Successful Anti-PD-1 Cancer Immunotherapy Requires T Cell-Dendritic Cell Crosstalk Involving the Cytokines IFN-γ and IL-12.

Anti-PD-1 immune checkpoint blockers can induce sustained clinical responses in cancer but how they function in vivo remains incompletely understood. Here, we combined intravital real-time imaging with single-cell RNA sequencing analysis and mouse models to uncover anti-PD-1 pharmacodynamics directly within tumors. We showed that effective antitumor responses required a subset of tumor-infiltrating dendritic cells (DCs), which produced interleukin 12 (IL-12). These DCs did not bind anti-PD-1 but produced IL-12 upon sensing interferon γ (IFN-γ) that was released from neighboring T cells. In turn, DC-derived IL-12 stimulated antitumor T cell immunity. These findings suggest that full-fledged activation of antitumor T cells by anti-PD-1 is not direct, but rather involves T cell:DC crosstalk and is licensed by IFN-γ and IL-12. Furthermore, we found that activating the non-canonical NF-κB transcription factor pathway amplified IL-12-producing DCs and sensitized tumors to anti-PD-1 treatment, suggesting a therapeutic strategy to improve responses to checkpoint blockade.

Human plasma cells engineered to secrete bispecifics drive effective in vivo leukemia killing.

Bispecific antibodies are an important tool for the management and treatment of acute leukemias. As a next step toward clinical translation of engineered plasma cells, we describe approaches for secretion of bispecific antibodies by human plasma cells. We show that human plasma cells expressing either fragment crystallizable domain-deficient anti-CD19 × anti-CD3 (blinatumomab) or anti-CD33 × anti-CD3 bispecific antibodies mediate T cell activation and direct T cell killing of B acute lymphoblastic leukemia or acute myeloid leukemia cell lines in vitro. We demonstrate that knockout of the self-expressed antigen, CD19, boosts anti-CD19-bispecific secretion by plasma cells and prevents self-targeting. Plasma cells secreting anti-CD19-bispecific antibodies elicited in vivo control of acute lymphoblastic leukemia patient-derived xenografts in immunodeficient mice co-engrafted with autologous T cells. In these studies, we found that leukemic control elicited by engineered plasma cells was similar to CD19-targeted chimeric antigen receptor-expressing T cells. Finally, the steady-state concentration of anti-CD19 bispecifics in serum 1 month after cell delivery and tumor eradication was comparable with that observed in patients treated with a steady-state infusion of blinatumomab. These findings support further development of ePCs for use as a durable delivery system for the treatment of acute leukemias, and potentially other cancers.

Host-functionalization of macrin nanoparticles to enable drug loading and control tumor-associated macrophage phenotype.

Macrophages are critical regulators of the tumor microenvironment and often present an immuno-suppressive phenotype, supporting tumor growth and immune evasion. Promoting a robust pro-inflammatory macrophage phenotype has emerged as a therapeutic modality that supports tumor clearance, including through synergy with immune checkpoint therapies. Polyglucose nanoparticles (macrins), which possess high macrophage affinity, are useful vehicles for delivering drugs to macrophages, potentially altering their phenotype. Here, we examine the potential of functionalized macrins, synthesized by crosslinking carboxymethyl dextran with L-lysine, as effective carriers of immuno-stimulatory drugs to tumor-associated macrophages (TAMs). Azide groups incorporated during particle synthesis provided a handle for click-coupling of propargyl-modified ß-cyclodextrin to macrins under mild conditions. Fluorescence-based competitive binding assays revealed the ability of ß-cyclodextrin to non-covalently bind to hydrophobic immuno-stimulatory drug candidates (Keq ~ 103 M-1), enabling drug loading within nanoparticles. Furthermore, transcriptional profiles of macrophages indicated robust pro-inflammatory reprogramming (elevated Nos2 and Il12; suppressed Arg1 and Mrc1 expression levels) for a subset of these immuno-stimulatory agents (UNC2025 and R848). Loading of R848 into the modified macrins improved the drug's effect on primary murine macrophages by three-fold in vitro. Intravital microscopy in IL-12-eYFP reporter mice (24 h post-injection) revealed a two-fold enhancement in mean YFP fluorescence intensity in macrophages targeted with R848-loaded macrins, relative to vehicle controls, validating the desired pro-inflammatory reprogramming of TAMs in vivo by cell-targeted drug delivery. Finally, in an intradermal MC38 tumor model, cyclodextrin-modified macrin NPs loaded with immunostimulatory drugs significantly reduced tumor growth. Therefore, efficient and effective repolarization of tumor-associated macrophages to an M1-like phenotype-via drug-loaded macrins-inhibits tumor growth and may be useful as an adjuvant to existing immune checkpoint therapies.

Phase II Trial of IL-12 Plasmid Transfection and PD-1 Blockade in Immunologically Quiescent Melanoma.

PURPOSE: Tumors with low frequencies of checkpoint positive tumor-infiltrating lymphocytes (cpTIL) have a low likelihood of response to PD-1 blockade. We conducted a prospective multicenter phase II trial of intratumoral plasmid IL-12 (tavokinogene telseplasmid; "tavo") electroporation combined with pembrolizumab in patients with advanced melanoma with low frequencies of checkpoint positive cytotoxic lymphocytes (cpCTL). PATIENTS AND METHODS: Tavo was administered intratumorally days 1, 5, and 8 every 6 weeks while pembrolizumab (200 mg, i.v.) was administered every 3 weeks. The primary endpoint was objective response rate (ORR) by RECIST, secondary endpoints included duration of response, overall survival and progression-free survival. Toxicity was evaluated by the CTCAE v4. Extensive correlative analysis was done. RESULTS: The combination of tavo and pembrolizumab was well tolerated with adverse events similar to those previously reported with pembrolizumab alone. Patients had a 41% ORR (n = 22, RECIST 1.1) with 36% complete responses. Correlative analysis showed that the combination enhanced immune infiltration and sustained the IL-12/IFNγ feed-forward cycle, driving intratumoral cross-presenting dendritic cell subsets with increased TILs, emerging T cell receptor clones and, ultimately, systemic cellular immune responses. CONCLUSIONS: The combination of tavo and pembrolizumab was associated with a higher than expected response rate in this poorly immunogenic population. No new or unexpected toxicities were observed. Correlative analysis showed T cell infiltration with enhanced immunity paralleling the clinical activity in low cpCTL tumors.

Recording the wild lives of immune cells.

Intravital microscopic imaging can uncover fundamental aspects of immune cell behavior in real time in both healthy and pathological states. Here, we discuss approaches for single-cell imaging of adaptive and innate immune cells to explore how they migrate, communicate, and mediate regulatory or effector functions in various tissues throughout the body. We further review how intravital single-cell imaging can be used to study drug effects on immune cells.

Arg1 expression defines immunosuppressive subsets of tumor-associated macrophages.

Tumor-associated macrophages (TAM) have attracted attention as they can modulate key cancer-related activities, yet TAM represent a heterogenous group of cells that remain incompletely characterized. In growing tumors, TAM are often referred to as M2-like macrophages, which are cells that display immunosuppressive and tumorigenic functions and express the enzyme arginase 1 (Arg1). Methods: Here we combined high resolution intravital imaging with single cell RNA seq to uncover the topography and molecular profiles of immunosuppressive macrophages in mice. We further assessed how immunotherapeutic interventions impact these cells directly in vivo. Results: We show that: i) Arg1+ macrophages are more abundant in tumors compared to other organs; ii) there exist two morphologically distinct subsets of Arg1 TAM defined by previously unknown markers (Gbp2b, Bst1, Sgk1, Pmepa1, Ms4a7); iii) anti-Programmed Cell Death-1 (aPD-1) therapy decreases the number of Arg1+ TAM while increasing Arg1- TAM; iv) accordingly, pharmacological inhibition of arginase 1 does not synergize with aPD-1 therapy. Conclusion: Overall, this research shows how powerful complementary single cell analytical approaches can be used to improve our understanding of drug action in vivo.

TLR7/8-agonist-loaded nanoparticles promote the polarization of tumour-associated macrophages to enhance cancer immunotherapy.

Tumour-associated macrophages are abundant in many cancers, and often display an immune-suppressive M2-like phenotype that fosters tumour growth and promotes resistance to therapy. Yet, macrophages are highly plastic and can also acquire an anti-tumorigenic M1-like phenotype. Here, we show that R848, an agonist of the toll-like receptors TLR7 and TLR8 identified in a morphometric-based screen, is a potent driver of the M1 phenotype in vitro and that R848-loaded ß-cyclodextrin nanoparticles (CDNP-R848) lead to efficient drug delivery to tumour-associated macrophages in vivo. As a monotherapy, the administration of CDNP-R848 in multiple tumour models in mice altered the functional orientation of the tumour immune microenvironment towards an M1 phenotype, leading to controlled tumour growth and protecting the animals against tumour rechallenge. When used in combination with the immune checkpoint inhibitor anti-PD-1, we observed improved immunotherapy response rates, including in a tumour model resistant to anti-PD-1 therapy alone. Our findings demonstrate the ability of rationally engineered drug-nanoparticle combinations to efficiently modulate tumour-associated macrophages for cancer immunotherapy.

Near infrared fluorescent imaging of choline kinase alpha expression and inhibition in breast tumors.

Choline kinase alpha (ChoKα) overexpression is associated with an aggressive tumor phenotype. ChoKα inhibitors induce apoptosis in tumors, however validation of their specificity is difficult in vivo. We report the use of optical imaging to assess ChoKα status in cells and in vivo using JAS239, a carbocyanine-based ChoKα inhibitor with inherent near infrared fluorescence. JAS239 attenuated choline phosphorylation and viability in a panel of human breast cancer cell lines. Antibody blockade prevented cellular retention of JAS239 indicating direct interaction with ChoKα independent of the choline transporters and catabolic choline pathways. In mice bearing orthotopic MCF7 breast xenografts, optical imaging with JAS239 distinguished tumors overexpressing ChoKα from their empty vector counterparts and delineated tumor margins. Pharmacological inhibition of ChoK by the established inhibitor MN58b led to a growth inhibition in 4175-Luc+ tumors that was accompanied by concomitant reduction in JAS239 uptake and decreased total choline metabolite levels as measured using magnetic resonance spectroscopy. At higher therapeutic doses, JAS239 was as effective as MN58b at arresting tumor growth and inducing apoptosis in MDA-MB-231 tumors, significantly reducing tumor choline below baseline levels without observable systemic toxicity. These data introduce a new method to monitor therapeutically effective inhibitors of choline metabolism in breast cancer using a small molecule companion diagnostic.

In vivo imaging reveals a tumor-associated macrophage-mediated resistance pathway in anti-PD-1 therapy.

Monoclonal antibodies (mAbs) targeting the immune checkpoint anti-programmed cell death protein 1 (aPD-1) have demonstrated impressive benefits for the treatment of some cancers; however, these drugs are not always effective, and we still have a limited understanding of the mechanisms that contribute to their efficacy or lack thereof. We used in vivo imaging to uncover the fate and activity of aPD-1 mAbs in real time and at subcellular resolution in mice. We show that aPD-1 mAbs effectively bind PD-1+ tumor-infiltrating CD8+ T cells at early time points after administration. However, this engagement is transient, and aPD-1 mAbs are captured within minutes from the T cell surface by PD-1- tumor-associated macrophages. We further show that macrophage accrual of aPD-1 mAbs depends both on the drug's Fc domain glycan and on Fcγ receptors (FcγRs) expressed by host myeloid cells and extend these findings to the human setting. Finally, we demonstrate that in vivo blockade of FcγRs before aPD-1 mAb administration substantially prolongs aPD-1 mAb binding to tumor-infiltrating CD8+ T cells and enhances immunotherapy-induced tumor regression in mice. These investigations yield insight into aPD-1 target engagement in vivo and identify specific Fc/FcγR interactions that can be modulated to improve checkpoint blockade therapy.

IRF3 and type I interferons fuel a fatal response to myocardial infarction.

Interferon regulatory factor 3 (IRF3) and type I interferons (IFNs) protect against infections and cancer, but excessive IRF3 activation and type I IFN production cause autoinflammatory conditions such as Aicardi-Goutières syndrome and STING-associated vasculopathy of infancy (SAVI). Myocardial infarction (MI) elicits inflammation, but the dominant molecular drivers of MI-associated inflammation remain unclear. Here we show that ischemic cell death and uptake of cell debris by macrophages in the heart fuel a fatal response to MI by activating IRF3 and type I IFN production. In mice, single-cell RNA-seq analysis of 4,215 leukocytes isolated from infarcted and non-infarcted hearts showed that MI provokes activation of an IRF3-interferon axis in a distinct population of interferon-inducible cells (IFNICs) that were classified as cardiac macrophages. Mice genetically deficient in cyclic GMP-AMP synthase (cGAS), its adaptor STING, IRF3, or the type I IFN receptor IFNAR exhibited impaired interferon-stimulated gene (ISG) expression and, in the case of mice deficient in IRF3 or IFNAR, improved survival after MI as compared to controls. Interruption of IRF3-dependent signaling resulted in decreased cardiac expression of inflammatory cytokines and chemokines and decreased inflammatory cell infiltration of the heart, as well as in attenuated ventricular dilation and improved cardiac function. Similarly, treatment of mice with an IFNAR-neutralizing antibody after MI ablated the interferon response and improved left ventricular dysfunction and survival. These results identify IRF3 and the type I IFN response as a potential therapeutic target for post-MI cardioprotection.

Choline kinase alpha-Putting the ChoK-hold on tumor metabolism.

It is well established that lipid metabolism is drastically altered during tumor development and response to therapy. Choline kinase alpha (ChoKα) is a key mediator of these changes, as it represents the first committed step in the Kennedy pathway of phosphatidylcholine biosynthesis and ChoKα expression is upregulated in many human cancers. ChoKα activity is associated with drug resistant, metastatic, and malignant phenotypes, and represents a robust biomarker and therapeutic target in cancer. Effective ChoKα inhibitors have been developed and have recently entered clinical trials. ChoKα's clinical relevance was, until recently, attributed solely to its production of second messenger intermediates of phospholipid synthesis. The recent discovery of a non-catalytic scaffolding function of ChoKα may link growth receptor signaling to lipid biogenesis and requires a reinterpretation of the design and validation of ChoKα inhibitors. Advances in positron emission tomography, magnetic resonance spectroscopy, and optical imaging methods now allow for a comprehensive understanding of ChoKα expression and activity in vivo. We will review the current understanding of ChoKα metabolism, its role in tumor biology and the development and validation of targeted therapies and companion diagnostics for this important regulatory enzyme. This comes at a critical time as ChoKα-targeting programs receive more clinical interest.

Magnetic resonance spectroscopy for detection of choline kinase inhibition in the treatment of brain tumors.

Abnormal choline metabolism is a hallmark of cancer and is associated with oncogenesis and tumor progression. Increased choline is consistently observed in both preclinical tumor models and in human brain tumors by proton magnetic resonance spectroscopy (MRS). Thus, inhibition of choline metabolism using specific choline kinase inhibitors such as MN58b may be a promising new strategy for treatment of brain tumors. We demonstrate the efficacy of MN58b in suppressing phosphocholine production in three brain tumor cell lines. In vivo MRS studies of rats with intracranial F98-derived brain tumors showed a significant decrease in tumor total choline concentration after treatment with MN58b. High-resolution MRS of tissue extracts confirmed that this decrease was due to a significant reduction in phosphocholine. Concomitantly, a significant increase in poly-unsaturated lipid resonances was also observed in treated tumors, indicating apoptotic cell death. MRI-based volume measurements demonstrated a significant growth arrest in the MN58b-treated tumors in comparison with saline-treated controls. Histologically, MN58b-treated tumors showed decreased cell density, as well as increased apoptotic cells. These results suggest that inhibition of choline kinase can be used as an adjuvant to chemotherapy in the treatment of brain tumors and that decreases in total choline observed by MRS can be used as an effective pharmacodynamic biomarker of treatment response.

Quantification of tumor fluorescence during intraoperative optical cancer imaging.

Intraoperative optical cancer imaging is an emerging technology in which surgeons employ fluorophores to visualize tumors, identify tumor-positive margins and lymph nodes containing metastases. This study compares instrumentation to measure tumor fluorescence. Three imaging systems (Spectropen, Glomax, Flocam) measured and quantified fluorescent signal-to-background ratios (SBR) in vitro, murine xenografts, tissue phantoms and clinically. Evaluation criteria included the detection of small changes in fluorescence, sensitivity of signal detection at increasing depths and practicality of use. In vitro, spectroscopy was superior in detecting incremental differences in fluorescence than luminescence and digital imaging (Ln[SBR] = 6.8 ± 0.6, 2.4 ± 0.3, 2.6 ± 0.1, p = 0.0001). In fluorescent tumor cells, digital imaging measured higher SBRs than luminescence (6.1 ± 0.2 vs. 4.3 ± 0.4, p = 0.001). Spectroscopy was more sensitive than luminometry and digital imaging in identifying murine tumor fluorescence (SBR = 41.7 ± 11.5, 5.1 ± 1.8, 4.1 ± 0.9, p = 0.0001), and more sensitive than digital imaging at detecting fluorescence at increasing depths (SBR = 7.0 ± 3.4 vs. 2.4 ± 0.5, p = 0.03). Lastly, digital imaging was the most practical and least time-consuming. All methods detected incremental differences in fluorescence. Spectroscopy was the most sensitive for small changes in fluorescence. Digital imaging was the most practical considering its wide field of view, background noise filtering capability, and sensitivity to increasing depth.

Direct inhibition of choline kinase by a near-infrared fluorescent carbocyanine.

Choline kinase alpha (ChoK) expression is increasingly being recognized as an important indicator of breast cancer prognosis; however, previous efforts to noninvasively measure ChoK status have been complicated by the spectral limitations of in vivo magnetic resonance spectroscopy (MRS) and the complex network of enzymes involved in choline metabolism. The most effective ChoK inhibitors are symmetric and contain quaternary ammonium groups within heterocyclic head groups connected by an aliphatic spacer. Characterization of these bis-pyridinium and bis-quinolinium compounds has led to phase I clinical trials to assess small-molecule inhibitors of ChoK for solid tumor treatment. We report the development of a novel carbocyanine dye, JAS239, whose bis-indolium structure conforms to the parameters established for ChoK specificity and whose spacer length confers fluorescence in the near-infrared (NIR) window. Fluorimetry and confocal microscopy were used to demonstrate that JAS239 rapidly enters breast cancer cells independent of the choline transporters, with accumulation in the cytosolic space where ChoK is active. Radio-tracing and (1)H MRS techniques were used to determine that JAS239 binds and competitively inhibits ChoK intracellularly, preventing choline phosphorylation while inducing cell death in breast cancer cell lines with similar efficacy to known ChoK inhibitors. Fluorescent molecules that report on ChoK status have potential use as companion diagnostics for noninvasive breast tumor staging, because NIR fluorescence allows for detection of real-time probe accumulation in vivo. Furthermore, their ability as novel ChoK inhibitors may prove effective against aggressive, therapy-resistant tumors.