Are More Potent Regulators of Humoral Alloimmunity after Kidney Transplant in Mice Compared to CD4+ Regulatory T Cells.

Adoptive cell therapy (ACT), especially with CD4+ regulatory T cells (CD4+ Tregs), is an emerging therapeutic strategy to minimize immunosuppression and promote long-term allograft acceptance, although much research remains to realize its potential. In this study, we investigated the potency of novel Ab-suppressor CXCR5+CD8+ T cells (CD8+ TAb-supp) in comparison with conventional CD25highFoxp3+CD4+ Tregs for suppression of humoral alloimmunity in a murine kidney transplant (KTx) model of Ab-mediated rejection (AMR). We examined quantity of peripheral blood, splenic and graft-infiltrating CD8+ TAb-supp, and CD4+ Tregs in KTx recipients and found that high alloantibody-producing CCR5 knockout KTx recipients have significantly fewer post-transplant peripheral blood and splenic CD8+ TAb-supp, as well as fewer splenic and graft-infiltrating CD4+ Tregs compared with wild-type KTx recipients. ACT with alloprimed CXCR5+CD8+ T cells reduced alloantibody titer, splenic alloprimed germinal center (GC) B cell quantity, and improved AMR histology in CCR5 knockout KTx recipients. ACT with alloprimed CD4+ Treg cells improved AMR histology without significantly inhibiting alloantibody production or the quantity of splenic alloprimed GC B cells. Studies with TCR transgenic mice confirmed Ag specificity of CD8+ TAb-supp-mediated effector function. In wild-type recipients, CD8 depletion significantly increased alloantibody titer, GC B cells, and severity of AMR pathology compared with isotype-treated controls. Anti-CD25 mAb treatment also resulted in increased but less pronounced effect on alloantibody titer, quantity of GC B cells, and AMR pathology than CD8 depletion. To our knowledge, this is the first report that CD8+ TAb-supp cells are more potent regulators of humoral alloimmunity than CD4+ Treg cells.

Comparative Inhibitory Effects of Tacrolimus, Cyclosporine, and Rapamycin on Human Anti-Pig Xenogeneic Mixed Lymphocyte Reactions.

Long-term immunosuppressive maintenance therapy is necessary to prevent the rejection of xenografts. However, it is still unclear which oral immunosuppressant is most suitable for pig-to-human xenotransplantation . A xenogeneic mixed lymphocyte reaction (MLR) system was established using peripheral blood mononuclear cells (PBMCs) isolated from wildtype (WT) or GTKO/CMAHKO/β4GalNT2KO (TKO) pigs as stimulator cells and human PBMCs as responder cells. Various concentrations of tacrolimus (Tac), cyclosporine (CsA), or rapamycin (Rapa) were added to the MLR system as interventions. The inhibitory effects of the three immunosuppressants on the proliferation and cytokine production of human T cells were studied and compared. The inhibitory effect of anti-CD154 mAb alone or in combination with Tac/CsA/Rapa on xenoreactive MLR was also investigated. PBMCs from both WT and TKO pigs stimulated significant proliferation of human T cells. Tac had a strong inhibitory effect on human T-cell proliferation stimulated by pig PBMCs. CsA inhibited human T-cell proliferation in a typical dose-dependent manner. When Tac and CsA concentrations reached 5 and 200ng/mL, respectively, the proliferation rates of CD3+/CD4+/CD8+ T cells were reduced almost to a negative level. Even at high concentrations, Rapa had only a moderate inhibitory effect on xenogeneic MLR. The inhibitory effects of these three immunosuppressants on xenogeneic T-cell responses were further confirmed by the detection of CD25 expression and supernatant cytokines (IL-2, IL-6, IFN-γ, TNF-α, IL-4, IL-10, and IL-17). Although anti-CD154 mAb monotherapy showed only moderate inhibitory effects on xenoreactive T-cell proliferation, low-dose anti-CD154 mAb combined with low-dose Tac, CSA, or Rapa could produce significant synergistic inhibitory effects. Tac is more efficient than CsA or Rapa in inhibiting xenogeneic T-cell responses in vitro. If used in combination with anti-CD154 mAb, all the three immunosuppressants can achieve satisfactory synergistic inhibitory effects.

Abstract B034: CD27 is a new promising T cell co-stimulatory target for cancer immunotherapy

Abstract Members of the tumor necrosis factor receptor superfamily (TNFRSF) are key co-stimulators of T cells. CD27, a member of the TNFRSF, is expressed only on the surface of lymphocytes, including naive and activated CD4+ and CD8+ T cells as well as NK cells. It enhances T cell activation, proliferation, and differentiation of effector and memory T cells after stimulation with its ligand, CD70. The costimulatory signal of CD27 is mediated via the NFkB pathway but also via the phosphatidylinositol 3 kinase and the protein kinase B pathways. CD27 signaling also influences the innate immune response via direct activation of NK cells and subsequent secretion of interferon-gamma (IFNg). Several published preclinical studies demonstrated that anti-CD27 agonistic monoclonal antibodies can promote T-cell activation and antitumor immunity making CD27 an attractive cancer immunotherapy target. Here we describe the characterization, preclinical development, and selection of our anti-CD27 fully human monoclonal antibody (mAb) lead candidate. We selected this candidate from a library of 147 anti-CD27 mAbs generated after immunization of humanized Trianni® mice with soluble human CD27 extracellular domain (hCD27-ECD). Anti-CD27 mAbs were tested in an accelerated stability study and showed excellent stability parameters for up to 7 days at 4 and 37°C in commonly used formulation buffers. The selected agonist anti-CD27 mAb demonstrated high affinity binding to both human and cynomolgus monkey CD27 and not to mouse CD27. It also demonstrated high specificity against CD27 with no cross-reactivity detected against other members of the TNFRSF. This lead candidate did not block the binding of CD27 natural ligand, CD70 and induced strong NFkB-mediated CD27 signaling in the absence or presence of cross-linking by Fc gamma receptors or secondary cross-linking antibodies. Moreover, this anti-CD27 mAb mediated NFkB activation is significantly potentiated by the addition of a sub-optimal amount of soluble CD70. The anti-CD27 lead mAb induced T cell proliferation and secretion of pro-inflammatory cytokines only in the presence of sub-optimal TCR stimulation in vitro using primary human T cells. It also activated NK cells demonstrated by CD69 expression induction. The anti-CD27 mAb lead candidate showed extended serum half-life in hCD27-KI mice. It also demonstrated a significant antitumor effect as a single agent in human CD27-Knockin mice (hCD27-KI) subcutaneously implanted with MC38 or in NOD-SCID mice subcutaneously implanted with Raji. These preclinical results establish that the selected anti-CD27 mAb is a promising drug candidate and we are actively pursuing its development. Citation Format: Thierry Guillaudeux, Yulia Ovechkina, Kurt Lustig, Shawn Iadonato. CD27 is a new promising T cell co-stimulatory target for cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B034.

CD137 (4-1BB) requires physically associated cIAPs for signal transduction and antitumor effects.

CD137 (4-1BB) is a member of the TNFR family that mediates potent T cell costimulatory signals upon ligation by CD137L or agonist monoclonal antibodies (mAbs). CD137 agonists attain immunotherapeutic antitumor effects in cancer mouse models, and multiple agents of this kind are undergoing clinical trials. We show that cIAP1 and cIAP2 are physically associated with the CD137 signaling complex. Moreover, cIAPs are required for CD137 signaling toward the NF-κB and MAPK pathways and for costimulation of human and mouse T lymphocytes. Functional evidence was substantiated with SMAC mimetics that trigger cIAP degradation and by transfecting cIAP dominant-negative variants. Antitumor effects of agonist anti-CD137 mAbs are critically dependent on the integrity of cIAPs in cancer mouse models, and cIAPs are also required for signaling from CARs encompassing CD137's cytoplasmic tail.

Enolase 1 of Candida albicans binds human CD4+ T cells and modulates naïve and memory responses.

To obtain a better understanding of the biology behind life-threatening fungal infections caused by Candida albicans, we recently conducted an in silico screening for fungal and host protein interaction partners. We report here that the extracellular domain of human CD4 binds to the moonlighting protein enolase 1 (Eno1) of C. albicans as predicted bioinformatically. By using different anti-CD4 monoclonal antibodies (mAb), we determined that C. albicans Eno1 (CaEno1) primarily binds to the extracellular domain 3 of CD4. Functionally, we observed that CaEno1 binding to CD4 activated lymphocyte-specific protein tyrosine kinase (LCK), which was also the case for anti-CD4 mAb tested in parallel. CaEno1 binding to naïve human CD4+ T cells skewed cytokine secretion towards a Th2 profile indicative of poor fungal control. Moreover, CaEno1 inhibited human memory CD4+ T cell recall responses. Therapeutically, CD4+ T cells transduced with a p41/Crf-1-specific TCR developed for adoptive T cell therapy were not inhibited by CaEno1 in vitro. Together, the interaction of human CD4+ T cells with CaEno1 modulated host CD4+ T cell responses in favor of the fungus. Thus, CaEno1 mediates not only immune evasion through its interference with complement regulators but also through direct modulation of CD4+ T cell responses. This article is protected by copyright. All rights reserved.

Establishing a novel and sensitive assay for bioactivity determination of anti-CD25 antibodies

Anti-CD25 antibodies have been approved for renal transplantation and has been used prior to and during transplantation by the Food and Drug Administration (FDA). However, no reported bioassays have been reflected the mechanism of action (MOA) of anti-CD25 antibodies. Here, we describe the development and validation of a reporter gene assay (RGA) based on the engineered C8166-STAT5RE-Luc cells expressing endogenous IL-2 receptors and a STAT5-inducible element-driven firefly luciferase in C8166 cell lines. The RGA was fully validated according to the International Conference on the Harmonization of Technical Requirements for the Registration of Pharmaceuticals for the Human Use-Q2 (ICH-Q2). After optimization, the assay showed excellent specificity, linearity, accuracy, precision, and robustness. Due to the MOA relatedness and the excellent assay performance, the RGA is suitable for exploring the critical quality attributes (CQAs), release inspection, comparability and stability of anti-CD25 mAbs.

CD4 T cell deficiency narrows virus-neutralizing IgG coverage for Polyomavirus and selects for antibody-escape variants

Abstract CD4 T cell deficiencies predispose patients to progressive multifocal leukoencephalopathy (PML), a brain disease caused by JC polyomavirus (JCPyV). PML is linked to immunomodulatory therapies that preferentially lower CD4 T cells in the CSF (natalizumab); but how CD4 T cells control JCPyV is undefined. “Blind spots” in the ability to neutralize JCPyV variants with mutations in the VP1 capsid protein were described in PML patients. We recently reported that a VP1 mAb selects for VP1-mutant escape variants both in vitroand in vivo; some of these variant viruses are neurovirulent. We hypothesized that alterations in the Ab response due to CD4 T cell insufficiency underlie emergence of Ab-escape mouse polyomavirus (MuPyV) variants. T-cell independent (TI) VP1-specific IgG (via anti-CD4 mAb depletion, CD40L blockade, MHCII KO mice, IL21R KO mice) neutralizes MuPyV but has reduced titer and avidity than anti-VP1 IgGs in CD4 T cell-sufficient mice. Serial passaging of MuPyV in vitrowith sera from TI mice selected an Ab-escape VP1 mutant containing amino acid deletions at residue 145–146 in an external loop of VP1 (MuPyV.145–146). In contrast to sera from healthy MuPyV-infected mice, TI sera failed to neutralize MuPyV.145–146. Depletion of CD4 T cells during persistent MuPyV infection, as a model of acquired CD4 T cell insufficiency (i.e. onset of immunomodulatory mAbs) via anti-CD4 mAb depletion, CD40L blockade, and anti-CD20 mAb B cell depletion also reduced anti-VP1 IgG titer and avidity. Ongoing studies seek to determine if a similar loss in recognition of the 145–146 VP1 epitope occurs. Our data shows that a narrowed antiviral IgG response due to CD4 T cell deficiency is a critical antecedent for outgrowth of Ab-escape PyV VP1 mutant viruses. Supported by grants from the NIH (5R01NS088367, 5R01NS092662, and R35NS127217) (AEL) Judy S. Finkelstein Memorial Award (KNA) T32 CA06395 (KNA)

Abstract 1546: ADCC enhanced anti-CD25 mAb (TST010) demonstrated antitumor activity via depleting Treg cells and increasing CD8+T/Treg ratio in preclinical tumor models

Abstract Regulatory T (Treg) cells are a distinctive lineage of CD4+ T cells as characterized by expression of FoxP3 and CD25 markers. Treg cells are involved in tumor development and progression by inhibiting antitumor immunity. Accumulation of Treg cells in the tumor microenvironment (TME) is often associated with an adverse prognosis in multiple cancers and also has implication for predicting response to immunotherapy. Treg cell depletion strategy has been explored in preclinical and clinical settings to induce effective antitumor immune responses in TME. CD25, a high-affinity binding subunit alpha of the IL-2 receptor, is constitutively and highly expressed on Treg cells but also transiently upregulated on effector T cells. The high level of CD25 expression on Treg cells could deprive of IL-2 for effector T cells and inhibit their proliferation. Thus, an effective approach to selectively suppress Treg cell function is to deplete Treg cells while avoid blocking IL-2 binding to CD25 for allowing T effect cell proliferation and activation. Using our immune tolerance breaking antibody platform, we generated a humanized IgG1 subtype anti-CD25 monoclonal antibody (TST010). TST010 bound to human CD25 with high affinity and selectivity but did not block IL-2 binding to CD25. No blocking activity to IL-2 signaling on human PBMC was further confirmed by the phosphorylation of Stat5 using FACS analysis. By reducing fucosylation during cell culture process, TST010 mAb gained the enhanced ADCC activity. In an ADCC reporter assay, TST010 showed its sub-nanomolar ADCC activity against CD25-overexpression SU-DHL-1 cell line and human induced Treg cells, but much less to human activated CD8+ T effect cells due to lower CD25 expression. Similarly, in a NK cytotoxicity assay, TST010 selectively killed human induced Treg cells in contrast to activated CD8+ T cells. In vivo antitumor activity of TST010 was evaluated in the syngeneic mouse tumor models. The mouse colon cancer MC38 model was developed on the human CD25 knock-in mice. In the MC38 tumor model, TST010 markedly reduced the Treg (FoxP3+CD4+) population in both peripheral blood and TME. Importantly, the CD8+T/Treg ratio in tumor was increased starting at 2 weeks post TST010 treatment, and it resulted in a significant single agent tumor growth inhibition at a 10 mg/kg dose. Furthermore, in the LLC tumor model which does not respond well to checkpoint inhibitors, the anti-CD25 mAb showed a good combination effect with a PD-L1 inhibitor. In summary, we have discovered a novel therapeutic anti-CD25 mAb that can deplete Treg cells without blocking IL-2 signaling. As demonstrated in our preclinical tumor models, it has a good potential to induce effective antitumor immune responses in TME and tumor growth inhibition especially in combination with PD-1/PD-L1 treatment. Citation Format: Di Sun, Huanhuan Guo, Hongjun Li, Xinlai Yao, Yiqing Xu, Shenjie Zhang, Shijie Zhou, Xiaoli Zi, Lisa Zheng, Yi Gu, Xueming Qian. ADCC enhanced anti-CD25 mAb (TST010) demonstrated antitumor activity via depleting Treg cells and increasing CD8+T/Treg ratio in preclinical tumor models [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 1546.

Abstract 3239: 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 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.

Synergistic effects of combined immunotherapy strategies in a model of multifocal hepatocellular carcinoma

Immune checkpoint-inhibitor combinations are the best therapeutic option for advanced hepatocellular carcinoma (HCC) patients, but improvements in efficacy are needed to improve response rates. We develop a multifocal HCC model to test immunotherapies by introducing c-myc using hydrodynamic gene transfer along with CRISPR-Cas9-mediated disruption of p53 in mouse hepatocytes. Additionally, induced co-expression of luciferase, EGFP, and the melanosomal antigen gp100 facilitates studies on the underlying immunological mechanisms. We show that treatment of the mice with a combination of anti-CTLA-4+ anti-PD1 mAbs results in partial clearance of the tumor with an improvement in survival. However, the addition of either recombinant IL-2 or an anti-CD137 mAb markedly improves both outcomes in these mice. Combining tumor-specific adoptive Tcell therapy to the aCTLA-4/aPD1/rIL2 or aCTLA-4/aPD1/aCD137 regimens enhances efficacy in a synergistic manner. As shown by multiplex tissue immunofluorescence and intravital microscopy, combined immunotherapy treatments enhance Tcell infiltration and the intratumoral performance of T lymphocytes.

Failure of Costimulatory Blockade-induced Regulatory T Cells to Sustain Long-term Survival of High Ischemic Allografts.

Costimulatory blockade-induced allograft tolerance has been achieved in rodent models, but these strategies do not translate well to nonhuman primate and clinical transplants. One confounder that may underlie this discrepancy is the greater ischemic inflammation imposed on the transplants. In mice, cardiac allografts subjected to prolonged cold ischemic storage (CIS) before transplant have increased ischemia-reperfusion injury, which amplifies infiltrating endogenous memory CD8 T-cell activation within hours after transplantation to mediate acute graft inflammation and cytotoxic lymphocyte-associated molecule-4 immunoglobulin-resistant rejection. This study tested strategies inhibiting memory CD8 T-cell activation within such high ischemic allografts to achieve long-term survival. A/J (H-2 a ) hearts subjected to 0.5 or 8 h of CIS were transplanted to C57BL/6 (H-2 b ) recipients and treatment with peritransplant costimulatory blockade. At 60 d posttransplant, regulatory T cells (Treg) were depleted in recipients of high ischemic allografts with anti-CD25 monoclonal antibody (mAb) or diphtheria toxin. Whereas peritransplant (days 0 and +1) anti-lymphocyte function-associated antigen-1 mAb and anti-CD154 mAb prolonged survival of >60% allografts subjected to minimal CIS for >100 d, only 20% of allografts subjected to prolonged CIS survived beyond day 80 posttransplant and rejection was accompanied by high titers of donor-specific antibody. Peritransplant anti-lymphocyte function-associated antigen-1, anti-tumor necrosis factor-α, and anti-CD154 mAb plus additional anti-CD154 mAb on days 14 and 16 obviated this donor-specific antibody and promoted Treg-mediated tolerance and survival of 60% of high ischemic allografts beyond day 100 posttransplant, but all allografts failed by day 120. These studies indicate a strategy inducing prolonged high ischemic allograft survival through Treg-mediated tolerance that is not sustained indefinitely.

NEO-TRA1: A CD25-Targeted De Novo non-Alpha Agonist of the IL-2 Receptor Selectively Expands Regulatory T Cells

Introduction: Regulatory T cells (Tregs) are crucial for maintaining immune tolerance and their dysfunction contributes to inflammatory and autoimmune conditions, such as graft versus host disease (GVHD). IL-2 is a pleiotropic cytokine that activates both immunosuppressive Tregs and inflammatory cells, including NK cells, cytotoxic T cells, and helper T cells. Although low dose IL-2 therapy effectively expands Tregs, this intervention is limited by coincident stimulation of conventional T cells and natural killer cells. Investigational therapeutics derived from directed mutagenesis of native IL-2 sequences have resulted in some improvements in Treg selectivity. To optimize the magnitude and selectivity of Treg expansion, we utilized de novo protein design technology to fine-tune the individual receptor interfaces of IL-2 mimetic proteins targeted to Tregs with CD25 antibodies. We found decoupling receptor agonism and targeting is an effective means of improving Treg stimulation and corresponding therapeutic candidates elicit robust expansion of Treg populations in vivo without detectable stimulation of inflammatory immune cells. Methods: We designed a series of de novo IL-2Ra independent agonists of the IL-2 receptor that demonstrate reduced binding affinity to CD122 and CD132, the beta and gamma chains of the IL-2 receptor complex, by up to 5 logs when compared to IL-2. These non-alpha IL-2 mimetic variants were fused to a humanized anti-CD25 mAb that lacks effector function for cis-targeting to Tregs. Candidate molecules were screened on primary human T cells to assess pSTAT5 signaling. Treg-specific molecules were administered to humanized NSG mice weekly (four times) and peripheral blood was collected for immune cell profiling via flow cytometry. Results: Screening of TRA candidates in primary human T cells identified several highly selective Treg agonists, including NEO-TRA1 (Fig1). At all concentrations, CD8+ and CD4+ non-Tregs showed low or negligible levels of pSTAT5 signaling upon treatment with NEO-TRA1. Nevertheless, CD4+CD25highCD127low Tregs show increasing pSTAT5 signaling at a wide range of concentrations starting at 20pM, and demonstrate preferential signaling in Tregs by NEO-TRA1. To establish the ability of our TRA to expand Treg in vivo, NSG mice engrafted with human hematopoietic stem cells to create a humanized immune system received weekly injections of NEO-TRA1, vehicle control, or a previously described Treg selective IL-2 mutein (IL-2v-Fc). Characterization of Day 13 CD4+CD25+Foxp3+ Treg percentages in peripheral blood mononuclear cells show that the Treg population expanded to ~30% of total CD4+ cells in mice receiving NEO-TRA1, while Treg percentages reached ~22% in mice receiving IL-2v-Fc (Fig2). Despite a slight decrease at Day 20, the Treg percentages remained higher in NEO-TRA1-treated cohorts than in control groups throughout the study. Conclusions: NEO-TRA1 exhibits potent expansion of Treg populations within primary human samples and in humanized mouse models. The observed profile of STAT5 phosphorylation demonstrates selective activation of Tregs over other lymphocyte populations. NEO-TRA1 is the first Treg agonist based on de novo protein design. NEO-TRA1 is both specific and potent and has high therapeutic potential for the treatment of GVHD and other autoimmune or inflammation diseases. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Depletion of Conventional Type-1 Dendritic Cells in Established Tumors Suppresses Immunotherapy Efficacy.

These findings reveal the intratumoral behavior of cDC1 dendritic cells in transgenic mouse models and demonstrate that the efficacy of immunotherapy regimens is precluded by elimination of these cells.