Famke L. Schneiders

Clinical experience with α-galactosylceramide (KRN7000) in patients with advanced cancer and chronic hepatitis B/C infection.

For over a century, research has sought ways to boost the immune system in order to eradicate tumors and viruses that exist after escaping immunosurveillance. For the treatment of cancer and hepatitis immunotherapeutic strategies have overall had limited clinical success. An urgent need exists therefore to introduce more effective therapeutic approaches. Invariant (i)NKT cells constitute a conserved T lymphocyte lineage with dominant immunoregulatory, antitumor and antiviral effector cell properties. iNKT specifically recognize the glycolipid α-galactosylceramide in the context of CD1d resulting in their activation. Activated iNKT can promote the development of a long-lasting Th1 biased proinflammatory immune response as demonstrated in multiple tumor-metastasis and viral infection models. Here, we will provide a brief overview of the preclinical data of α-galactosylceramide that formed the basis for subsequent clinical trials in patients with advanced cancer and chronic hepatitis B/C, and elaborate on our own clinical experience with α-galactosylceramide in these patient groups.

Bispecific antibody platforms for cancer immunotherapy

Over the past decades advances in bioengineering and expanded insight in tumor immunology have resulted in the emergence of novel bispecific antibody (bsAb) constructs that are capable of redirecting immune effector cells to the tumor microenvironment. (Pre-) clinical studies of various bsAb constructs have shown impressive results in terms of immune effector cell retargeting, target dependent activation and the induction of anti-tumor responses. This review summarizes recent advances in the field of bsAb-therapy and limitations that were encountered. Furthermore, we will discuss potential future developments that can be expected to take the bsAb approach successfully forward.

Arming the Melanoma Sentinel Lymph Node through Local Administration of CpG-B and GM-CSF: Recruitment and Activation of BDCA3/CD141+ Dendritic Cells and Enhanced Cross-Presentation

Sluijter and colleagues report that intradermal injection of combined CpG/GM-CSF at the primary melanoma excision site prior to removal of sentinel lymph nodes (SLN) led to recruitment of BDCA3+ conventional dendritic cell (cDC) precursors from blood and enhanced DC maturation with selective increase of SLN-resident CLEC9A/BDCA3/CD141+ cDCs. Melanoma-induced suppression of dendritic cells (DC) in the sentinel lymph node (SLN) interferes with the generation of protective antitumor immunity. In an effort to strengthen immune defense against metastatic spread, we performed a three-arm phase II study comprising 28 patients with stage I–II melanoma randomized to receive intradermal injections around the primary tumor excision site of saline or low-dose CpG-B, alone or combined with GM-CSF, before excision of the SLNs. After pathologic examination, 5 patients were diagnosed with stage III melanoma based on the presence of tumor cells in the SLNs. Combined CpG/GM-CSF administration resulted in enhanced maturation of all identifiable conventional (cDC) and plasmacytoid (pDC) DC subsets and selectively induced increased frequencies of SLN-resident BDCA3/CD141+ cDC subsets that also expressed the C-type lectin receptor CLEC9A. Correlative in vivo analyses and in vitro studies provided evidence that these subsets were derived from BDCA3+ cDC precursors in the blood that were recruited to the SLNs in a type I IFN-dependent manner and subsequently matured under the combined influence of CpG and GM-CSF. In line with their reported functional abilities, frequencies of in vivo CpG/GM-CSF–induced BDCA3/CD141+ DCs correlated with increased ex vivo cross-presenting capacity of SLN suspensions. Combined local CpG/GM-CSF delivery thus supports protective antimelanoma immunity through concerted activation of pDC and cDC subsets and recruitment of BDCA3+ cDC subsets with T cell–stimulatory and cross-priming abilities. Cancer Immunol Res; 3(5); 495–505. ©2015 AACR.

Activated iNKT cells promote Vγ9Vδ2-T cell anti-tumor effector functions through the production of TNF-α

Vγ9Vδ2-T cells constitute a proinflammatory lymphocyte subpopulation with established antitumor activity. Phosphoantigens activate Vγ9Vδ2-T cells in vivo and in vitro. We studied whether the antitumor activity of Vγ9Vδ2-T cells can be potentiated by invariant NKT cells (iNKT), an important immunoregulatory T cell subset. When activated by the glycolipid α-galactosylceramide (α-GalCer), iNKT produce large amounts of cytokines involved in antitumor immune responses. Monocyte-derived dendritic cells were loaded with both phosphoantigens (using aminobisphosphonates) and α-GalCer during maturation and subsequently co-cultured with Vγ9Vδ2-T and iNKT cells. Aminobisphosphonates dose-dependently enhanced Vγ9Vδ2-T cell activation, and this was potentiated by α-GalCer-induced iNKT co-activation. iNKT co-activation also enhanced the IFN-γ production and cytolytic potential of Vγ9Vδ2-T cells against tumor cells. Using transwell experiments and neutralizing antibodies cross-talk between iNKT and Vγ9Vδ2-T cells was found to be mediated by TNF-α. Our data provide a rationale for combining both activating ligands to improve Vγ9Vδ2-T cell based approaches in cancer-immunotherapy.

CD1d-Restricted Antigen Presentation by Vγ9Vδ2-T Cells Requires Trogocytosis

Schneiders and colleagues show that phosphoantigen (pAg)-activated Vγ9Vδ2-T cells were able to present glycolipid Ag α-galactosylceramide to iNKT cells not from de novo synthesis of antigen-presenting molecules but from trogocytosed CD1d-containing membrane fragments from pAg-expressing cells. CD1d-restricted invariant natural killer T cells (iNKT) constitute an important immunoregulatory T-cell subset that can be activated by the synthetic glycolipid α-galactosylceramide (α-GalCer) and play a dominant role in antitumor immunity. Clinical trials with α-GalCer–pulsed monocyte-derived dendritic cells (moDC) have shown anecdotal antitumor activity in advanced cancer. It was reported that phosphoantigen (pAg)-activated Vγ9Vδ2-T cells can acquire characteristics of professional antigen-presenting cells (APC). Considering the clinical immunotherapeutic applications, Vγ9Vδ2-T APC can offer important advantages over moDC, potentially constituting an attractive novel APC platform. Here, we demonstrate that Vγ9Vδ2-T APC can present antigens to iNKT. However, this does not result from de novo synthesis of CD1d by Vγ9Vδ2-T, but critically depends on trogocytosis of CD1d-containing membrane fragments from pAg-expressing cells. CD1d-expressing Vγ9Vδ2-T cells were able to activate iNKT in a CD1d-restricted and α-GalCer–dependent fashion. Although α-GalCer–loaded moDC outperformed Vγ9Vδ2-T APC on a per cell basis, Vγ9Vδ2-T APC possess unique features with respect to clinical immunotherapeutic application that make them an interesting platform for consideration in future clinical trials. Cancer Immunol Res; 2(8); 732–40. ©2014 AACR.

mTOR Inhibition Per Se Induces Nuclear Localization of FOXP3 and Conversion of Invariant NKT (iNKT) Cells into Immunosuppressive Regulatory iNKT Cells

CD1d-restricted activation of invariant NKT (iNKT) cells results in the abundant production of various types of cytokines and the subsequent modulation of immune responses. This has been shown to be relevant in several clinical disorders, including cancer, autoimmunity, and graft tolerance. Although it is well known that the suppressive function of regulatory T cells is critically dependent on the FOXP3 gene, FOXP3 can also be expressed by conventional human T cells upon activation, indicating the lack of specificity of FOXP3 as a marker for suppressive cells. In this study, we report that the mammalian target of rapamycin (mTOR) inhibitor rapamycin and IL-10, but not TGF-β, can induce FOXP3 expression in iNKT cell lines. Importantly, however, FOXP3+ iNKT cells only acquired suppressive abilities when cultured in the presence of the mTOR inhibitor rapamycin. Suppression of responder T cell proliferation by FOXP3+ iNKT cells was found to be cell contact–dependent and was accompanied by a reduced capacity of iNKT cells to secrete IFN-γ. Notably, imaging flow cytometry analysis demonstrated predominant nuclear localization of FOXP3 in suppressive FOXP3+ iNKT cells, whereas nonsuppressive FOXP3+ iNKT cells showed a predominance of cytoplasmically localized FOXP3. In conclusion, whereas IL-10 can enhance FOXP3 expression in iNKT cells, mTOR inhibition is solely required for promoting nuclear localization of FOXP3 and the induction of suppressive FOXP3+ iNKT cells.

Exploiting the CD1d-iNKT Cell Axis for Potentiation of DC-Based Cancer Vaccines

Invariant natural killer T cells (iNKT) and dendritic cells (DC) play a central role in tumor immunity through downstream activation of immune effector cells by pro-inflammatory cytokines. Evidence is accumulating that the CD1d-iNKT cell axis can be effectively used to potentiate DC-based cancer vaccines. Here, we provide a detailed methodology for the generation of (CD1d-expressing) monocyte-derived DC (moDC) and their subsequent loading with the iNKT cell agonist α-galactosylceramide (α-GalCer) or their direct ligation by agonistic anti-CD1d monoclonal antibodies.

Clinical Trials with α-Galactosylceramide (KRN7000) in Advanced Cancer

For over a century, research has sought ways to boost the immune system of cancer patients in order to eradicate tumors that arose after escaping presumptive immunosurveillance. With increasing knowledge of the immune system, immunotherapeutic strategies to break tolerance to the tumor evolved from largely nonspecific to more specific and increasingly potent forms of cancer vaccination. Overall however, immunotherapeutic strategies for the treatment of cancer have had limited clinical success and an urgent need exists, therefore, to introduce more effective, knowledge-based therapeutic approaches. Invariant natural killer T (iNKT) cells constitute an evolutionary conserved T lymphocyte lineage with dominant immunoregulatory and antitumor effector cell properties. iNKT specifically recognize the glycolipid α-galactosylceramide (α-GalCer/KRN7000) in the context of the CD1d antigen-presenting molecule resulting in their activation. Activated iNKT have been shown to promote the development of a long-lasting Th1-biased proinflammatory antitumor immune response in a variety of murine tumor-metastasis models of liver, lung and lymph nodes, including colon and lung carcinoma, lymphoma, sarcoma, and melanoma, suggesting broad clinical applicability. Here, we will provide an overview of the preclinical data of α-GalCer that formed the basis for subsequent clinical trials in advanced cancer patients, review these clinical trials, focusing on our own experience with α-GalCer, and discuss future perspectives.

A bispecific nanobody approach to leverage the potent and widely applicable tumor cytolytic capacity of Vγ9Vδ2-T cells

ABSTRACT Though Vγ9Vδ2-T cells constitute only a small fraction of the total T cell population in human peripheral blood, they play a vital role in tumor defense and are therefore of major interest to explore for cancer immunotherapy. Vγ9Vδ2-T cell-based cancer immunotherapeutic approaches developed so far have been generally well tolerated and were able to induce significant clinical responses. However, overall results were inconsistent, possibly due to the fact that these strategies induced systemic activation of Vγ9Vδ2-T cells without preferential accumulation and targeted activation in the tumor. Here we show that a novel bispecific nanobody-based construct targeting both Vγ9Vδ2-T cells and EGFR induced potent Vγ9Vδ2-T cell activation and subsequent tumor cell lysis both in vitro and in an in vivo mouse xenograft model. Tumor cell lysis was independent of KRAS and BRAF tumor mutation status and common Vγ9Vδ2-T cell receptor sequence variations. In combination with the conserved monomorphic nature of the Vγ9Vδ2-TCR and the facile replacement of the tumor-specific nanobody, this immunotherapeutic approach can be applied to a large group of cancer patients.

Vγ9Vδ2-T cells as antigen presenting cells for iNKT cell based cancer immunotherapy

CD1d-restricted invariant natural killer T cells (iNKT) constitute an important immunoregulatory T-cell subset involved in the induction of antitumor immune responses. Here, we provide a view on the recent observation that Vγ9Vδ2-T cells, through trogocytosis of CD1d-containing membrane fragments, have the capacity to act as antigen presenting cells for iNKT.