Pharmacokinetics and Pharmacodynamics of T-Cell Bispecifics in the Tumour Interstitial Fluid.

The goal of this study is to investigate the pharmacokinetics in plasma and tumour interstitial fluid of two T-cell bispecifics (TCBs) with different binding affinities to the tumour target and to assess the subsequent cytokine release in a tumour-bearing humanised mouse model. Pharmacokinetics (PK) as well as cytokine data were collected in humanised mice after iv injection of cibisatamab and CEACAM5-TCB which are binding with different binding affinities to the tumour antigen carcinoembryonic antigen (CEA). The PK data were modelled and coupled to a previously published physiologically based PK model. Corresponding cytokine release profiles were compared to in vitro data. The PK model provided a good fit to the data and precise estimation of key PK parameters. High tumour interstitial concentrations were observed for both TCBs, influenced by their respective target binding affinities. In conclusion, we developed a tailored experimental method to measure PK and cytokine release in plasma and at the site of drug action, namely in the tumour. Integrating those data into a mathematical model enabled to investigate the impact of target affinity on tumour accumulation and can have implications for the PKPD assessment of the therapeutic antibodies.

Impaired lymphatic function accelerates cancer growth.

Increased lymphangiogenesis is a common feature of cancer development and progression, yet the influence of impaired lymphangiogenesis on tumor growth is elusive. C3HBA breast cancer and KHT-1 sarcoma cell lines were implanted orthotopically in Chy mice, harboring a heterozygous inactivating mutation of vascular endothelial growth factor receptor-3, resulting in impaired dermal lymphangiogenesis. Accelerated tumor growth was observed in both cancer models in Chy mice, coinciding with reduced peritumoral lymphangiogenesis. An impaired lymphatic washout was observed from the peritumoral area in Chy mice with C3HBA tumors, and the number of macrophages was significantly reduced. While fewer macrophages were detected, the fraction of CD163+ M2 macrophages remained constant, causing a shift towards a higher M2/M1 ratio in Chy mice. No difference in adaptive immune cells was observed between wt and Chy mice. Interestingly, levels of pro- and anti-inflammatory macrophage-associated cytokines were reduced in C3HBA tumors, pointing to an impaired innate immune response. However, IL-6 was profoundly elevated in the C3HBA tumor interstitial fluid, and treatment with the anti-IL-6 receptor antibody tocilizumab inhibited breast cancer growth. Collectively, our data indicate that impaired lymphangiogenesis weakens anti-tumor immunity and favors tumor growth at an early stage of cancer development.

Determination of lymph flow in murine oral mucosa using depot clearance of near-infrared-labeled albumin.

The lymphatic vessels are playing an important role in inflammation since they return extravasated fluid, proteins, and cells back into the circulation and regulate immune cell trafficking. The oral mucosa, including gingiva, is well supplied with lymphatic vessels and is frequently challenged with inflammatory insults. Lymphatic vessels in gingiva protect against periodontal disease development, but quantification of lymph flow in this area has so far never been performed, due to lack of reliable methods. Mice of FVB strain (n=17) were anesthetized with isoflurane and placed on a jaw retraction board allowing the mouth to be kept open and stable. Albumin conjugated with Alexa680-fluorochrome (with or without LPS from Porphyromonas gingivalis) was injected superficially in oral mucosa mesio-buccal to the left first molar in each mouse. 60 min post-injection the mouse was transferred to an OptixMX3 optical imager where the total fluorescence was measured in the posterior facial area. The measurements continued further every 60 min for 7h for each mouse. The mice were awake and active between measurements. The in vivo washout of Alexa680-albumin was calculated using the natural logarithm of the relative values creating a negative slope for each mouse. Statistical analysis of variance was performed. The injection and distribution site for tracer was verified with India ink and shown to be in the interstitium below the oral mucosal epithelium, in an area well supplied with initial lymphatic vessels. Washout of the tracer Alexa680-albumin was log-linear, and the basal lymph flow calculated from depot clearance averaged -0.28 ± 0.08%/min (n=8). The clearance was significantly faster (-0.30 ± 0.08%/min, n=9) in acutely inflamed oral mucosa (p=0.0326). We developed a method that can successfully quantify the lymph flow in oral mucosa in steady state conditions and under acute perturbation. By use of this method, new information about the lymphatic function in oral mucosa during physiological and pathological conditions can be achieved.

A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules.

The central nervous system (CNS) is considered an organ devoid of lymphatic vasculature. Yet, part of the cerebrospinal fluid (CSF) drains into the cervical lymph nodes (LNs). The mechanism of CSF entry into the LNs has been unclear. Here we report the surprising finding of a lymphatic vessel network in the dura mater of the mouse brain. We show that dural lymphatic vessels absorb CSF from the adjacent subarachnoid space and brain interstitial fluid (ISF) via the glymphatic system. Dural lymphatic vessels transport fluid into deep cervical LNs (dcLNs) via foramina at the base of the skull. In a transgenic mouse model expressing a VEGF-C/D trap and displaying complete aplasia of the dural lymphatic vessels, macromolecule clearance from the brain was attenuated and transport from the subarachnoid space into dcLNs was abrogated. Surprisingly, brain ISF pressure and water content were unaffected. Overall, these findings indicate that the mechanism of CSF flow into the dcLNs is directly via an adjacent dural lymphatic network, which may be important for the clearance of macromolecules from the brain. Importantly, these results call for a reexamination of the role of the lymphatic system in CNS physiology and disease.