Fibroblast activation protein targeted radiotherapy induces an immunogenic tumor microenvironment and enhances the efficacy of PD-1 immune checkpoint inhibition.

PURPOSE: FAP is a membrane-bound protease under investigation as a pan-cancer target, given its high levels in tumors but limited expression in normal tissues. FAP-2286 is a radiopharmaceutical in clinical development for solid tumors that consists of two functional elements: a FAP-targeting peptide and a chelator used to attach radioisotopes. Preclinically, we evaluated the immune modulation and anti-tumor efficacy of FAP-2287, a murine surrogate for FAP-2286, conjugated to the radionuclide lutetium-177 (177Lu) as a monotherapy and in combination with a PD-1 targeting antibody. METHODS: C57BL/6 mice bearing MCA205 mouse FAP-expressing tumors (MCA205-mFAP) were treated with 177Lu-FAP-2287, anti-PD-1, or both. Tumor uptake of 177Lu- FAP-2287 was assessed by SPECT/CT scanning, while therapeutic efficacy was measured by tumor volume and survival. Immune profiling of tumor infiltrates was evaluated through flow cytometry, RNA expression, and immunohistochemistry analyses. RESULTS: 177Lu-FAP-2287 rapidly accumulated in MCA205-mFAP tumors leading to significant tumor growth inhibition (TGI) and longer survival time. Significant TGI was also observed from anti-PD-1 and the combination. In flow cytometry analysis of tumors, 177Lu-FAP-2287 increased CD8+ T cell infiltration which was maintained in the combination with anti-PD-1. The increase in CD8+ T cells was accompanied by an induction of STING-mediated type I interferon response and higher levels of co-stimulatory molecules such as CD86. CONCLUSION: In a preclinical model, FAP-targeted radiotherapy enhanced anti-PD-1-mediated TGI by modulating the TME and increasing the recruitment of tumor-infiltrating CD8+ T cells. These findings provide a rationale for clinical studies of combined 177Lu-FAP-2286 radiotherapy and immune checkpoint inhibition in FAP-positive tumors.

Investigating Generation of Antibodies against the Lipid Nanoparticle Vector Following COVID-19 Vaccination with an mRNA Vaccine.

Despite the success of mRNA-based vaccines against infectious diseases (including COVID-19), safety concerns have been raised relating to the lipid nanoparticles (LNPs) used to deliver the mRNA cargo. Antibodies against the polyethylene glycol (PEG) coating on these non-viral vectors are present in the general population and can in some instances induce allergic reactions. Furthermore, treatment with PEGylated therapeutics may increase the plasma concentration of such anti-PEG antibodies. The widespread use of PEGylated nanoparticles for mRNA vaccines concerns researchers and clinicians about a potential rise in future cases of allergic reactions against mRNA vaccines and cross-reactions with other PEGylated therapeutics. To determine if vaccination with Comirnaty increased the plasma concentration of antibodies against LNPs, we investigated the blood plasma concentration of anti-LNP antibodies in healthy individuals before and after vaccination with the mRNA-based COVID-19 vaccine Comirnaty (BNT162b2). Blood samples were acquired from 21 healthy adults before vaccination, 3-4 weeks after the first vaccination dose but before the second dose, and 2-6 months after the second (booster) dose. The blood plasma concentration of antibodies recognizing the LNPs was analyzed using a microscopy-based assay capable of measuring antibody-binding to individual authentic LNPs. No significant increase in anti-LNP antibodies was observed after two doses of Comirnaty. The LNPs used for intramuscular delivery of mRNA in the vaccine against COVID-19, Comirnaty, do, therefore, not seem to induce the generation of anti-vector antibodies.

[68Ga]Ga-NODAGA-E[(cRGDyK)]2 angiogenesis PET following myocardial infarction in an experimental rat model predicts cardiac functional parameters and development of heart failure.

BACKGROUND: Angiogenesis has increasingly been a target for imaging and treatment over the last decade. The integrin αvß3 is highly expressed in cells during angiogenesis and are therefore a promising target for imaging. In this study, we aimed to investigate the PET tracer [68Ga]Ga-RGD as a marker of angiogenesis following MI and its ability to predict cardiac functional parameters. METHODS: First, the real-time interaction between [68Ga]Ga-RGD and integrin αvß3 was investigated using surface plasmon resonance (SPR). Second, an animal study was performed to investigate the [68Ga]Ga-RGD uptake in the infarcted area after one and four weeks following MI in a rat model (MI = 68, sham surgery = 36). Finally, the specificity of the [68Ga]Ga-RGD tracer was evaluated ex vivo using histology, autoradiography, gamma counting and flow cytometry. RESULTS: SPR showed that [68Ga]Ga-RGD has a high affinity for integrin αvß3, forming a strong and stable binding. PET/CT showed a significantly higher uptake of [68Ga]Ga-RGD in the infarcted area compared to sham one week (p < 0.001) and four weeks (p < 0.001) after MI. The uptake of [68Ga]Ga-RGD after one week correlated to end diastolic volume (r = 0.74, p < 0.001) and ejection fraction (r = - 0.71, p < 0.001) after four weeks. CONCLUSION: This study demonstrates that [68Ga]Ga-RGD has a high affinity for integrin αvß3, which enables the evaluation of angiogenesis and remodeling. The [68Ga]Ga-RGD uptake after one week indicates that [68Ga]Ga-RGD may be used as an early predictor of cardiac functional parameters and possible development of heart failure after MI. These encouraging data supports the clinical translation and future use in MI patients.

Unravelling Heterogeneities in Complement and Antibody Opsonization of Individual Liposomes as a Function of Surface Architecture.

Coating nanoparticles with poly(ethylene glycol) (PEG) is widely used to achieve long-circulating properties after infusion. While PEG reduces binding of opsonins to the particle surface, immunogenic anti-PEG side-effects show that PEGylated nanoparticles are not truly "stealth" to surface active proteins. A major obstacle for understanding the complex interplay between opsonins and nanoparticles is the averaging effects of the bulk assays that are typically applied to study protein adsorption to nanoparticles. Here, a microscopy-based method for directly quantifying opsonization at the single nanoparticle level is presented. Various surface coatings are investigated on liposomes, including PEG, and show that opsonization by both antibodies and complement C3b is highly dependent on the surface chemistry. It is further demonstrated that this opsonization is heterogeneous, with opsonized and non-opsonized liposomes co-existing in the same ensemble. Surface coatings modify the percentage of opsonized liposomes and/or opsonin surface density on the liposomes, with strikingly different patterns for antibodies and complement. Thus, this assay provides mechanistic details about opsonization at the single nanoparticle level previously inaccessible to established bulk assays.

Charged Polarons and Molecules in a Bose-Einstein Condensate.

Ultracold hybrid ion-atom gases represent an exciting frontier for quantum simulation offering a new set of functionalities and control. Here, we study a mobile ion immersed in a Bose-Einstein condensate and show that the long-range nature of the ion-atom interaction gives rise to an intricate interplay between few- and many-body physics. This leads to the existence of several polaronic and molecular states due to the binding of an increasing number of bosons to the ion, which is well beyond what can be described by a short-range pseudopotential. We use a complementary set of techniques including a variational ansatz and field theory to describe this rich physics and calculate the full spectral response of the ion. It follows from thermodynamic arguments that the ion-atom interaction leads to a mesoscopic dressing cloud of the polarons, and a simplified model demonstrates that the spectral weight of the molecules scale with increasing powers of the density. We finally calculate the quantum dynamics of the ion after a quench experiment.

Novel nucleotide-packaging vaccine delivers antigen and poly(I:C) to dendritic cells and generate a potent antibody response in vivo.

An issue with many current vaccines is the dependency on broadly inflammatory adjuvants, such as aluminum hydroxide or aluminum salts that affect many immune- and non-immune cells. These adjuvants are not necessarily activating all antigen-presenting cells (APCs) that take up the antigen and most likely they also activate APCs with no antigen uptake, as well as many non-immune cells. Conjugation of antigen and adjuvant would enable the use of smaller amounts of adjuvant and avoid unnecessary tissue damage and activation of bystander cells. It would ensure that all APCs that take up the antigen would also become activated and avoid that immature and non-activated APCs present the antigen to T cells without a co-stimulatory signal, leading to tolerogenesis. We have developed a novel vaccine that co-deliver antigen and a nucleotide adjuvant to the same APC and lead to a strong activation response in dendritic cells and macrophages. The vaccine is constructed as a fusion-protein with an antigen fused to the DNA/RNA-binding domain from the Hc2 protein from Chlamydia trachomatis. We have found that the fusion protein is able to package polyinosinic:polycytidylic acid (poly(I:C)) or dsDNA into small particles. These particles were taken up by macrophages and dendritic cells and led to strong activation and maturation of these cells. Immunization of mice with the fusion protein packaged poly(I:C) led to a stronger antibody response compared to immunization with a combination of poly(I:C) and antigen without the Hc2 DNA/RNA-binding domain.

An in situ depot for the sustained release of a TLR7/8 agonist in combination with a TGFß inhibitor promotes anti-tumor immune responses.

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-ß inhibitor in preclinical tumor models in female mice.

Studying how administration route and dose regulates antibody generation against LNPs for mRNA delivery with single-particle resolution.

Following the recent approval of both siRNA- and mRNA-based therapeutics, nucleic acid therapies are considered a game changer in medicine. Their envisioned widespread use for many therapeutic applications with an array of cellular target sites means that various administration routes will be employed. Concerns exist regarding adverse reactions against the lipid nanoparticles (LNPs) used for mRNA delivery, as PEG coatings on nanoparticles can induce severe antibody-mediated immune reactions, potentially being boosted by the inherently immunogenic nucleic acid cargo. While exhaustive information is available on how physicochemical features of nanoparticles affects immunogenicity, it remains unexplored how the fundamental choice of administration route regulates anti-particle immunity. Here, we directly compared antibody generation against PEGylated mRNA-carrying LNPs administered by the intravenous, intramuscular, or subcutaneous route, using a novel sophisticated assay capable of measuring antibody binding to authentic LNP surfaces with single-particle resolution. Intramuscular injections in mice were found to generate overall low and dose-independent levels of anti-LNP antibodies, while both intravenous and subcutaneous LNP injections generated substantial and highly dose-dependent levels. These findings demonstrate that before LNP-based mRNA medicines can be safely applied to new therapeutic applications, it will be crucial to carefully consider the choice of administration route.

Accelerated blood clearance and hypersensitivity by PEGylated liposomes containing TLR agonists.

Systemic administration of toll-like receptor (TLR) agonists have demonstrated impressive preclinical results as an anti-cancer therapy due to their potent innate immune-stimulatory properties. The clinical advancement has, however, been hindered by severe adverse effects due to systemic activation of the immune system. Liposomal drug delivery systems may modify biodistribution, cellular uptake, and extend blood circulation, and thus, potentially enable systemic administration of TLR agonists at therapeutic doses. In this study, we investigated potential barriers for the administration of TLR agonists formulated in polyethylene glycosylated (PEGylated) liposomes with regards to liposome formulation, TLR agonist, administration route, administration schedule, biodistribution, blood clearance, and anti-PEG antibodies. We found that administration of TLR agonists formulated in PEGylated liposomes led to high anti-PEG antibody titers, which upon multiple intravenous administrations, resulted in accelerated blood clearance and acute hypersensitivity reactions. The latter was found to be associated with anti-PEG IgG antibody and not anti-PEG IgM antibody opsonization. This study highlights the need to carefully design and evaluate nanoparticle delivery systems for immunotherapy as anti-nanoparticle immune responses may challenge the therapeutic application.

Development of a 64Cu-labeled CD4+ T cell targeting PET tracer: evaluation of CD4 specificity and its potential use in collagen-induced arthritis.

BACKGROUND: CD4+ T cells are central inflammatory mediators in the pathogenesis of autoimmune rheumatoid arthritis (RA), as they are one of the dominating cell types in synovial inflammation. Molecular imaging of CD4+ T cells has potential role for early detection and monitoring of RA. Here, we developed a new radiotracer for in vivo immunoPET imaging of murine CD4+ T cells and tested it in the collagen-induced arthritis (CIA) mouse model of human RA. RESULTS: The tracer, [64Cu]Cu-NOTA-CD4-F(ab)'2 ([64Cu]Cu-NOTA-CD4), was generated from F(ab)'2 fragments of R-anti-mouse CD4 antibodies conjugated to the 2-S-(isothiocyanatbenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) chelator and radiolabeled with copper-64. Accumulation of the tracer and isotype control was evaluated in the CIA model and mice receiving whole-body irradiation (WBI) (5 Gy). The potential of [64Cu]Cu-NOTA-CD4 for response assessment was evaluated in CIA induced mice treated with dexamethasone (DXM). Imaging data were compared with flow cytometry and immunohistochemistry (IHC) of inflammatory cells including CD4+ T cells. [64Cu]Cu-NOTA-CD4 showed increased accumulation in T cell-rich tissues compared with isotype control (p < 0.0001). In addition, reduced accumulation of [64Cu]Cu-NOTA-CD4 was observed in T cell-depleted tissue (p < 0.0001). Flow cytometry and IHC confirmed the increased infiltration of CD4+ T cells in CIA mice. CONCLUSIONS: We developed and evaluated a new radiotracer, [64Cu]Cu-NOTA-CD4, for immunoPET imaging of murine CD4+ T cells. [64Cu]Cu-NOTA-CD4 was successfully synthesized by F(ab)'2 fragments of R-anti-mouse CD4 antibodies conjugated to a chelator and radiolabeled with copper-64. We found that our novel CD4 PET tracer can be used for noninvasive visualization of murine CD4+ T cells.

Mechanisms of selective monocyte targeting by liposomes functionalized with a cationic, arginine-rich lipopeptide.

Stimulation of monocytes with immunomodulating agents can harness the immune system to treat a long range of diseases, including cancers, infections and autoimmune diseases. To this end we aimed to develop a monocyte-targeting delivery platform based on cationic liposomes, which can be utilized to deliver immunomodulators and thus induce monocyte-mediated immune responses while avoiding off-target side-effects. The cationic liposome design is based on functionalizing the liposomal membrane with a cholesterol-anchored tri-arginine peptide (TriArg). We demonstrate that TriArg liposomes can target monocytes with high specificity in both human and murine blood and that this targeting is dependent on the content of TriArg in the liposomal membrane. In addition, we show that the mechanism of selective monocyte targeting involves the CD14 co-receptor, and selectivity is compromised when the TriArg content is increased, resulting in complement-mediated off-target uptake in granulocytes. The presented mechanistic findings of uptake by peripheral blood leukocytes may guide the design of future drug delivery systems utilized for immunotherapy. STATEMENT OF SIGNIFICANCE: Monocytes are attractive targets for immunotherapies of cancers, infections and autoimmune diseases. Specific delivery of immunostimulatory drugs to monocytes is typically achieved using ligand-targeted drug delivery systems, but a simpler approach is to target monocytes using cationic liposomes. To achieve this, however, a deep understanding of the mechanisms governing the interactions of cationic liposomes with monocytes and other leukocytes is required. We here investigate these interactions using liposomes incorporating a cationic arginine-rich lipopeptide. We demonstrate that monocyte targeting can be achieved by fine-tuning the lipopeptide content in the liposomes. Additionally, we reveal that the CD14 receptor is involved in the targeting process, whereas the complement system is not. These mechanistic findings are critical for future design of monocyte-targeting liposomal therapies.

In vivo detection of urokinase-type plasminogen activator receptor (uPAR) expression in arterial atherogenesis using [64Cu]Cu-DOTA-AE105 positron emission tomography (PET).

BACKGROUND AND AIMS: Urokinase-type plasminogen activator receptor (uPAR) is associated with extracellular matrix (ECM) degradation and cancer aggressiveness. Its role in arterial atherogenesis as a molecular imaging target is not well-established. The aim of this study was to non-invasively visualize uPAR expression in atherosclerosis by a novel uPAR-targeting positron emission tomography (PET) tracer [64Cu]Cu-DOTA-AE105. METHODS: We used molecular biology to investigate uPAR expression by analyzing human atherosclerotic plaques and cultured cells. A retrospective analysis was performed on patients, who underwent combined PET/CT (n = 10) to measure [64Cu]Cu-DOTA-AE105 uptake in five large arteries, divided into a high and low-risk group based on coronary artery calcium score (CAC score). RESULTS: The in vitro assay for THP-1 monocytes displayed a significantly upregulated uPAR expression upon stimulation (5.2-fold upregulation, p < 0.0001 by a one-way ANOVA followed by Tukey's test) by single-cell flowcytometric analysis. Freshly excised human atherosclerotic plaques underwent flow cytometric and microarray analyses manifesting 73.9 ± 2.9% of mononuclear phagocyte system (MPS) cells expressing uPAR and had a greater than 7-fold higher gene expression of plasminogen activator urokinase receptor (PLAUR, p = 0.002), integrin subunit alpha X (ITGAX, p = 0.0008), and cluster of differentiation 163 (CD163, p < 0.0001). The tissue-to-background ratios (TBRmax) in five large arteries showed a higher [64Cu]Cu-DOTA-AE105 uptake in the group with high CAC score compared to the group with low CAC score (2.4 ± 0.1 vs 1.7 ± 0.1, p = 0.057), significantly higher in the ascending aorta (2.7 ± 0.1 vs 2.0 ± 0.1, p = 0.038) and the abdominal aorta (3.2 ± 0.2 vs 2.0 ± 0.2, p = 0.038) by a non-parametric Mann-Whitney test. CONCLUSIONS: uPAR is abundantly expressed by MPS cells in atherosclerotic plaques and can be visualized by the novel PET tracer [64Cu]Cu-DOTA-AE105 that may non-invasively detect extracellular matrix remodeling during atherogenesis.

Cell surface-tethered IL-12 repolarizes the tumor immune microenvironment to enhance the efficacy of adoptive T cell therapy.

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.

Flow Cytometric Evaluation of the Ongoing Angiogenic Response in Rat Cardiac Tissue Following Myocardial Infarction.

Angiogenesis is involved in regeneration of cardiac tissue following acute myocardial infarction (MI), a disease often investigated in rat models. Therefore, the ability to thoroughly evaluate the angiogenic response following experimentally induced MI in rats, and distinguish it from inflammation, is desired. This would enable evaluation of the angiogenic potential of new therapeutics and improve knowledge on MI pathophysiology. Due to the complex response to MI involving multiple cell types and the limited selection of rat-specific antibodies, careful optimization is crucial to capture this complexity. Here, we present an 8-color flow cytometry-based multicolor panel that will enable quantification of the ongoing angiogenic response as well as characterize the cells involved. A detailed description of tissue preparation, immunostaining, and gating strategy is provided. © 2021 Wiley Periodicals LLC. Basic Protocol: Cardiac tissue preparation and staining to investigate the ongoing angiogenic response in rat cardiac tissue following myocardial infarction Support Protocol: Titration of all antibodies in the presented panel.

Conventional Treatment of Glioblastoma Reveals Persistent CD44+ Subpopulations.

Glioblastoma (GBM) is the most frequent and devastating primary tumor of the central nervous system with a median survival of 12 to 15 months after diagnosis. GBM is highly difficult to treat due to its delicate location, inter- and intra-tumoral heterogeneity, and high plasticity in response to treatment. In this study, we intracranially implanted primary GBM cells into mice which underwent conventional GBM treatments, including irradiation, temozolomide, and a combination. We obtained single cell suspensions through a combination of mechanical and enzymatic dissociation of brain tissue and investigated in detail the changes in GBM cells in response to conventional treatments in vivo using multi-color flow cytometry and cluster analysis. CD44 expression was elevated in all treatment groups, which was confirmed by subsequent immunohistochemistry. High CD44 expression was furthermore shown to correlate with poor prognosis of GBM and low-grade glioma (LGG) patients. Together, these results indicate a key role for CD44 in glioma pathogenesis.

CD11c-targeted Delivery of DNA to Dendritic Cells Leads to cGAS- and STING-dependent Maturation.

Immunotherapeutic activation of tumor-specific T cells has proven to be an interesting approach in anticancer treatment. Particularly, anti-CTLA-4 and anti-PD-1/PD-L1 treatment looks promising, and conceivably, even better clinical results might be obtained if such treatment could be combined with boosting the existing tumor-specific T-cell response. One way to achieve this could be by increasing the level of maturation of dendritic cells locally and in the draining lymph nodes. When exposed to cancer cells, dendritic cells may spontaneously mature because of danger-associated molecular patterns derived from the tumor cells. Double-stranded DNA play a particularly important role in the activation of the dendritic cells, through engagement of intracellular DNA-sensors, and signaling through the adaptor protein STING. In the present study, we have investigated the maturational response of human monocyte-derived dendritic cells (moDC) and human monocytic THP-1 cells to targeted and untargeted DNA. We used an anti-CD11c antibody conjugated with double-stranded DNA to analyze the maturation status of human moDCs, as well as maturation using a cGAS KO and STING KO THP-1 cell maturation model. We found that dendritic cells can mature after exposure to cytoplasmic double-stranded DNA delivered through CD11c-mediated endocytosis. Moreover, we show that THP-1 cells matured using IL-4, GM-CSF, and ionomycin upregulate DC-maturation markers after CD11c-targeted delivery of double-stranded DNA. This upregulation is completely abrogated in cGAS KO and STING KO cells.

Folate receptor targeting of radiolabeled liposomes reduces intratumoral liposome accumulation in human KB carcinoma xenografts.

BACKGROUND: Active, ligand-mediated, targeting of functionalized liposomes to folate receptors (FRs) overexpressed on cancer cells could potentially improve drug delivery and specificity. Studies on folate-targeting liposomes (FTLs) have, however, yielded varying results and generally fail to display a clear benefit of FR targeting. METHOD: Tumor accumulating potential of FTLs and NTLs were investigated in a FR overex-pressing xenograft model by positron emission tomography/computed tomography imaging. RESULTS: Tumors displayed significantly lower activity of FTLs than NTLs. Furthermore, FTLs displayed worse circulating properties and increased liver-accumulation than NTLs. CONCLUSION: This study underlines that long-circulating properties of liposomes must be achieved to take advantage of EPR-dependent tumor accumulation which may be lost by functionalization. FR-functionalization negatively affected both tumor accumulation and circulation properties.