Acquired resistance is a major limiting factor for durable T cell therapies in solid tumors. Antigen escape pathways such as insufficient antigen coverage or loss of target antigen remain major resistance mechanisms that need to be addressed in order to expand the field of T cell therapies.Interleukin-12 (IL-12) is a potent stimulator of innate and adaptive immune cells that holds strong potential for cancer immunotherapy, but its clinical utility has been limited by high systemic toxicities. We have previously shown that tethering an IL-12 immunocytokine to the surface of T cells prior to adoptive cell transfer (ACT) safely improves anti-tumor efficacy by promoting T cell function specifically in the tumor. Here, we demonstrate that cell-tethered IL-12 delivers adjuvant activity that leads to priming and expansion of by-stander, tumor-specific T cells, and thereby counteract common immune escape pathways.
Methods
Adjuvant activity of IL12-tethered pmel T cells, reactive towards the gp100 antigen of B16 tumors, was evaluated in the B16-OVA syngeneic mouse model. Notably, adoptive transfer of IL12-tethered pmel T cells, but not pmel T cells alone, resulted in proliferation of endogenous tumor infiltrating lymphocytes. To assess whether this reflected tumor-specific T cell responses, we used dextramer staining against non-targeted, tumor-specific antigens and found that both abundance and activation increased following cell-tethered IL-12 treatment. Encouraged by these findings, the OT-1 model was used to track epitope spreading to tumor-specific naïve T cells. Following treatment with IL-12-tethered PMEL T cells, we tracked the proliferation and tumor engraftment of labelled, naïve OT-I T cells, which are reactive towards the non-targeted OVA antigen.
Results
Cell-tethered-IL12, but neither ACT nor ACT and systemically administered IL-12, induced proliferation and engraftment OT-1 T cells in tumor-draining lymph nodes (tdLNs) and tumors of B16-OVA-bearing mice. This effect was antigen-dependent as the OT-I T cells were not primed in B16.F10 (OVA antigen-negative) tumors. Mechanistically, this priming was associated with IL-12-induced increases in activation and tdLN infiltration of cross-presenting dendritic cells (cDC1) as well as increased presentation of the SIINFEKL epitope of OVA specifically on this subset of dendritic cells.
Conclusions
Together, our findings suggest that tethering IL-12 to tumor-specific T cells prior to adoptive transfer promotes epitope spreading through the combination of tumor cell-killing induced by the ACT therapy and IL-12-induced activation of cDC1s in the tdLN. This adjuvant activity from T cell-tethered IL-12 holds promise for overcoming antigen escape pathways that limit the efficacy of antigen-specific T cells against heterogeneous tumors
Adoptive cell therapy (ACT), based on treatment with autologous tumour infiltrating lymphocyte (TIL)-derived or genetically modified chimeric antigen receptor (CAR) T cells, has become a potentially curative therapy for subgroups of patients with melanoma and hematological malignancies. To further improve response rates, and to broaden the applicability of ACT to more types of solid malignancies, it is necessary to explore and define strategies that can be used as adjuvant treatments to ACT. Stimulation of endogenous dendritic cells (DCs) alongside ACT can be used to promote epitope spreading and thereby decrease the risk of tumour escape due to target antigen downregulation, which is a common cause of disease relapse in initially responsive ACT treated patients. Addition of check-point blockade to ACT and DC stimulation might further enhance response rates by counteracting an eventual inactivation of infused and endogenously primed tumour-reactive T cells. This review will outline and discuss therapeutic strategies that can be utilized to engage endogenous DCs alongside ACT and check-point blockade, to strengthen the anti-tumour immune response.
// Lise M. Lindahl 1 , Simon Fredholm 2 , Claudine Joseph 2 , Boye Schnack Nielsen 3 , Lars Jønson 4 , Andreas Willerslev-Olsen 2 , Maria Gluud 2 , Edda Blümel 2 , David L. Petersen 2 , Nina Sibbesen 2 , Tengpeng Hu 2 , Claudia Nastasi 2 , Thorbjørn Krejsgaard 2 , Ditte Jæhger 2 , Jenny L. Persson 5 , Nigel Mongan 6 , Mariusz A. Wasik 7 , Ivan V. Litvinov 8 , Denis Sasseville 9 , Sergei B. Koralov 10 , Charlotte M. Bonefeld 2 , Carsten Geisler 2 , Anders Woetmann 2 , Elisabeth Ralfkiaer 11 , Lars Iversen 1 , Niels Odum 2 1 Department of Dermatology, Aarhus University Hospital, Aarhus, Denmark 2 Department of Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark 3 Bioneer A/S, Hørsholm, Denmark 4 Department of Molecular Medicine, Copenhagen University Hospital, Copenhagen, Denmark 5 Clinical Research Center, Lund University, Malmö, Sweden 6 School of Veterinary Medicine and Science, University of Nottingham, Loughborough, United Kingdom 7 Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA 8 Division of Dermatology, Ottawa Hospital Research Institute, University of Ottawa, Ottawa, Ontario, Canada 9 Division of Dermatology, McGill University Health Centre, Montréal, Quebec, Canada 10 Department of Pathology, New York University School of Medicine, New York, NY, USA 11 Department of Pathology, Copenhagen University Hospital, Copenhagen, Denmark Correspondence to: Lars Iversen, email: lars.iversen@clin.au.dk Niels Odum, email: ndum@sund.ku.dk Keywords: miR-21, in situ, STAT5, IL-2, cutaneous T-cell lymphoma (CTCL) Received: March 23, 2016 Accepted: June 03, 2016 Published: June 18, 2016 ABSTRACT In cutaneous T cell lymphomas (CTCL), miR-21 is aberrantly expressed in skin and peripheral blood and displays anti-apoptotic properties in malignant T cells. It is, however, unclear exactly which cells express miR-21 and what mechanisms regulate miR-21. Here, we demonstrate miR-21 expression in situ in both malignant and reactive lymphocytes as well as stromal cells. qRT-PCR analysis of 47 patients with mycosis fungoides (MF) and Sezary Syndrome (SS) confirmed an increased miR-21 expression that correlated with progressive disease. In cultured malignant T cells miR-21 expression was inhibited by Tofacitinib (CP-690550), a clinical-grade JAK3 inhibitor. Chromatin immunoprecipitation (ChIP) analysis showed direct binding of STAT5 to the miR-21 promoter. Cytokine starvation ex vivo triggered a decrease in miR-21 expression, whereas IL-2 induced an increased miR-21 expression in primary SS T cells and cultured cytokine-dependent SS cells (SeAx). siRNA-mediated depletion of STAT5 inhibited constitutive- and IL-2-induced miR-21 expression in cytokine-independent and dependent T cell lines, respectively. IL-15 and IL-2 were more potent than IL-21 in inducing miR-21 expression in the cytokine-dependent T cells. In conclusion, we provide first evidence that miR-21 is expressed in situ in CTCL skin lesions, induced by IL-2 and IL-15 cytokines, and is regulated by STAT5 in malignant T cells. Thus, our data provide novel evidence for a pathological role of IL-2Rg cytokines in promoting expression of the oncogenic miR-21 in CTCL.
Cancer curing immune responses against heterogeneous solid cancers require that a coordinated immune activation is initiated in the antigen avid but immunosuppressive tumor microenvironment (TME). The plastic TME, and the poor systemic tolerability of immune activating drugs are, however, fundamental barriers to generating curative anticancer immune responses. Here, we introduce the CarboCell technology to overcome these barriers by forming an intratumoral sustained drug release depot that provides high payloads of immune stimulatory drugs selectively within the TME. The CarboCell thereby induces a hot spot for immune cell training and polarization and further drives and maintains the tumor-draining lymph nodes in an anticancer and immune activated state. Mechanistically, this transforms cancerous tissues, consequently generating systemic anticancer immunoreactivity. CarboCell can be injected through standard thin-needle technologies and has inherent imaging contrast which secure accurate intratumoral positioning. In particular, here we report the therapeutic performance for a dual-drug CarboCell providing sustained release of a Toll-like receptor 7/8 agonist and a transforming growth factor-β inhibitor in preclinical tumor models in female mice.
Immune-activating cytokines such as interleukin-12 (IL-12) hold strong potential for cancer immunotherapy but have been limited by high systemic toxicities. We describe here an approach to safely harness cytokine biology for adoptive cell therapy through uniform and dose-controlled tethering onto the surface of the adoptively transferred cells. Tumor-specific T cells tethered with IL-12 showed superior antitumor efficacy across multiple cell therapy models compared to conventional systemic IL-12 coadministration. Mechanistically, the IL-12–tethered T cells supported a strong safety profile by driving interferon-γ production and adoptively transferred T cell activity preferentially in the tumor. Immune profiling revealed that the tethered IL-12 reshaped the suppressive tumor immune microenvironment, including triggering a pronounced repolarization of monocytic myeloid-derived suppressor cells into activated, inflammatory effector cells that further supported antitumor activity. This tethering approach thus holds strong promise for harnessing and directing potent immunomodulatory cytokines for cell therapies while limiting systemic toxicities.
Therapeutic cancer vaccines represent an intriguing approach to cancer immunotherapy and they have been widely explored for the last decade. As opposed to standard modalities, such as surgery and chemotherapy, an effective vaccine-based immune response may provide protection against metastatic disease. Peptide based vaccines can elicit a highly targeted immune response and include a simple, fast and cost-effective production due to recent developments in solid phase peptide synthesis. Recent development within the field of COVID-19 vaccines has highlighted the use of lipid nanoparticles as an effective drug delivery system for vaccination. Incorporation of peptide antigens into engineered micro- and nanoparticles enables induction of a potent T cell response, partly attributed to prolonged and improved antigen presentation by dendritic cells after particle internalization. Peptide-based vaccines are often based on delivery of high-affinity T cell model epitopes. However, the therapeutic relevance of vaccination with low-affinity epitopes is gaining increasing support following the observation that high-affinity epitopes can promote T cell exhaustion resulting from excessive T cell receptor stimulation. Here, we characterize and evaluate a novel lipid nanoparticle (LNP) vaccine platform that is suited for delivery of both high- and low-affinity epitopes in the setting of therapeutic cancer vaccination.
Methods
LNPs were formulated to carry high- or low-affinity peptide epitopes from Ovalbumin (OVA) in conjunction with the TLR7 agonist 1V270. The peptides were anchored to the surface of the LNPs via a reducible DSPE-PEG2000 linker system. The therapeutic vaccine platform was evaluated in vivo both as a monotherapy and in combination with adoptive transfer of OT-I T cells in the syngeneic B16-OVA murine melanoma model.
Results
The LNP vaccine promotes efficient antigen-release and ensures high, continuous antigen-presentation by antigen-presenting cells. While the LNPs can be administered via multiple routes, intratumoral vaccination favors enhanced particle uptake in dendritic cells in the tumor. Formulated with either high- or low-affinity epitopes, intratumorally delivered vaccine particles promote superior tumor-infiltration of adoptively transferred T cells, which translates into potent anti-tumor efficacy in vivo. Finally, we show that vaccination with both CD8+ and CD4+ epitopes can delay tumor growth and prolong survival in an antigen-dependent manner.
Conclusions
This study presents a versatile and multi-purpose LNP vaccine platform that ensures effective delivery of high- and low-affinity epitopes. Intratumoral administration promotes vaccine particle uptake by intratumoral dendritic cells, which is followed by T cell infiltration and anti-tumor efficacy in vivo.
Ethics Approval
All animal procedures were approved by the Danish National Animal Experiments Inspectorate.
Adoptive T cell therapy (ACT) is often accompanied by supporting immunomodulatory drugs to protect T cells from the suppressive tumor microenvironment (TME) [1]. However, systemic administration of these immunomodulators can cause serious side effects and fail to distribute optimally to exert sufficient lymphocyte stimulation within the tumor and lymphoid compartments. Loading T cells with adjuvant drugs or cytokines prior to cell transfer provides a solution to this issue, showing the potential to use T cells as vehicles to carry immunomodulatory molecules to target sites [2]. SHP2 is an important hub connecting several intracellular oncogenic signaling pathways including PD-1/PD-L1, representing a notable target for cancer immunotherapy. SHP2 inhibition has been shown to elicited tumor regression by improving CD8+ T cells activity [3]. Herein we present a lipid nanoparticle system encapsulating an SHP2 inhibitor (SHP2i) that allows high T cell loading capacity and enhances their therapeutic activity.
Methods
Remote-loading gradients were used to achieve high encapsulation efficiency of SHP2i into the lipid nanoparticle platform. Mouse cytotoxic T cells were loaded with SHP2i, and loading efficiency and release rates from the T cells were evaluated in vitro. Flow cytometry was used to assess T cell viability, proliferation, and phenotype. In vivo biodistribution of loaded T cells was evaluated by labeling lipid nanoparticles with gadolinium and T cells with Cell-trace-marker, which were measured with ICP-MS and Flow respectively. The therapeutic anti-tumor efficacy of the loaded T cells was demonstrated on EG.7-OVA tumor-bearing mice.
Results
The developed formulation allowed high T cell loading efficiency of SHPi and extended-release over 5 days. Loading T cells with lipid formulated SHP2i did not compromise cell viability and proliferation and resulted in T cells retaining a central memory phenotype than unloaded counterparts. Adoptively transferred T cells loaded with lipid nanoparticles showed the same distribution and proliferation behavior as the unloaded T cells in vivo, accumulating into tumor tissue three days post cell infusion. Loaded OT.I T cells significantly improved tumor growth inhibition and overall survival than OT.I T cells alone, with 5 out of 6 mice completely tumor-free, resulting in durable long-term responders.
Conclusions
Loading T cells with liposomal SHP2i before ACT allowed specific and controlled delivery of immunomodulatory drugs by T cells. The loaded T cells showed improved anti-tumor efficacy. The developed lipid formulation allows the loading of a variety of immunomodulatory drugs to T cells, which serve both as a drug delivery vehicle and enhance the tumor efficacy of the transferred cells.
References
Waldman AD, Fritz JM, Lenardo MJ. A guide to cancer immunotherapy: from T cell basic science to clinical practice. Nat. Rev. Immunol. 2020. p. 651–68. Combes F, Meyer E, Sanders NN. Immune cells as tumor drug delivery vehicles. J Control Release. Elsevier; 2020;327:70–87. Yuan X, Bu H, Zhou J, Yang CY, Zhang H. Recent Advances of SHP2 Inhibitors in Cancer Therapy: Current Development and Clinical Application. J Med Chem. 2020;63:11368–96.
Ethics Approval
The study has been approved by the Danish Animal Experiments Inspectorate with the permit number 2020-15-0201-00482. The participants gave informed consent before taking part.
The efficacy of anti-programmedcelldeath1therapy (aPD-1), which was recently approved for basal cell carcinoma (BCC) treatment, can be enhanced by adjuvant ablative fractional laser (AFL) in syngeneic murine tumor models. In this explorative study, we aimed to assess locally applied AFL as an adjuvant to systemic aPD-1 treatment in a clinically relevant autochthonous BCC model. BCC tumors (n = 72) were induced in Ptch1+/-K14-CreER2p53fl/fl-mice (n = 34), and the mice subsequently received aPD-1 alone, AFL alone, aPD-1+AFL, or no treatment. The outcome measures included mouse survival time, tumor clearance, tumor growth rates, and tumor immune infiltration. Both aPD-1 and AFL alone significantly increased survival time relative to untreated controls (31 d and 34.5 d, respectively vs. 14 d, p = 0.0348-0.0392). Complementing aPD-1 with AFL further promoted survival (60 d, p = 0.0198 vs. aPD-1) and improved tumor clearance and growth rates. The BCCs were poorly immune infiltrated, but aPD-1 with adjuvant AFL and AFL alone induced substantial immune cell infiltration in the tumors. Similar to AFL alone, combined aPD-1 and AFL increased neutrophil counts (4-fold, p = 0.0242), the proportion of MHCII-positive neutrophils (p = 0.0121), and concordantly, CD4+ and CD8+ T-cell infiltration (p = 0.0061-0.0242). These descriptive results suggest that the anti-tumor response that is generated by aPD-1 with adjuvant AFL is potentially promoted by increased neutrophil and T-cell engraftment in tumors. In conclusion, local AFL shows substantial promise as an adjuvant to systemic aPD-1 therapy in a clinically relevant preclinical BCC model.
Abstract Despite the promise of adoptive cell therapy (ACT) in the treatment of some cancer types, success against solid tumors remains elusive due to multiple immune escape pathways. T cell therapies that affect tumor microenvironment or provide adjuvant activity in addition to tumor cell lysis hold strong potential for overcoming immune escape in solid tumors. Interleukin-12 (IL-12) is a potent stimulator of innate and adaptive immune cells that holds strong potential for cancer immunotherapy, but its clinical utility has been limited by high systemic toxicities. We have previously shown that tethering an IL-12 immunocytokine to the surface of T cells prior to adoptive cell transfer (ACT) safely improves anti-tumor efficacy by promoting T cell function specifically in the tumor. Here, we demonstrate that tethered IL-12 further delivers adjuvant activity by initiating an immune cascade involving natural killer cell-mediated activation of cross-priming dendritic cells to resulting in broader anti-tumor T cell responses. In a preclinical cell therapy model tumor-specific T cells tethered with IL-12, but not the T cells alone, triggered proliferation of tumor-specific endogenous tumor infiltrating lymphocytes (TIL). This was accompanied by increased activation and infiltration of cross-presenting dendritic cells (cDC1) in tumor-draining lymph nodes (tdLN). Analyses of cDC1 subsets revealed upregulation of CD86 expression on both migratory and resident cDC1s within the tdLN, two cell types that are critical for both tumor antigen transport to tdLN and activation of naïve CD8 T cells, respectively. This activity was specific to cDC1s, as increased activation or accumulation of type 2 dendritic cells (cDC2) was not observed. Mechanistically, the IL-12 tethered T cells upregulated expression of FLT3 ligand on NK cells, a key cytokine for cDC1 recruitment and expansion. NK cell depletion ablated cDC1 tdLN infiltration and endogenous tumor infiltrating T cell expansion, indicating that recruitment and activation of endogenous tumor-specific T cells is at least partially driven by tethered IL-12-mediated activation of NK cells to recruit and expand the cross-priming dendritic cells. Together, this demonstrates that our surface tethered cytokine technology provides powerful adjuvant activity to adoptively transferred T cells and promotes activation of endogenous anti-tumor adaptive immune response, thus promoting a broad antigenic repertoire. This approach thus holds potential to help overcome challenges of antigen escape and tumor heterogeneity that limit efficacy of T cell therapies against solid tumors. This abstract is also being presented as PO078. Citation Format: Kate L. Stokes, Ditte E. Jaehger, Alvin Pratama, Holmfridur R. Halldorsdottir, Gulzar Ahmad, Jon D. Nardozzi, Katharine L. Sackton, Thomas L. Andresen, Douglas S. Jones. Adoptive transfer of T cells surface-tethered with IL-12 activates a natural killer/dendritic cell axis to promote antigen spreading for enhanced anti-tumor efficacy [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PR012.
