Role of the immunoglobulin constant region in the antitumor activity of antibodies to cytotoxic T-lymphocyte antigen-4 (CTLA-4).

9055 Background: Anti-CTLA-4 therapy enhances antitumor T-cell responses by both cell-intrinsic and cell-extrinsic mechanisms. Effector T-cell (Teff) activation is increased directly by interfering with CTLA-4-B7 interactions that negatively regulate T cells and by interfering with CTLA-4 expressed on regulatory T cells (Tregs), which function to inhibit immune responses. To analyze in greater detail the mechanism of action of anti-CTLA-4 antibodies (Abs), we examined the role of the immunoglobulin constant region in the antitumor activity of anti-CTLA-4 Abs in mouse tumor models. Methods: The activity of anti-CTLA-4 Abs with different mouse IgG constant regions were compared in several mouse tumor models, and intratumoral and peripheral T cells were analyzed by FACS. DNA samples from 488 patients with metastatic melanoma who received ipilimumab in a phase III clinical trial, MDX010-20, were analyzed for polymorphisms in the IgG fragment c receptor (FcR) at FCGR3A (V158F) and FCGR2A (H131R) loci. Results: In subcutaneous MC38 and CT26 colon tumor models, an anti-CTLA-4 Ab containing the mouse IgG2a constant region exhibited enhanced antitumor activity compared to an anti-CTLA-4 Ab containing the IgG2b constant region, while anti-CTLA-4 Abs containing mouse IgG1 or a mutated mouse IgG1 D265A constant region showed no activity. Anti-CTLA-4-IgG2a caused a dramatic reduction of Tregs at the tumor site that resulted in a greater Teff/Treg ratio. In contrast, all isotypes resulted in expansion of Tregs in the periphery. These results point to an important role for FcR in the action of anti-CTLA-4 Abs. In patients from study MDX010-20, there was no association between the 2 FcR polymorphisms (FCGR3A and FCGR2A) and overall survival. Conclusions: These preclinical studies reveal a novel dual activity of anti-CTLA-4 Abs consisting of intratumoral reduction of Tregs together with activation of Teff cells. This effect is mediated by the constant region and is presumably due to antibody-dependent cellular cytotoxicity. The data suggest that FcR-bearing cells at the tumor site, as well as the presence of intratumoral Tregs, may be important factors in the antitumor activity of anti-CTLA-4 Abs.

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Nonclinical evaluation of the combination of mouse IL-21 and anti- mouse CTLA-4 or PD-1 blocking antibodies in mouse tumor models.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.3019 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. 3019-3019 ◽

Cited By ~ 2

Author(s):

Maria Jure-Kunkel ◽

Mark Selby ◽

Katherine Lewis ◽

Gregg Masters ◽

Jose Valle ◽

...

Keyword(s):

T Cell ◽

Antitumor Activity ◽

Nk Cell ◽

Phase Iii ◽

Tumor Growth Inhibition ◽

Clinical Activity ◽

Mouse Tumor ◽

Synergistic Activity ◽

Tumor Models ◽

Mouse Tumor Models

3019 Background: Interleukin 21 (IL-21), a γc chain family cytokine, is produced primarily by CD4+ T cells and has many effects on immune cells, including enhancing CD8+ T cell and NK cell proliferation and cytotoxicity. Recombinant IL-21 (rIL-21) therapy resulted in objective responses in ~20% of melanoma and renal cell carcinoma patients. In mouse models, monoclonal antibody (mAb) blockade of CTLA-4 prolongs antigen-specific T cell responses, while blockade of programmed death 1 (PD-1) reverses tumor induced T cell suppression. Ipilimumab, a CTLA-4 blocking mAb, significantly improved overall survival in patients with metastatic melanoma in 2 phase III trials, and in phase I studies a PD-1 blocking mAb (nivolumab) has antitumor activity in various cancers. Side effect profiles for each mAb have been related to their mechanism and are generally manageable. It was hypothesized that combination of IL-21 plus CTLA-4 or PD-1 blockade may enhance antitumor responses, potentially leading to improved clinical activity. Methods: Preclinical studies were conducted to test the antitumor activity of mouse IL-21 (mIL-21) in combination with an anti-mouse PD-1 (mPD-1) mAb (4H2-IgG1) or with an anti-mCTLA-4 blocking mAb (9D9-IgG2b) in syngeneic mouse tumor models, including MC38, CT-26, EMT-6, and B16F10. mIL-21 was tested at doses ranging from 50-200 μg/dose, administered up to 3d/wk. mCTLA-4 mAb or mPD-1 mAb were administered 3-4x total at 200-300 μg/dose. Results: Combination treatments produced enhanced antitumor activity vs. monotherapy. In the MC38 model, mIL-21 treatment led to 30% median tumor growth inhibition (TGI) by d29, while mPD-1 mAb produced 60% median TGI and 1/10 tumor-free mice. Combination of both agents led to synergistic antitumor activity, with complete regressions (CR) in 7/10 mice and 99.9% median TGI (p=0.046). CTLA-4 mAb + mIL-21 also produced synergistic activity in the MC38 model. By d21, mIL-21 monotherapy induced 34% TGI while CTLA-4 mAb resulted in 28% TGI, with no CR in either group. Combination resulted in 6/8 mice with CR and 86% TGI (p<0.05). Conclusions: These results support the use of rIL-21+nivolumab and rIL-21+ipilimumab in recently initiated clinical trials.

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843 Development and engineering of human sialidase for degradation of immunosuppressive sialoglycans to treat cancer

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.843 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A884-A884

Author(s):

Li Peng ◽

Lizhi Cao ◽

Sujata Nerle ◽

Robert LeBlanc ◽

Abhishek Das ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Immune Cells ◽

First Generation ◽

Single Agent ◽

Mouse Tumor ◽

Cytokine Release ◽

Tumor Models ◽

Cell Immunity ◽

Mouse Tumor Models

BackgroundSialoglycans, a type of glycans with a terminal sialic acid, have emerged as a critical glyco-immune checkpoint that impairs antitumor response by inhibiting innate and adaptive immunity. Upregulation of sialoglycans on tumors has been observed for decades and correlates with poor clinical outcomes across many tumor types. We previously showed that targeted desialylation of tumors using a bifunctional sialidase x antibody molecule, consisting of sialidase and a tumor-associated antigen (TAA)-targeting antibody, has led to robust single-agent efficacy in mouse tumor models. In addition to tumor cells, most immune cells present substantially more abundant sialoglycans than non-hematological healthy cells, which may also contribute to immunosuppression. Therefore, we studied the impact of immune cell desialylation and evaluated the therapeutic potential of a newly developed sialidase-Fc fusion (Bi-Sialidase), which lacks a TAA-targeting moiety and consists of engineered human neuraminidase 2 (Neu2) and human IgG1 Fc region, in preclinical mouse tumor models.MethodsThe first generation Neu2 variant was further optimized to improve titers and stability to constructed Bi-Sialidase. Bi-Sialidase’s desialylation potency and impact on immune responses were studied in vitro using various human immune functional assays, including T-cell activation, allogeneic mixed lymphocyte reaction, antibody-dependent cellular cytotoxicity, macrophages polarization/activation, neutrophil activation, and peripheral blood mononuclear cell (PBMC) cytokine release assays. We evaluated its antitumor efficacy in mouse tumor models. Bi-Sialidase’s safety profile was characterized by conducting rat and non-human primate (NHP) toxicology studies.ResultsThe optimized Bi-Sialidase achieved a titer of 2.5 g/L from a 15-day fed-batch Chinese hamster ovary cell culture; in contrast, the wild-type and first-generation Neu2 had no production or a low titer (<0.1 g/L) under similar conditions, respectively. We demonstrated that Bi-Sialidase led to dose-dependent desialylation of immune cells and potentiated T-cell immunity, without impacting NK, macrophage, or neutrophil activation by desialylating immune cells. Activated and exhausted T cells upregulated surface sialoglycans and Bi-Sialidase-mediated desialylation reinvigorated exhausted-like T cells as measured by IFNg production. Bi-Sialidase treatment also enhanced DC priming and activation of naïve T cells by desialylating both T cells and DCs. Furthermore, Bi-Sialidase showed single-agent antitumor activity in multiple mouse tumor models, including MC38, CT26, A20, and B16F10. Importantly, Bi-Sialidase did not cause cytokine release in human PBMC assays and was tolerated to up to 100 mg/kg in rats and NHPs, demonstrating a wide safety margin.ConclusionsBi-Sialidase with an optimized Neu2 offers a novel immunomodulatory approach to enhancing T-cell immunity by desialylating immunosuppressive sialoglycans for cancer treatment.

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Finding the right balance: aOX40 immunotherapy enhances effector T cell function without impairing regulatory T cells intrinsic suppressive capacity in mouse tumor models

Journal for ImmunoTherapy of Cancer ◽

10.1186/2051-1426-3-s2-p77 ◽

2015 ◽

Vol 3 (S2) ◽

Author(s):

Amy Moran ◽

Fanny Polesso ◽

Minhazur Sarker ◽

David Parker ◽

Susan E Murray ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Cell Function ◽

Mouse Tumor ◽

Tumor Models ◽

Effector T Cell ◽

Suppressive Capacity ◽

The Right ◽

Mouse Tumor Models

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PD-1 blockade enhances elotuzumab efficacy in mouse tumor models

Blood Advances ◽

10.1182/bloodadvances.2017004382 ◽

2017 ◽

Vol 1 (12) ◽

pp. 753-765 ◽

Cited By ~ 24

Author(s):

Natalie A. Bezman ◽

Amy Jhatakia ◽

Alper Y. Kearney ◽

Ty Brender ◽

Mark Maurer ◽

...

Keyword(s):

T Cell ◽

Antitumor Activity ◽

Mouse Models ◽

Cell Activity ◽

Antitumor Efficacy ◽

Mouse Tumor ◽

Tumor Models ◽

Key Points ◽

Mouse Tumor Models

Key PointsThe combination of elotuzumab and an anti–PD-1 antibody leads to enhanced antitumor efficacy in mouse models. Enhanced antitumor activity is likely due to the promotion of tumor-infiltrating NK and T-cell activity.

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Abstract 2577: RLI, a sushi-IL-15Rα/IL-15 fusion protein, is a potent immunomodulatory agent on NK and CD8+ T cells and synergizes with anti-PD1 treatment in preclinical mouse tumor models

10.1158/1538-7445.am2014-2577 ◽

2014 ◽

Author(s):

Mélanie Desbois ◽

Coralie Beal ◽

Clelia Coutzac ◽

Magali Terme ◽

Geraldine Teppaz ◽

...

Keyword(s):

T Cells ◽

Fusion Protein ◽

Cd8 T Cells ◽

Mouse Tumor ◽

Tumor Models ◽

Immunomodulatory Agent ◽

Mouse Tumor Models

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778 Modulating tumor microenvironment with arginase-1 specific T cells

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.778 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A813-A813

Author(s):

Evelina Martinenaite ◽

Mia Aaboe Jørgensen ◽

Rasmus Erik Johansson Mortensen ◽

Shamaila Munir Ahmad ◽

Stine Emilie Weis-Banke ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Microenvironment ◽

Memory T Cells ◽

Ethics Committee ◽

Mouse Tumor ◽

Immune Modulators ◽

Arginase 1 ◽

Mouse Tumor Models

BackgroundIO112 is an immune modulatory cancer therapy under preclinical development to target arginase-1-expressing tumor cells and immune inhibitory myeloid cells, such as myeloid derived suppressor cells (MDSCs), and tumor associated macrophages (TAMs). Arginase-1 acts as a metabolic immune regulator at the tumor site by reducing availability of L-arginine to the infiltrating immune cells thus reducing T cell functionality and proliferation. Previously, we demonstrated that IO112 triggers activation of spontaneous CD4+ and CD8+ T-cell responses against arginase-1, found in both cancer patients and healthy individuals.1 These T cells are present in the memory T cell compartment, and are activated in arginase-1 inducing conditions, such as presence of TH2 cytokines IL-4 or IL-13 in vitro.2 3 In this study we aimed to explore the role of arginase-1-specific T cells as immune modulators in immune homeostasis and tumor microenvironment for the development of IO112 immunomodulatory therapy.MethodsHuman arginase-1-specific T cells were isolated and expanded for functional characterization of reactivity against arginase-1 expressing target cells as well as subsequent phenotyping of the targeted arginase-1 positive cells. Syngeneic C57BL/6 mouse tumor models were used to assess the therapeutic efficacy of IO112.ResultsWe show that arginase-1-specific memory T cells specifically recognize arginase-1 expressing cells, such as mRNA transfected autologous dendritic cells (DCs) and B cells as well as M2 polarized macrophages in vitro. In addition, activated arginase-1-specific T cells produce pro-inflammatory cytokines IFNγ and TNFα. Secretion of TH1 cytokines by these T cells suggests that they may act as potent immune modulators in the tumor microenvironment, since many arginase-1 expressing myeloid cells are not terminally differentiated and they can be re-polarized to an immunostimulatory, M1-like phenotype. We also observed that targeting of M2-polarized arginase-1 expressing monocytic leukemia cell line THP-1 with arginase-1-specific CD4+ T cells induces upregulation of PD-L1 on the THP-1 cells. Furthermore, we demonstrate anti-tumor activity of IO112 in syngeneic mouse tumor models (B16 and MC38), both as monotherapy and in combination with anti-PD-1 treatment. The therapeutic effect was associated with increased immune infiltration in the IO112-treated mice compared to the control.ConclusionsWe demonstrate that arginase-1 specific T cells can influence the polarization of arginase-1-expressing immune cells. Our study provides evidence that IO112 immune therapy against arginase-1 is an attractive way of modulating the immune suppressive tumor microenvironment for therapeutic benefit. With this rationale, we are currently undertaking Investigational New Drug (IND) application enabling studies to explore this approach in a clinical setting.ReferencesMartinenaite E, Mortensen REJ, Hansen M, Holmström MO, Ahmad SM, Jørgensen NGD, Met Ö, Donia M, Svane IM, Andersen MH. Frequent adaptive immune responses against arginase-1. Oncoimmunology 2018;7(3):e1404215.Martinenaite E, Ahmad SM, Svane IM, Andersen MH. Peripheral memory T cells specific for Arginase-1. Cell Mol Immunol 2019;16(8):718–719.Martinenaite E, Ahmad SM, Bendtsen SK, Jørgensen MA, Weis-Banke SE, Svane IM, Andersen MH. Arginase-1-based vaccination against the tumor microenvironment: the identification of an optimal T-cell epitope. Cancer Immunol Immunother 2019;68(11):1901–1907.Ethics ApprovalThis study was approved by the Scientific Ethics Committee for The Capital Region of Denmark and Danish Ethics Committee on experimental animal welfare.

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Abstract 3758: TLR9 agonist SNA-induced innate and adaptive immune responses in tumor microenvironment enhance checkpoint inhibitor antitumor activity in mouse tumor models

10.1158/1538-7445.am2018-3758 ◽

2018 ◽

Author(s):

SubbaRao Nallagatla ◽

Bart R. Anderson ◽

Richard Kang ◽

Ekambar R. Kandimalla

Keyword(s):

Tumor Microenvironment ◽

Antitumor Activity ◽

Immune Responses ◽

Checkpoint Inhibitor ◽

Mouse Tumor ◽

Tumor Models ◽

Adaptive Immune Responses ◽

Tlr9 Agonist ◽

Adaptive Immune ◽

Mouse Tumor Models

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Tumor suppression of novel anti–PD-1 antibodies mediated through CD28 costimulatory pathway

Journal of Experimental Medicine ◽

10.1084/jem.20182359 ◽

2019 ◽

Vol 216 (7) ◽

pp. 1525-1541 ◽

Cited By ~ 9

Author(s):

Craig Fenwick ◽

Juan-Luis Loredo-Varela ◽

Victor Joo ◽

Céline Pellaton ◽

Alex Farina ◽

...

Keyword(s):

T Cells ◽

Antitumor Activity ◽

Cd8 T Cells ◽

Tumor Suppression ◽

Tumor Model ◽

Mouse Tumor ◽

The Novel ◽

Evolutionarily Conserved ◽

Mouse Tumor Models ◽

Costimulatory Pathway

Classical antagonistic antibodies (Abs) targeting PD-1, such as pembrolizumab and nivolumab, act through blockade of the PD-1–PDL-1 interaction. Here, we have identified novel antagonistic anti–PD-1 Abs not blocking the PD-1–PDL-1 interaction. The nonblocking Abs recognize epitopes on PD-1 located on the opposing face of the PDL-1 interaction and overlap with a newly identified evolutionarily conserved patch. These nonblocking Abs act predominantly through the CD28 coreceptor. Importantly, a combination of blocking and nonblocking Abs synergize in the functional recovery of antigen-specific exhausted CD8 T cells. Interestingly, nonblocking anti–PD-1 Abs have equivalent antitumor activity compared with blocker Abs in two mouse tumor models, and combination therapy using both classes of Abs enhanced tumor suppression in the mouse immunogenic tumor model. The identification of the novel nonblocker anti–PD-1 Abs and their synergy with classical blocker Abs may be instrumental in potentiating immunotherapy strategies and antitumor activity.

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