Oncolytic viruses (OVs) represent a potential therapeutic strategy in cancer treatment. However, there is currently a lack of comprehensive quantitative models characterizing clinical OV kinetics and distribution to the tumor. In this work, we present a mechanistic modeling framework for V937 OV, after intratumoral (i.t.) or intravascular (i.v.) administration in patients with cancer. A minimal physiologically-based pharmacokinetic model was built to characterize biodistribution of OVs in humans. Viral dynamics was incorporated at the i.t. cellular level and linked to tumor response, enabling the characterization of a direct OV killing triggered by the death of infected tumor cells and an indirect killing induced by the immune response. The model provided an adequate description of changes in V937 mRNA levels and tumor size obtained from phase I/II clinical trials after V937 administration. The model showed prominent role of viral clearance from systemic circulation and infectivity in addition to known tumor aggressiveness on clinical response. After i.v. administration, i.t. exposure of V937 was predicted to be several orders of magnitude lower compared with i.t. administration. These differences could be overcome if there is high virus infectivity and/or replication. Unfortunately, the latter process could not be identified at the current clinical setting. This work provides insights on selecting optimal OV considering replication rate and infectivity.

Background: The immunomodulation of the antitumor response driven by immunocheckpoint inhibitors (ICIs) such as PD-L1 (Programmed Death Ligand-1) monoclonal antibody (alpha-PD-L1) have shown relevant clinical outcomes in a subset of patients. This fact has led to the search for rational combinations with other therapeutic agents such as Doxorubicin (Dox), which cytotoxicity involves an immune activation that may enhance ICI response. Therefore, this study aims to evaluate the combination of chemotherapy and ICI by developing Dox Immunoliposomes functionalized with monovalent-variable fragments (Fab') of alpha-PD-L1. Results: Immunoliposomes were assayed in vitro and in vivo in a B16 OVA melanoma murine cell line over-expressing PD-L1. Here, immune system activation in tumor, spleen and lymph nodes, together with the antitumor efficacy were evaluated. Results showed that immunoliposomes bound specifically to PD-L1(+) cells, yielding higher cell interaction and Dox internalization, and decreasing up to 30-fold the IC50, compared to conventional liposomes. This mechanism supported a higher in vivo response. Indeed, immunoliposomes promoted full tumor regression in 20% of mice and increased in 1 month the survival rate. This formulation was the only treatment able to induce significant (p < 0.01) increase of activated tumor specific cytotoxic T lymphocytes at the tumor site. Conclusion: PD-L1 targeted liposomes encapsulating Dox have proved to be a rational combination able to enhance the modulation of the immune system by blocking PD-L1 and selectively internalizing Dox, thus successfully providing a dual activity offered by both, chemo and immune therapeutic strategies.

V937 is an investigational novel oncolytic non-genetically modified Kuykendall strain of Coxsackievirus A21 which is in clinical development for the treatment of advanced solid tumor malignancies. V937 infects and lyses tumor cells expressing the intercellular adhesion molecule I (ICAM-I) receptor. We integrated in vitro and in vivo data from six different preclinical studies to build a mechanistic model that allowed a quantitative analysis of the biological processes of V937 viral kinetics and dynamics, viral distribution to tumor, and anti-tumor response elicited by V937 in human xenograft models in immunodeficient mice following intratumoral and intravenous administration. Estimates of viral infection and replication which were calculated from in vitro experiments were successfully used to describe the tumor response in vivo under various experimental conditions. Despite the predicted high clearance rate of V937 in systemic circulation (t(1/2) = 4.3 min), high viral replication was observed in immunodeficient mice which resulted in tumor shrinkage with both intratumoral and intravenous administration. The described framework represents a step towards the quantitative characterization of viral distribution, replication, and oncolytic effect of a novel oncolytic virus following intratumoral and intravenous administrations in the absence of an immune response. This model may further be expanded to integrate the role of the immune system on viral and tumor dynamics to support the clinical development of oncolytic viruses.

Background Anti-programmed cell death 1 (PD-1)/programmed death-ligand 1 (PD-L1) monoclonal antibodies (mAbs) show remarkable clinical anti-tumour efficacy. However, rational combinations are needed to extend the clinical benefit to primary resistant tumours. The design of such combinations requires the identification of the kinetics of critical immune cell populations in the tumour microenvironment. Methods In this study, we compared the kinetics of immune cells in the tumour microenvironment upon treatment with immunotherapy combinations with different anti-tumour efficacies in the non-inflamed tumour model TC-1/A9. Tumour-bearing C57BL/6J mice were treated with all possible combinations of a human papillomavirus (HPV) E7 long peptide, polyinosinic-polycytidylic acid (PIC) and anti-PD-1 mAb. Tumour growth and kinetics of the relevant immune cell populations were assessed over time. The involvement of key immune cells was confirmed by depletion with mAbs and immunophenotyping with multiparametric flow cytometry. Results The maximum anti-tumour efficacy was achieved after intratumoural administration of HPV E7 long peptide and PIC combined with the systemic administration of anti-PD-1 mAb. The intratumoural immune cell kinetics of this combination was characterised by a biphasic immune response. An initial upsurge of proinflammatory myeloid cells led to a further rise in effector CD8(+) T lymphocytes at day 8. Depletion of either myeloid cells or CD8(+) T lymphocytes diminished the anti-tumour efficacy of the combination. Conclusions The anti-tumour efficacy of a successful immunotherapy combination in a non-inflamed tumour model relies on an early inflammatory process that remodels the myeloid cell compartment.

Simple Summary: The clinical efficacy of immunotherapies when treating cold tumors is still low, and different treatment combinations are needed when dealing with this challenging scenario. In this work, a middle-out strategy was followed to develop a model describing the antitumor efficacy of different immune-modulator combinations, including an antigen, a toll-like receptor-3 agonist, and an immune checkpoint inhibitor in mice treated with non-inflamed tumor cells. Our results support that clinical response requires antigen-presenting cell activation and also relies on the amount of CD8 T cells and tumor resistance mechanisms present. This mathematical model is a very useful platform to evaluate different immuno-oncology combinations in both preclinical and clinical settings. Immune checkpoint inhibitors, administered as single agents, have demonstrated clinical efficacy. However, when treating cold tumors, different combination strategies are needed. This work aims to develop a semi-mechanistic model describing the antitumor efficacy of immunotherapy combinations in cold tumors. Tumor size of mice treated with TC-1/A9 non-inflamed tumors and the drug effects of an antigen, a toll-like receptor-3 agonist (PIC), and an immune checkpoint inhibitor (anti-programmed cell death 1 antibody) were modeled using Monolix and following a middle-out strategy. Tumor growth was best characterized by an exponential model with an estimated initial tumor size of 19.5 mm(3) and a doubling time of 3.6 days. In the treatment groups, contrary to the lack of response observed in monotherapy, combinations including the antigen were able to induce an antitumor response. The final model successfully captured the 23% increase in the probability of cure from bi-therapy to triple-therapy. Moreover, our work supports that CD8(+) T lymphocytes and resistance mechanisms are strongly related to the clinical outcome. The activation of antigen-presenting cells might be needed to achieve an antitumor response in reduced immunogenic tumors when combined with other immunotherapies. These models can be used as a platform to evaluate different immuno-oncology combinations in preclinical and clinical scenarios.

Immunotherapy has changed the paradigm of cancer treatments. In this way, several combinatorial strategies based on monoclonal antibodies (mAb) such as anti (a)-PD-1 or anti (a)-PD-L1 are often reported to yield promising clinical benefits. However, the pharmacokinetic (PK) behavior of these mAbs is a critical issue that requires selective analytical techniques. Indeed, few publications report data on a-PD1/a-PD-L1 exposure and its relationship with therapeutic or toxic effects. In this regard, preclinical assays allow the time profiles of antibody plasma concentrations to be characterized rapidly and easily, which may help to increase PK knowledge. In this study, we have developed and validated two in-house ELISAs to quantify a-PD-1 and a-PD-L1 in plasma collected from tumor-bearing mice. The linear range for the a-PD-1 assay was 2.5-125 ng/mL and 0.11-3.125 ng/mL for the a-PD-L1 assay, whereas the intra-and inter-day precision was lower than 20% for both analytes. The PK characterization revealed a significant decrease in drug exposure after administration of multiple doses. Plasma half-life for a-PD-1 was slightly shorter (22.3 h) than for a-PD-L1 (46.7 h). To our knowledge, this is the first reported preclinical ELISA for these immune checkpoint inhibitors, which is sufficiently robust to be used in different preclinical models. These methods can help to understand the PK behavior of these antibodies under different scenarios and the relationship with response, thus guiding the choice of optimal doses in clinical settings.

Nanoparticles such as liposomes are able to overcome cancer treatment challenges such as multidrug resistance by increasing the bioavailability of the encapsulated drug, bypassing drug pumps or through targeting resistant cells. Here, we merge enhanced drug delivery by nanotechnology with tumor cell membrane modulation combined in a single formulation. This is achieved through the incorporation of Short chain sphingolipids (SCSs) in the liposomal composition, which permeabilizes cell membranes to amphiphilic drugs such as Doxorubicin (Dxr). To study the mechanism and capability of SCS-containing nanodevices to overcome Dxr resistance, a sensitive uterine sarcoma cell line, MES-SA, and a resistant derived cell line, MES-SA/MX2, were used. The mechanism of resistance was explored by lipidomics and flow cytometry, revealing significant differences in lipid composition and in P glycoprotein (Pgp) expression.In vitroassays show that SCS liposomes were able to reverse cell resistance, and importantly, display a higher net effect on resistant than sensitive cells. SCS lipids modulated the cell membrane of MES-SA/MX2 drug resistant cells, while Pgp expression was not affected. Furthermore, SCS-modified liposomes were evaluated in a sarcoma xenograft model on drug accumulation, pharmacokinetics and efficacy. SCS liposomes improved Dxr levels in tumor nuclei of MES-SA/MX2 tumor cells, which was accompanied by a delay in tumor growth of the resistant model. Here we show that Dxr accumulation in tumor cells by SCS-modified liposomes was especially improved in Dxr resistant cells, rendering Dxr as effective as in sensitive cells. Moreover, this phenomenon translated to improved efficacy when Dxr liposomes where modified with SCSs in the drug resistant tumor model, while no benefit was seen in the sensitive tumors.

Aim: Development of EGF-liposomes (LP-EGF) for selective molecules delivery in tumors expressing EGFR. Material & methods: In vitro cellular interaction of EGF-LP and nontargeted liposomes (LP-N) was assayed at 37 and 4°C in cells expressing different EGFR levels. Receptor-mediated uptake was investigated by competition with a monoclonal antibody anti-EGFR. Selective intracellular drug delivery and efficacy was tested by oxaliplatin encapsulation. In vivo biodistribution of LP-N and LP-EGF was done in xenograft model. Results: LP-EGF was internalized by an active and selective mechanism through EGFR without receptor activation. Oxaliplatin LP-EGF decreased IC50 between 48 and 13% in cell EGFR+. LP-EGF was accumulated in tumor over 72 h postdosing, while LP-N in spleen. Conclusion: LP-EGF represents an attractive nanosystem for cancer therapy or diagnosis.