Depletion Of Treg Cells Research Articles

The Effect of Antibody Fragments on CD25 Targeted Regulatory T Cell Near-Infrared Photoimmunotherapy.

Regulatory T (Treg) cells play a major role in immune suppression permitting tumors to evade immune surveillance. Depletion of intratumoral Treg cells can result in tumor regression. However, systemic depletion of Tregs may also induce autoimmune adverse events. Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cell-specific cancer therapy that locally kills specific cells in the tumor. Antibody-photoabsorber (IRDye700DX) conjugates (APC) are injected and bind to the tumor, and subsequent administration of NIR light to the tumor results in rapid cell death only in targeted cells. CD25-targeted NIR-PIT has been shown to induce spatially selective depletion of tumor-associated Treg cells. In this study, we compared the efficacy of an antibody fragment, anti-CD25-F(ab')2, and a full antibody, anti-CD25-IgG, as agents for NIR-PIT. Tumor-bearing mice were divided into four groups: (1) no treatment; (2) anti-CD25-IgG-IR700 i.v. only; (3) anti-CD25-F(ab')2-IR700 i.v. with NIR light exposure; and (4) anti-CD25-IgG-IR700 i.v. with NIR light exposure. Although both CD25-targeted NIR-PITs resulted in significant tumor growth inhibition, the anti-CD25-F(ab')2-IR700 based NIR-PIT was superior to the anti-CD25-IgG-IR700 NIR-PIT. The anti-CD25-F(ab')2-IR700 demonstrated faster clearance from the body than the anti-CD25-IgG-IR700. Sustained circulation of anti-CD25-IgG-IR700 may block IL-2 binding on the activated effector T-cells decreasing immune response. In conclusion, anti-CD25-F(ab')2 based NIR-PIT was more effective in reducing tumor growth than anti-CD25-IgG based NIR-PIT. Absence of the Fc portion of the APC leads to faster clearance and therefore promotes a superior activated T cell response in tumors.

Recent advances with Treg depleting fusion protein toxins for cancer immunotherapy.

T regulatory cells (Tregs) are an important T cell population for immune tolerance, prevention of autoimmune diseases and inhibition of antitumor immunity. The tumor-promoting role played by Tregs in cancer has prompted numerous approaches to develop immunotherapeutics targeting Tregs. One approach to depletion of Treg cells is retargeting the highly potent cytotoxic activity of bacterial toxins. These agents capitalize on the well-characterized bacterial toxins, diphtheria toxin and Pseudomonas aeruginosa exotoxin A-both of which harbor membrane translocation domains and enzymatic domains that catalytically halt protein synthesis within intoxicated eukaryotic cells and act at picomolar or subpicomolar concentrations. In this review, we summarize the preclinical and clinical development of several Treg-depleting cancer immunotherapies based on these two bacterial toxins.

Mogamulizumab Treatment Elicits Autoantibodies Attacking the Skin in Patients with Adult T-Cell Leukemia-Lymphoma.

The anti-CCR4 mAb, mogamulizumab, offers therapeutic benefit to patients with adult T-cell leukemia-lymphoma (ATL), but skin-related adverse events (AE) such as erythema multiforme occur frequently. The purpose of this study was to determine the mechanisms by which mogamulizumab causes skin-related AEs in patients with ATL. We investigated whether autoantibodies were present in patients' sera using flow cytometry to determine binding to keratinocytes and melanocytes (n = 17), and immunofluorescence analysis of tissue sections. We analyzed the IgM heavy chain repertoire in peripheral blood mononuclear cells before and after mogamulizumab or other chemotherapy by next-generation sequencing (NGS; n = 16). Autoantibodies recognizing human keratinocytes or melanocytes were found in the sera of 6 of 8 patients suffering from mogamulizumab-induced erythema multiforme. In one patient, complement-dependent cytotoxicity (CDC) mediated by autoantibodies against keratinocytes or melanocytes was proportionally related to the severity of the erythema multiforme. The presence of autoantibodies in the epidermis was confirmed in all biopsy specimens of mogamulizumab-induced erythema multiforme (n = 12). Furthermore, colocalization of autoantibodies and C1q, suggesting the activation of CDC, was observed in 67% (8/12). In contrast, no autoantibody or C1q was found in ATL tumor skin lesions (n = 13). Consistent with these findings, NGS demonstrated that IgM germline genes had newly emerged and expanded, resulting in IgM repertoire skewing at the time of erythema multiforme. Mogamulizumab elicits autoantibodies playing an important role in skin-related AEs, possibly associated with regulatory T-cell depletion. This is the first report demonstrating the presence of skin-directed autoantibodies after mogamulizumab treatment.

Targeting Treg cells in cancer immunotherapy.

Foxp3-expressing regulatory T (Treg) cells, which are indispensable for preventing autoimmunity, also suppress effective tumor immunity. Treg cells abundantly infiltrate into tumor tissues, which is often associated with poor prognosis in cancer patients. Removal of Treg cells enhances anti-tumor immune responses but may also elicit autoimmunity. A key issue in devising Treg-targeting cancer immunotherapy is, therefore, how to specifically deplete Treg cells infiltrating into tumor tissues without affecting tumor-reactive effector Tcells, while suppressing autoimmunity. This can be achieved by differentially controlling Treg and effector Tcells by various ways. In this review, we discuss how tumor-infiltrating Foxp3+ Treg cells hamper effective anti-tumor immune responses especially in tumor tissues and how they can be specifically targeted for augmenting tumor immunity but not autoimmunity.

Hijacking antibody-induced CTLA-4 lysosomal degradation for safer and more effective cancer immunotherapy

It remains unclear why the clinically used anti-CTLA-4 antibodies, popularly called checkpoint inhibitors, have severe immunotherapy-related adverse effects (irAEs) and yet suboptimal cancer immunotherapeutic effects (CITE). Here we report that while irAE-prone Ipilimumab and TremeIgG1 rapidly direct cell surface CTLA-4 for lysosomal degradation, the non-irAE-prone antibodies we generated, HL12 or HL32, dissociate from CTLA-4 after endocytosis and allow CTLA-4 recycling to cell surface by the LRBA-dependent mechanism. Disrupting CTLA-4 recycling results in robust CTLA-4 downregulation by all anti-CTLA-4 antibodies and confers toxicity to a non-irAE-prone anti-CTLA-4 mAb. Conversely, increasing the pH sensitivity of TremeIgG1 by introducing designed tyrosine-to-histidine mutations prevents antibody-triggered lysosomal CTLA-4 downregulation and dramatically attenuates irAE. Surprisingly, by avoiding CTLA-4 downregulation and due to their increased bioavailability, pH-sensitive anti-CTLA-4 antibodies are more effective in intratumor regulatory T-cell depletion and rejection of large established tumors. Our data establish a new paradigm for cancer research that allows for abrogating irAE while increasing CITE of anti-CTLA-4 antibodies.

Abstract 4098: A heavy chain only antibody against CTLA4 with outstanding preclinical efficacy and safety profile

Abstract Anti-CTLA-4 antibodies are one of the only three types of immune checkpoint inhibitors (together with anti-PD-1 and anti-PD-L1 antibodies) with proven monotherapy value in cancer therapy. Despite demonstrated efficacy, their broad application in monotherapies and combinational therapies are hampered by the safety profiles. Evidence in animal models suggests that efficacy of anti-CTLA-4 antibodies is at least partially due to depletion of regulatory T cells (Treg) via ADCC function, whereas clinically adverse events are positively correlated with systemic exposure. Here we embarked to develop the next generation anti-CTLA-4 antibody with better efficacy and safety profile. Fully human Heavy Chain only antibody (HCAb) 4003-2 was generated using the Harbour HCAb transgenic mouse platform. 4003-2 specifically binds to CTLA-4 and blocks its binding to B7.1/B7.2. It was optimized to have enhanced ADCC function that affords superior ability to deplete Treg cells both in vitro and in vivo. 4003-2 showed in murine tumor models significantly more potent anti-tumor activity than other CTLA4 antibodies, at least partly driven by substantially enhanced depletion of intra-tumoral Treg cells. In addition, due to its shorter serum half-life, 4003-2 has less systemic drug exposure in vivo at efficacious doses, suggesting potentially improvement of safety profile in clinical applications. The augmented efficacy and safety profile of 4003-2 present it as an excellent candidate for the development of next generation anti-CTLA4 therapy. Citation Format: George Liu, Xin Gan, Yun He, Jiuqiao Zhao, Huayan Duan, Yiping Rong. A heavy chain only antibody against CTLA4 with outstanding preclinical efficacy and safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4098.

Local T regulatory cells depletion by an integrated nanodrug system for efficient chem-immunotherapy of tumor

T regulatory (Treg) cell is a major immunosuppressive factor that restrains the antitumor effect of immunotherapy, because it gets more after the immune activation and is hardly to be eliminated. Here, an acid-sensitive integrated nanodrug system is designed to activate antitumor immune response as well as locally deplete Treg cells with low side effect. The nanosystem is synthetized by coordinating doxorubicin (DOX) and pentoxifylline (PTX) with Zn ions, then stabilized via liposome encapsulation (denoted as DTX@Lipo). DTX@Lipo can activate antitumor immune effect by chemotherapy of DOX. Besides, the release of PTX inhibits c-Rel expression, leading to the reduction of Treg cells in tumor site. Owing to the good tumor accumulation and local drug release ability, DTX@Lipo exhibits better biosafety and convenience than traditional antibody blockade method for Treg cells depletion. According to the results of in vivo experiments, the nanodrug system can significantly increase the ratio between effector T (Teff) cells and Treg cells locally, resulting in an immunoactivated tumor microenvironment. Importantly, it exhibits significant antitumor effect when combined with PD-1 blockade therapy, providing great potential for tumor therapy.

Regulatory T (Treg) cells in cancer: Can Treg cells be a new therapeutic target?

Regulatory T (Treg) cells suppress abnormal/excessive immune responses to self‐ and nonself‐antigens to maintain immune homeostasis. In tumor immunity, Treg cells are involved in tumor development and progression by inhibiting antitumor immunity. There are several Treg cell immune suppressive mechanisms: inhibition of costimulatory signals by CD80 and CD86 expressed by dendritic cells through cytotoxic T‐lymphocyte antigen‐4, interleukin (IL)‐2 consumption by high‐affinity IL‐2 receptors with high CD25 (IL‐2 receptor α‐chain) expression, secretion of inhibitory cytokines, metabolic modulation of tryptophan and adenosine, and direct killing of effector T cells. Infiltration of Treg cells into the tumor microenvironment (TME) occurs in multiple murine and human tumors. Regulatory T cells are chemoattracted to the TME by chemokine gradients such as CCR4‐CCL17/22, CCR8‐CCL1, CCR10‐CCL28, and CXCR3‐CCL9/10/11. Regulatory T cells are then activated and inhibit antitumor immune responses. A high infiltration by Treg cells is associated with poor survival in various types of cancer. Therefore, strategies to deplete Treg cells and control of Treg cell functions to increase antitumor immune responses are urgently required in the cancer immunotherapy field. Various molecules that are highly expressed by Treg cells, such as immune checkpoint molecules, chemokine receptors, and metabolites, have been targeted by Abs or small molecules, but additional strategies are needed to fine‐tune and optimize for augmenting antitumor effects restricted in the TME while avoiding systemic autoimmunity. Here, we provide a brief synopsis of these cells in cancer and how they can be controlled to achieve therapeutic outcomes.

Menopause and FOXP3+ Treg cell depletion eliminate female protection against T cell-mediated angiotensin II hypertension.

Although it is known that the prevalence and severity of hypertension increases in women after menopause, the contribution of T cells to this process has not been explored. Although the immune system is both necessary and required for the development of angiotensin II (ANG II) hypertension in men, we have demonstrated that premenopausal women are protected from T cell-mediated hypertension. The goal of the current study was to test the hypotheses that 1) female protection against T cell-mediated ANG II hypertension is eliminated following progression into menopause and 2) T regulatory cells (Tregs) provide premenopausal protection against ANG II-induced hypertension. Menopause was induced in Rag-1-/- mice (via 4-vinylcyclohexene diepoxide), and all mice received a 14-day ANG II infusion. Donor CD3+ T cells were adoptively transferred 3 wk before ANG II infusion. In the absence of T cells, systolic blood pressure responses to ANG II were similar to those seen in premenopausal mice (Δ12 mmHg). After adoptive transfer of T cells, ANG II significantly increased systolic blood pressure in postmenopausal females (Δ28 mmHg). A significant increase in F4/80 positive renal macrophages, an increase in renal inflammatory gene expression, along with a reduction in renal expression of mannose receptor C-type 1, a marker for M2 macrophages, accompanied the increase in systolic blood pressure (SBP). Flow cytometric analysis identified that Tregs were significantly decreased in the spleen and kidneys of Rag-1-/- menopausal mice versus premenopausal females, following ANG II infusion. In a validation study, an anti-CD25 antibody was used to deplete Tregs in premenopausal mice, which induced a significant increase in SBP. These results demonstrate that premenopausal protection against T cell-mediated ANG II hypertension is eliminated once females enter menopause, suggesting that a change in hormonal status upregulates macrophage-induced proinflammatory and T cell-dependent responses. Furthermore, we are the first to report that the presence of Tregs are required to suppress ANG II hypertension in premenopausal females.NEW & NOTEWORTHY Whether progression into menopause eliminated female protection against T cell-mediated hypertension was examined. Menopausal mice without T cells remained protected against angiotensin II (ANG II) hypertension; however, in the presence of T cells, blood pressure responses to ANG II increased significantly in menopause. Underlying mechanisms examined were anti-inflammatory protection provided by T regulatory cells in premenopausal females and renal inflammatory processes involving macrophage infiltration and cytokine activation.

A novel TNFR2 antibody induces T cell co-stimulation and promotes durable anti-tumor immunity

Abstract TNFR2, an emerging therapeutic target in immuno-oncology, is upregulated upon T cell activation and is expressed by tumor-infiltrating effector and regulatory T (Treg) cells. We generated a novel monoclonal antibody, Y9, specific to murine TNFR2 and investigated its mechanism of action. In vitro, Y9 stimulation of purified T cells increased proliferation and effector function, indicating that Y9 acts as an agonist and can provide co-stimulation. In vivo, Y9 treatment of mice with established tumors resulted in complete tumor clearance across a variety of models. The activity of Y9 on immune cells was confirmed by its decreased activity in mice depleted of NK or CD8+ T cells. Unlike the proposed Treg depletion mechanism of other therapeutic antibodies, depletion of Treg cells is not the primary mechanism of action of Y9 treatment. Instead, decreased TNFR2 and other co-inhibitory receptor surface expression was observed following treatment. Y9 activity depended on FcγR binding, which facilitated enhanced agonist activity. Based on this evidence, we developed MM-401, a human antibody targeting TNFR2. MM-401 has agonistic activity; upon incubation of MM-401 with human CD4+ and CD8+ T cells, we observed upregulation of activation markers and cytokine production comparable to MEDI6469 (anti-OX40). We also observed that MM-401 promotes antibody-dependent cellular cytotoxicity (ADCC) in an NK cell-mediated in vitro assay and a reduction in the number of Treg cells in human ovarian cancer ascites. These data suggest that MM-401 could also promote anti-tumor immunity by mediating ADCC, as well as by direct co-stimulation of T cell responses, and justifies the continued development of MM-401 as a novel cancer immunotherapy.

Interleukin-2/antibody complex expanding Foxp3+ regulatory T cells exacerbates Th2-mediated allergic airway inflammation

Foxp3+ regulatory CD4+ T (Treg) cells play an essential role in preventing overt immune responses against self and innocuous foreign antigens. Selective expansion of endogenous Treg cells in response to the administration of interleukin (IL)-2/antibody complex, such as the IL-2/JES6-1 complex (IL-2C) in mice, is considered an attractive therapeutic approach to various immune disorders. Here, we investigated the therapeutic potential of IL-2C in allergic airway inflammation models. IL-2C treatment ameliorated Th17-mediated airway inflammation; however, unexpectedly, IL-2C treatment exacerbated Th2-mediated allergic airway inflammation by inducing the selective expansion of Th2 cells and type-2 innate lymphoid cells. We also found that IL-2 signaling is required for the expansion of Th2 cells in lymphoproliferative disease caused by Treg cell depletion. Our data suggest that IL-2C is selectively applicable to the treatment of allergic airway diseases depending on the characteristics of airway inflammation.

Unleashing Type-2 Dendritic Cells to Drive Protective Antitumor CD4+ T Cell Immunity

Differentiation of proinflammatory CD4+ conventional Tcells (Tconv) is critical for productive antitumor responses yet their elicitation remains poorly understood. We comprehensively characterized myeloid cells in tumor draining lymph nodes (tdLN) of mice and identified two subsets of conventional type-2 dendritic cells (cDC2) that traffic from tumor to tdLN and present tumor-derived antigens to CD4+ Tconv, but then fail to support antitumor CD4+ Tconv differentiation. Regulatory Tcell (Treg) depletion enhanced their capacity to elicit strong CD4+ Tconv responses and ensuing antitumor protection. Analogous cDC2 populations were identified in patients, and as in mice, their abundance relative to Treg predicts protective ICOS+ PD-1lo CD4+ Tconv phenotypes and survival. Further, in melanoma patients with lowTreg abundance, intratumoral cDC2 density alonecorrelates with abundant CD4+ Tconv and with responsiveness to anti-PD-1 therapy. Together, this highlights a pathway that restrains cDC2 and whose reversal enhances CD4+ Tconv abundance and controls tumor growth.

PC-3 A reappraisal of CTLA-4 checkpoint blockade hypothesis in cancer immunotherapy

The classic checkpoint blockade hypothesis states that cancer immunity is restrained by 2 distinct checkpoints: the first is the CTLA-4:B7 interaction that limits priming of naive T cells in lymphoid organs, while the second is the PD-1/B7-H1(PD-L1) interaction that results in exhaustion of effector T cells within the tumor microenvironment. Ipilimumab, a clinically-used so-called immune checkpoint inhibitor (ICI) has been FDA approved for the treatment of metastatic melanoma and might get clinical approval for additional oncological indications, most likely in combination with other drugs, for example anti-PD-1/PD-L1. It is assumed that anti-CTLA-4 antibodies cause tumor rejection by blocking negative signaling from the B7-CTLA-4 interactions. Surprisingly, at concentrations considerably higher than plasma levels achieved by Ipilimumab blocks neither B7 transendocytosis by CTLA-4 nor CTLA-4 binding to immobilized or cell-associated B7. In Ctla4 h/m mice expressing both human and mouse CTLA4 genes, anti-CTLA-4 antibodies that bind to human but not mouse CTLA-4 efficiently induce Treg-depletion and Fc receptor-dependent tumor rejection. The blocking antibody L3D10 is comparable to the non-blocking Ipilimumab in causing tumor rejection. Remarkably, L3D10 progenies (HL12/HL32) that lost blocking activity during humanization remain fully competent in Treg depletion and tumor rejection. We also set out to test 3 commercially available anti-mouse Ctla-4 mAbs(4F10/9H10/9D9) that had been shown to induce tumor rejection for their ability to block the B7-Ctla-4 interaction under physiologically relevant conditions, these data also argue against a role for blocking the B7-Ctla-4 interaction in the induction of antitumor immunity by anti-mouse Ctla-4 mAbs. Since these mAbs showed comparable immunotherapeutic effect and comparable depletion of Treg cells in the tumor microenvironment. Thus, both anti-human CTLA-4 mAbs and anti-mouse CTLA-4 mAbs cause tumor rejection by mechanisms that are independent of checkpoint blockade but dependent on host Fc receptor. These data have important implications for the development of the next generation of immunotherapeutic anti-CTLA-4 mAbs and call for a reappraisal of the checkpoint blockade hypothesis.

PD-L1 Ameliorates Murine Acute Graft-Versus-Host Disease by Suppressing Effector But Not Regulatory T Cells Function.

There is increasing evidence that interaction between programmed death 1 (PD-1) and its ligands PD-1 (PD-L1) plays a critical role in the pathology of acute graft-versus-host disease (aGVHD). However, the role of PD-L1 in the development of aGVHD has been controversial in recent mouse studies. In this study, we carried out studies in a murine aGVHD model to clarify the role of PD-L1 in aGVHD pathogenesis. We found that systemic overexpression of PD-L1 by hydrodynamic gene transfer (HGT) method in vivo ameliorates aGVHD-induced lethality in mice. Systemic overexpression of PD-L1 inhibits the donor T cells activation, effector memory status, as well as Th1 and Th17 cells responses in vivo. In addition, PD-L1 Ig treatment significantly suppressed T cells' proliferation, promoted T cells' apoptosis, and reduced pro-inflammatory cytokines expression by effector T cells in vitro in the stimulation of anti-CD3/CD28 and allogeneic dendritic cells. However, we found that PD-L1 overexpression did not affect Treg cells' differentiation in vivo and in vitro, depletion of Treg cells in PD-L1 HGT recipients did not aggravate aGVHD mortality. Therefore, our results demonstrated that systemic treatment with PD-L1 protein ameliorates aGVHD by suppressing effector but not regulatory T cell function. Our findings suggest that systemic treatment with PD-L1 may be a potential strategy to prevent or ameliorate aGVHD.

Phase II study of sitravatinib in combination with nivolumab in patients undergoing nephrectomy for locally-advanced clear cell renal cell carcinoma (ccRCC).

TPS686 Background: Sitravatinib is a receptor tyrosine kinase inhibitor (RTKI) that targets multiple closely related RTK pathways including VEGFR, PDGFR, KIT, MET and the TAM receptors (TYRO3, AXL and MERTK) Nivolumab is a monoclonal antibody against PD-1 and releases PD-1-mediated inhibition of T-cell proliferation and cytokine production. Together, sitravatinib and nivolumab may cooperate to elicit greater anti-tumor activity than either agent alone, as sitravatinib is predicted to enhance several steps in the cancer immunity cycle that may augment nivolumab’s efficacy. Mechanisms by which sitravatinib may augment an antitumor immune response include enhanced antigen presentation; depletion of immunosuppressive regulatory T-cells (Tregs) and myeloid-derived suppressor cells (MDSCs) via inhibition of split kinases VEGFR and KIT; and shifting tumor-associated macrophages from an immunosuppressive M2 to a pro-immunogenic M1 phenotype via inhibition of TAM RTKs. Each of these factors converge on promoting T effector cell expansion, tumor infiltration and an antigen-specific anti-tumor immune response. Methods: This open-label, non-randomized, preoperative window of opportunity Phase 2 study evaluates tolerability and clinical activity of sitravatinib in combination with nivolumab in pts with locally-advanced ccRCC undergoing nephrectomy. Study treatment consists of 2 weeks of sitravatinib monotherapy followed by 4 weeks of the combination. Sitravatinib is administered orally daily at 120 mg; nivolumab intravenously every 2 weeks at 240 mg. The primary objective is to evaluate clinical activity using percentage of pts achieving a presurgical point-in-time objective response. Secondary objectives include evaluation of safety and tolerability, and determination of the immune effects of sitravatinib monotherapy and the combination through serial tissue and blood collections (temporal changes in PD-L1 expression, selected cytokines and immune cell populations including MDSCs, Tregs and ratio of M1:M2 macrophages). The study is open for enrollment and recruitment is ongoing. Clinical trial information: NCT03680521.

Abstract A192: Non-IL-2 blocking anti-CD25-targeting antibodies: depletion of regulatory T-cells driving optimal effector response for rejection of established tumors

Abstract 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.

Abstract A177: Impact of regulatory T-cells on carcinogenesis of oral squamous cell carcinoma

Abstract Although some head-and-neck squamous cell carcinomas (HNSCCs) are associated with human papillomavirus (HPV) infection, a large proportion of HNSCCs are HPV-negative and are characterized by mutational signatures associated with smoking, suggesting that HPV-negative HNSCCs are induced by carcinogen exposure. In many human cancers, including HNSCC, regulatory T (Treg) cells are found at elevated densities and are thought to be a major barrier to the generation of robust anti-tumor T-cell responses. However, recent studies indicate that Treg cells can serve diverse functions in non-lymphoid sites. Therefore, it is critical to elucidate the function of Treg cells infiltrating HNSCC lesions. To study the functional impact of Treg cells on the development and progression of HPV-negative HNSCC, we utilized an autochthonous mouse model of carcinogen-induced oral squamous cell carcinoma (SCC), in which mice are exposed long-term to the carcinogen 4-nitroquinoline-1-oxide (4-NQO) in the drinking water. Immunohistochemistry and flow cytometric analysis revealed elevated densities of CD3+ T-cells and Foxp3+ Treg cells in premalignant dysplastic lesions and SCC lesions within the tongue epithelium, suggesting that many lesions exhibit a T-cell-inflamed phenotype. Single-cell TCR sequencing demonstrated the presence of recurrent Treg cell clones within carcinogen-induced lesions, suggesting clonal expansion of Treg cells in the tumor microenvironment. Surprisingly, the systemic depletion of Treg cells at the later stages of carcinogen exposure did not induce tumor regression, but instead induced the opposite effect, driving increased incidence and burden of SCC. These findings suggest that Treg cells may play an unexpected role in restricting the progression of carcinogen-induced SCC in the oral cavity, prompting a re-examination of the common paradigm that Treg cells promote tumorigenesis by suppressing antitumor immunity. Clinically, our findings suggest that novel therapies could be developed to combat HNSCC by triggering the recruitment or activation of tumor-associated Treg cells, and suggest that checkpoint blockade therapies that induce intratumoral Treg cell depletion may have unintended adverse consequences. Citation Format: Jaime L. Chao, Peter A. Savage. Impact of regulatory T-cells on carcinogenesis of oral squamous cell carcinoma [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 A177.

Abstract A141: Intratumoral depletion of regulatory T-cells using CD25-targeted photodynamic therapy induces antitumoral immune responses

Abstract Tumor immunotherapy aims to overcome the immunosuppressive microenvironment within tumors, and various approaches have been developed. Tumor-associated T regulatory cells (Tregs) suppress the activation and expansion of tumor antigen-specific effector T-cells, thus providing a permissive environment for tumor growth. Therefore, optimal strategies need to be established to deplete tumor-infiltrated Tregs because systemic depletion of Tregs can result in reduced anti-tumor effector cells and autoimmunity. Here, to deplete Tregs selectively in tumors, we intratumorally injected anti-CD25 antibodies conjugated to Chlorin e6 (Ce6), a photosensitizer that absorbs light to generate reactive oxygen species. Local depletion of tumor-associated Tregs by photodynamic therapy (PDT) inhibited tumor growth, which was likely due to the altered tumor immune microenvironment that was characterized by increased infiltration of CD8+ effector T-cells and the expression of IFN-γ and CD107a, which is cytolytic granule exocytosis marker, in tumor tissues. Furthermore, PDT-induced intratumoral Treg depletion did not influence adaptive immune responses in a murine influenza infection model. Thus, our results show that intratumoral Treg-targeted PDT could specifically modulate tumor microenvironments by depleting Tregs and could be used as a novel cancer immunotherapy technique. Citation Format: Heung Kyu Lee, Dong Sun Oh. Intratumoral depletion of regulatory T-cells using CD25-targeted photodynamic therapy induces antitumoral immune responses [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 A141.

Pilot Study of Regulatory T-Cell Depletion in the Setting of Autologous Stem Cell Transplantation for Multiple Myeloma

Introduction High dose melphalan with autologous stem cell transplantation (ASCT) is a therapy mainstay in multiple myeloma (MM). However, the median PFS after ASCT is under 5 years even with contemporary agents. This may be due to immune dysfunction after ASCT. Regulatory T cells (Tregs, CD4+CD25+FoxP3+) reconstitute rapidly after ASCT, can inhibit antitumor immune responses, and are a target for non-cross-resistant therapy in the MRD-positive state. Methods We conducted a randomized pilot study to evaluate 2 methods of Treg depletion in MM patients undergoing ASCT. No Treg depletion was performed in the control ASCT arm. An anti-CD25 mAb (basiliximab 20 mg IV) was given day +1 post-ASCT in the in vivo Treg depletion (IVTRD) arm. In the ex vivo Treg depletion (EVTRD) arm, CD4+CD25+ Tregs were depleted from ASC grafts with anti-CD25 microbeads and CliniMACS device (Miltenyi). Primary endpoints were: 1) evaluate efficiency of Treg depletion, 2) measure kinetics of Treg depletion and recovery and 3) determine toxicities with the two methods of Treg depletion. Secondary endpoints were engraftment rates and disease response. Results Fifteen patients were enrolled, 5 in each arm. One patient in the IVTRD arm was removed from study due to mobilization failure. ASC were collected following filgrastim/plerixafor. The conditioning regimen consisted of melphalan 200 mg/m2. All patients engrafted; median times to neutrophil/platelet engraftment were similar between arms. 4/5 patients in the EVTRD arm had neutropenic fever (vs 2/5 in control ASCT arm and 2/4 in IVTRD arm). Engraftment syndrome occurred in 1 patient in the EVTRD arm. Conclusion Median ex vivo depletion of CD4+CD25+ Tregs from 5 ASC grafts was 93% (figure 1). Baseline frequencies of peripheral blood (PB) CD4+FoxP3+ Tregs were similar between all arms (p=NS). A reduction in Treg frequency was seen in the EVTRD arm compared to controls at day +14 (p=0.0001) and +21 (p=0.025, figure 2). The IVTRD arm had lower Treg frequency at day +28 compared to controls (p=0.053). These data suggest that in vivo and ex vivo Treg depletion are feasible and result in significant reduction and delay in Treg recovery post-ASCT for MM. These methods could serve as a platform for using post-transplant immunotherapies to improve post-ASCT outcomes.

Role of regulatory T cells in experimental autoimmune glomerulonephritis.

Anti-glomerular basement membrane (anti-GBM) disease is characterized by antibodies and T cells directed against the Goodpasture antigen, the noncollagenous domain of the α3-chain of type IV collagen [α3(IV)NC1] of the GBM. Consequences are the deposition of autoantibodies along the GBM and the development of crescentic glomerulonephritis (GN) with rapid loss of renal function. Forkhead box protein P3 (Foxp3)+ regulatory T (Treg) cells are crucial for the maintenance of peripheral tolerance to self-antigens and the prevention of immunopathology. Here, we use the mouse model of experimental autoimmune GN to characterize the role of Treg cells in anti-GBM disease. Immunization of DBA/1 mice with α3(IV)NC1 induced the formation of α3(IV)NC1-specific T cells and antibodies and, after 8-10 wk, the development of crescentic GN. Immunization resulted in increased frequencies of peripheral Treg cells and renal accumulation of these cells in the stage of acute GN. Depletion of Treg cells during immunization led to enhanced generation of α3(IV)NC1-specific antibodies and T cells and to aggravated GN. In contrast, depletion or expansion of the Treg cell population in mice with established autoimmunity had only minor consequences for renal inflammation and did not alter the severity of GN. In conclusion, our results indicate that in anti-GBM disease, Treg cells restrict the induction of autoimmunity against α3(IV)NC1. However, Treg cells are inefficient in preventing crescentic GN after autoimmunity has been established.