Antibody desolvation with sodium chloride and acetonitrile generates bioactive protein nanoparticles.

About 30% of the FDA approved drugs in 2021 were protein-based therapeutics. However, therapeutic proteins can be unstable and rapidly eliminated from the blood, compared to conventional drugs. Furthermore, on-target but off-tumor protein binding can lead to off-tumor toxicity, lowering the maximum tolerated dose. Thus, for effective treatment therapeutic proteins often require continuous or frequent administration. To improve protein stability, delivery and release, proteins can be encapsulated inside drug delivery systems. These drug delivery systems protect the protein from degradation during (targeted) transport, prevent premature release and allow for long-term, sustained release. However, thus far achieving high protein loading in drug delivery systems remains challenging. Here, the use of protein desolvation with acetonitrile as an intermediate step to concentrate monoclonal antibodies for use in drug delivery systems is reported. Specifically, trastuzumab, daratumumab and atezolizumab were desolvated with high yield (∼90%) into protein nanoparticles below 100 nm with a low polydispersity index (<0.2). Their size could be controlled by the addition of low concentrations of sodium chloride between 0.5 and 2 mM. Protein particles could be redissolved in aqueous solutions and redissolved antibodies retained their binding activity as evaluated in cell binding assays and exemplified for trastuzumab in an ELISA.

EGFR-selective activation of CD27 co-stimulatory signaling by a bispecific antibody enhances anti-tumor activity of T cells.

A higher density of tumor infiltrating lymphocytes (TILs) in the tumor microenvironment, particularly cytotoxic CD8+ T cells, is associated with improved clinical outcome in various cancers. However, local inhibitory factors can suppress T cell activity and hinder anti-tumor immunity. Notably, TILs from various cancer types express the co-stimulatory Tumor Necrosis Factor receptor CD27, making it a potential target for co-stimulation and re-activation of tumor-infiltrated and tumor-reactive T cells. Anti-cancer therapeutics based on exploiting CD27-mediated T cell co-stimulation have proven safe, but clinical responses remain limited. This is likely because current monoclonal antibodies fail to effectively activate CD27 signaling, as this receptor requires higher-order receptor cross-linking. Here, we report on a bispecific antibody, CD27xEGFR, that targets both CD27 and the tumor antigen, epidermal growth factor receptor (EGFR). By targeting EGFR, which is commonly expressed on carcinomas, CD27xEGFR induced cancer cell-localized crosslinking and activation of CD27. The design of CD27xEGFR includes an Fc-silent domain, which is designed to minimize potential toxicity by reducing Fc gamma receptor-mediated binding and activation of immune cells. CD27xEGFR bound to both of its targets simultaneously and triggered EGFR-restricted co-stimulation of T cells as measured by T cell proliferation, T cell activation markers, cytotoxicity and IFN-γ release. Further, CD27xEGFR augmented T cell cytotoxicity in a panel of artificial antigen-presenting carcinoma cell line models, leading to Effector-to-Target ratio-dependent elimination of cancer cells. Taken together, we present the in vitro characterization of a novel bispecific antibody that re-activates T cell immunity in EGFR-expressing cancers through targeted co-stimulation of CD27.

Tumor infiltrating CD8/CD103/TIM-3-expressing lymphocytes in epithelial ovarian cancer co-express CXCL13 and associate with improved survival.

Reactivation of tumor infiltrating T lymphocytes (TILs) with immune checkpoint inhibitors or co-stimulators has proven to be an effective anti-cancer strategy for a broad range of malignancies. However, epithelial ovarian cancer (EOC) remains largely refractory to current T cell-targeting immunotherapeutics. Therefore, identification of novel immune checkpoint targets and biomarkers with prognostic value for EOC is warranted. Combining multicolor immunofluorescent staining's with single cell RNA-sequencing analysis, we here identified a TIM-3/CXCL13-positive tissue-resident memory (CD8/CD103-positive) T cell (Trm) population in EOC. Analysis of a cohort of ~175 patients with high-grade serous EOC revealed TIM-3-positive Trm were significantly associated with improved patient survival. As CXCL13-positive CD8-positive T cells have been strongly linked to patient response to anti-PD1 immune checkpoint blockade, combinatorial TIM-3 and PD-1 blockade therapy may be of interest for the (re)activation of anti-cancer immunity in EOC.

Towards Immunotherapy-Induced Normalization of the Tumor Microenvironment.

Immunotherapies modulate the function of immune cells to eradicate cancer cells through various mechanisms. These therapies are successful across a spectrum of cancers, but they are curative only in a subset of patients. Indeed, a major obstacle to the success of immunotherapies is the immunosuppressive nature of the tumor microenvironment (TME), comprising the stromal component and immune infiltrate of tumors. Importantly, the TME in most solid cancers is characterized by sparsely perfused blood vessels resulting from so-called pathological angiogenesis. In brief, dysregulated development of new vessels results in leaky tumor blood vessels that inefficiently deliver oxygen and other nutrients. Moreover, the occurrence of dysregulated fibrosis around the lesion, known as pathological desmoplasia, further compresses tumor blood vessels and impairs blood flow. TME normalization is a clinically tested treatment strategy to reverse these tumor blood vessel abnormalities resulting in stimulated antitumor immunity and enhanced immunotherapy efficacy. TME normalization includes vascular normalization to reduce vessel leakiness and reprogramming of cancer-associated fibroblast to decompress vessels. How immunotherapies themselves normalize the TME is poorly understood. In this review, we summarize current concepts and progress in TME normalization. Then, we review observations of immunotherapy-induced TME normalization and discuss the considerations for combining vascular normalizing and immunotherapies. If TME could be more completely normalized, immunotherapies could be more effective in more patients.

CD40- and 41BB-specific antibody fusion proteins with PDL1 blockade-restricted agonism.

Background: A strategy to broaden the applicability of checkpoint inhibitors is the combined use with antibodies targeting the immune stimulatory receptors CD40 and 41BB. However, the use of anti-CD40 and anti-41BB antibodies as agonists is problematic in two ways. First, anti-CD40 and anti-41BB antibodies need plasma membrane-associated presentation by FcγR binding to exert robust agonism but this obviously limits their immune stimulatory efficacy by triggering ADCC, CDC or anti-inflammatory FcγRIIb activities. Second, off tumor activation of CD40 and 41BB may cause dose limiting systemic inflammation. Methods: To overcome the FcγR-dependency of anti-41BB and anti-CD40 antibodies, we genetically fused such antibodies with a PDL1-specific blocking scFv as anchoring domain to enable FcγR-independent plasma membrane-associated presentation of anti-CD40- and anti-41BB antibodies. By help of GpL-tagged variants of the resulting bispecific antibodies, binding to their molecular targets was evaluated by help of cellular binding studies. Membrane PDL1-restricted engagement of CD40 and 41BB but also inhibition of PDL1-induced PD1 activation were evaluated in coculture assays with PDL1-expressing tumor cell lines and 41BB, CD40 and PD1 responsible cell lines or T-cells. Results: The binding properties of the bispecific antibody fusion proteins remained largely unchanged compared to their parental molecules. Upon anchoring to membrane PDL1, the bispecific antibody fusion proteins activated CD40/41BB signaling as efficient as the parental anti-CD40/anti-41BB antibodies when bound to FcγRs or cells expressing membrane-bound CD40L/41BBL. PD1 inhibition remained intact and the anti-41BB fusion protein thus showed PDL1-restricted costimulation of T-cells activated in vitro with anti-CD3 or a BiTe. Conclusions: Targeting of anti-CD40 and anti-41BB fusion proteins to membrane PDL1 with a blocking PDL1 scFv links PD1-PDL1 checkpoint blockade intrinsically with engagement of CD40 or 41BB.

Dexamethasone Increases Cisplatin-Loaded Nanocarrier Delivery and Efficacy in Metastatic Breast Cancer by Normalizing the Tumor Microenvironment.

Dexamethasone is a glucocorticoid steroid with anti-inflammatory properties used to treat many diseases, including cancer, in which it helps manage various side effects of chemo-, radio-, and immunotherapies. Here, we investigate the tumor microenvironment (TME)-normalizing effects of dexamethasone in metastatic murine breast cancer (BC). Dexamethasone normalizes vessels and the extracellular matrix, thereby reducing interstitial fluid pressure, tissue stiffness, and solid stress. In turn, the penetration of 13 and 32 nm dextrans, which represent nanocarriers (NCs), is increased. A mechanistic model of fluid and macromolecule transport in tumors predicts that dexamethasone increases NC penetration by increasing interstitial hydraulic conductivity without significantly reducing the effective pore diameter of the vessel wall. Also, dexamethasone increases the tumor accumulation and efficacy of ∼30 nm polymeric micelles containing cisplatin (CDDP/m) against murine models of primary BC and spontaneous BC lung metastasis, which also feature a TME with abnormal mechanical properties. These results suggest that pretreatment with dexamethasone before NC administration could increase efficacy against primary tumors and metastases.

Glucose transporter 1-mediated vascular translocation of nanomedicines enhances accumulation and efficacy in solid tumors.

Nanomedicine modification with ligands directed to receptors on tumor blood vessels has the potential for selectively enhancing nanomedicine accumulation in malignant tissues by overcoming the vascular barrier of tumors. Nevertheless, the development of broadly applicable ligand approaches capable of promoting the transvascular transport of nanomedicines in a wide spectrum of tumors has been elusive so far. By considering the indispensable and persistent glycolytic fueling of tumors, we developed glucose-installed polymeric micelles loading cisplatin (Gluc-CDDP/m) targeting the glucose transporter 1 (GLUT1), which is overexpressed in most tumors and present on vascular endothelial cells, toward improving the delivery efficiency and therapeutic efficacy. The design of the glucose ligands on Gluc-CDDP/m was engineered to control the conjugation via the carbon 6 of the glucose moieties, as well as the ligand density on the poly (ethylene glycol) (PEG) shell of the micelles. The series of micelles was then studied in vitro and in vivo against GLUT1-high human squamous cell carcinoma of the head and neck OSC-19 cells and GLUT1-low human glioblastoma-astrocytoma U87MG cells. Our results showed that precisely tuning the micelles to have glucose ligands on 25% of their PEG chains increased the efficacy against the tumors by significantly enhancing the tumor accumulation, even in GLUT1-low U87MG tumors. The enhancement of the intratumoral levels of these micelles was hindered by concomitant administration of glucose, or the GLUT1 inhibitor STF-31, confirming a GLUT1/glucose-mediated increment of the accumulation. Intravital confocal laser scanning microscopy imaging of tumor tissues further demonstrated the rapid extravasation and penetration of Gluc-CDDP/m in OSC-19 tumors compared to non-targeted CDDP/m. These findings indicate GLUT1-targeting as a promising approach for overcoming the vascular barrier and boosting the delivery of nanomedicine in tumors.

Characterisation of microbial activity in the framework of natural attenuation without groundwater monitoring wells?: a new Direct-Push probe.

At many contaminated field sites in Europe, monitored natural attenuation is a feasible site remediation option. Natural attenuation includes several processes but only the microbial degradation leads to real contaminant removal and very few methods are accepted by the authorities providing real evidence of microbial contaminant degradation activity. One of those methods is the recently developed in situ microcosm approach (BACTRAP®). These in situ microcosms consist of perforated stainless steel cages or PTFE tubes filled with an activated carbon matrix that is amended with 13C-labelled contaminants; the microcosms are then exposed within groundwater monitoring wells. Based on this approach, natural attenuation was accepted by authorities as a site remediation option for the BTEX-polluted site Zeitz in Germany. Currently, the in situ microcosms are restricted to the use inside groundwater monitoring wells at the level of the aquifer. The (classical) system therefore is only applicable on field sites with a network of monitoring wells, and only microbial activity inside the monitoring wells at the level of the aquifer can be assessed. In order to overcome these limitations, a new Direct-Push BACTRAP probe was developed on the basis of the Geoprobe® equipment. With respect to the mechanical boundary conditions of the DP technique, these new probes were constructed in a rugged and segmented manner and are adaptable to various sampling concepts. With this new probe, the approach can be extended to field sites without existing monitoring wells, and microbial activity was demonstrated to be measureable even under very dry conditions inside the vadose zone above the aquifer. In a field test, classical and Direct-Push BACTRAPs were applied in the BTEX-contaminated aquifer at the ModelPROBE reference site Zeitz (Germany). Both types of BACTRAPs were incubated in the centre and at the fringe of the BTEX plume. Analysis of phospholipid fatty acid (PLFA) patterns showed that the bacterial communities on DP-BACTRAPs were more similar to the soil than those found on classical BACTRAPs. During microbial degradation of the (13)C-labelled substrate on the carrier material of the microcosms, the label was only slightly incorporated into bacterial biomass, as determined by PLFA analysis. This provides clear indication for decreased in situ natural attenuation potential in comparison to earlier sampling campaigns, which is presumably caused by a large-scale source remediation measure in the meantime. In conclusion, Direct-Push-based BACTRAPs offer a promising way to monitor natural attenuation or remediation success at field sites which are currently inaccessible by the technique due to the lack of monitoring wells or due to a main contamination present within the vadose zone.