Phosphatidylserine-mediated oral tolerance.

Phosphatidylserine (PS) is an anionic phospholipid exposed on the surface of apoptotic cells. The exposure of PS typically recruits and signals phagocytes to engulf and silently clear these dying cells to maintain tolerance via immunological ignorance. However, recent and emerging evidence has demonstrated that PS converts an "immunogen" into a "tolerogen", and PS exposure on the surface of cells or vesicles actively promotes a tolerogenic environment. This tolerogenic property depends on the biophysical characteristics of PS-containing vesicles, including PS density on the particle surface to effectively engage tolerogenic receptors, such as TIM-4, which is exclusively expressed on the surface of antigen-presenting cells. We harnessed the cellular and molecular mechanistic insight of PS-mediated immune regulation to design an effective oral tolerance approach. This immunotherapy has been shown to prevent/reduce immune response against life-saving protein-based therapies, food allergens, autoantigens, and the antigenic viral capsid peptide commonly used in gene therapy, suggesting a broad spectrum of potential clinical applications. Given the good safety profile of PS together with the ease of administration, oral tolerance achieved with PS-based nanoparticles has a very promising therapeutic impact.

Pharmaceutical Aspects and Clinical Evaluation of COVID-19 Vaccines.

COVID-19, the disease caused by the novel severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2), was first detected in December 2019 and has since morphed into a global pandemic claiming over 2.4 million human lives and severely impacting global economy. The race for a safe and efficacious vaccine was thus initiated with government agencies as well as major pharmaceutical companies as frontrunners. An ideal vaccine would activate multiple arms of the adaptive immune system to generate cytotoxic T cell responses as well as neutralizing antibody responses, while avoiding pathological or deleterious immune responses that result in tissue damage or exacerbation of the disease. Developing an effective vaccine requires an inter-disciplinary effort involving virology, protein biology, biotechnology, immunology and pharmaceutical sciences. In this review, we provide a brief overview of the pathology and immune responses to SARS-CoV-2, which are fundamental to vaccine development. We then summarize the rationale for developing COVID-19 vaccines and provide novel insights into vaccine development from a pharmaceutical science perspective, such as selection of different antigens, adjuvants, delivery platforms and formulations. Finally, we review multiple clinical trial outcomes of novel vaccines in terms of safety and efficacy.

Biological Function and Immunotherapy Utilizing Phosphatidylserine-based Nanoparticles.

Phosphatidylserine (PS) is a naturally occurring anionic phospholipid that is primarily located in the inner leaflet of eukaryotic cell membranes. The role of PS during apoptosis is one of the most studied biological functions of PS. Externalization of PS during apoptosis mediates an "eat me" signal for phagocytic uptake, leading to clearance of apoptotic cells and thus maintain self-tolerance by immunological ignorance. However, an emerging view is that PS exposure-mediated cellular uptake is not an immunologically silent event, but rather promoting an active tolerance towards self and foreign proteins. This biological property of PS has been exploited by parasites and viruses in order to evade immune surveillance of the host immune system. Further, this novel immune regulatory property of PS that results in tolerance induction can be harnessed for clinical applications, such as to treat autoimmune conditions and to reduce immunogenicity of therapeutic proteins. This review attempts to provide an overview of the biological functions of PS in the immune response and its potential therapeutic applications.

Sialic Acid-Dependent Inhibition of T Cells by Exosomal Ganglioside GD3 in Ovarian Tumor Microenvironments.

The tumor microenvironment is rendered immunosuppressive by a variety of cellular and acellular factors that represent potential cancer therapeutic targets. Although exosomes isolated from ovarian tumor ascites fluids have been previously reported to induce a rapid and reversible T cell arrest, the factors present on or within exosomes that contribute to immunosuppression have not been fully defined. In this study, we establish that GD3, a ganglioside expressed on the surface of exosomes isolated from human ovarian tumor ascites fluids, is causally linked to the functional arrest of T cells activated through their TCR. This arrest is inhibited by Ab blockade of exosomal GD3 or by the removal of GD3+ exosomes. Empty liposomes expressing GD3 on the surface also inhibit the activation of T cells, establishing that GD3 contributes to the functional arrest of T cells independent of factors present in exosomes. Finally, we demonstrate that the GD3-mediated arrest of the TCR activation is dependent upon sialic acid groups, because their enzymatic removal from exosomes or liposomes results in a loss of inhibitory capacity. Collectively, these data define GD3 as a potential immunotherapeutic target.

Factor VIII associated with lipidic nanoparticles retains efficacy in the presence of anti-factor VIII antibodies in hemophilia A mice.

The development of inhibitory antibodies against factor VIII (FVIII) is a major challenge in hemophilia A (HA) therapy. Such antibodies develop in nearly 30% of patients receiving replacement FVIII, abrogating therapeutic efficacy. This work evaluated whether B-domain deleted FVIII encapsulated in phosphatidylinositol containing lipid nanoparticles (PI-BDD FVIII) could serve as an efficacious FVIII replacement therapy in the presence of inhibitors. The HA mice were given clinically relevant doses of FVIII to develop inhibitors. The efficacy of free and PI-BDD FVIII was studied in inhibitor-positive HA mice using a tail clip assay. Mathematical modeling of these data was conducted to evaluate the hypothesis that lipid association sterically shields the protein from inhibitor binding. The immunization protocol resulted in a mean inhibitory titer level of 198 ± 52 BU/ml. Free BDD FVIII was ineffective at controlling blood loss in inhibitor-positive HA mice as early as 2 h post dose. In contrast, PI-BDD FVIII treated animals retained partial hemostatic efficacy as long as 18 h post dose. Mathematical modeling supports the hypotheses that a greater fraction of lipid-associated FVIII remains unbound to inhibitors and that PI-BDD FVIII has lower binding affinity to inhibitors than does the free protein. In addition, the modeling approaches extend current efforts to model the impact of immunogenicity on PK and the therapeutically meaningful endpoint of efficacy, thereby addressing an important knowledge gap, particularly in the FVIII scientific literature. Clinical translation of these findings could result in a significant improvement in the quality of care of inhibitor-positive HA patients. Copyright © 2016 John Wiley & Sons, Ltd.

Effects of hypertonic buffer composition on lymph node uptake and bioavailability of rituximab, after subcutaneous administration.

The subcutaneous administration of biologics is highly desirable; however, incomplete bioavailability after s.c. administration remains a major challenge. In this work we investigated the effects of excipient dependent hyperosmolarity on lymphatic uptake and plasma exposure of rituximab as a model protein. Using Swiss Webster (SW) mice as the animal model, we compared the effects of NaCl, mannitol and O-phospho-L-serine (OPLS) on the plasma concentration of rituximab over 5 days after s.c. administration. An increase was observed in plasma concentrations in animals administered rituximab in hypertonic buffer solutions, compared with isotonic buffer. Bioavailability, as estimated by our pharmacokinetic model, increased from 29% in isotonic buffer to 54% in hypertonic buffer containing NaCl, to almost complete bioavailability in hypertonic buffers containing high dose OPLS or mannitol. This improvement in plasma exposure is due to the improved lymphatic trafficking as evident from the increase in the fraction of dose trafficked through the lymph nodes in the presence of hypertonic buffers. The fraction of the dose trafficked through the lymphatics, as estimated by the model, increased from 0.05% in isotonic buffer to 13% in hypertonic buffer containing NaCl to about 30% for hypertonic buffers containing high dose OPLS and mannitol. The data suggest that hypertonic solutions may be a viable option for improving s.c. bioavailability.

Exposure to factor VIII protein in the presence of phosphatidylserine induces hypo-responsiveness toward factor VIII challenge in hemophilia A mice.

Administration of recombinant factor VIII (FVIII), an important co-factor in blood clotting cascade, elicits unwanted anti-FVIII antibodies in hemophilia A (HA) patients. Previously, FVIII associated with phosphatidylserine (PS) showed significant reduction in the anti-FVIII antibody response in HA mice. The reduction in the immune response to FVIII-PS could be due either to a failure of the immune system to recognize the antigen (i.e. immunological ignorance) or to an active induction of an antigen-specific nonresponsiveness (i.e. immunological tolerance). If it were a result of tolerance, one would predict that pre-exposure to FVIII-PS would render the mice hypo-responsive to a subsequent FVIII challenge. Here, we have demonstrated that naive HA mice that were pretreated with FVIII-PS showed a significantly reduced FVIII immune response to further challenge with native FVIII and that this decreased responsiveness could be adoptively transferred to other mice. An increase in number of FoxP3-expressing CD4(+) regulatory T-cells (Treg) was observed for the FVIII-PS-immunized group as compared with animals that received FVIII alone, suggesting the involvement of Treg in PS-mediated hypo-responsiveness. The PS-mediated reduction in antibody response was reversed by the co-administration of function-blocking anti-TGF-ß antibody with FVIII-PS. The decreased response to FVIII induced by FVIII-PS was determined to be antigen-specific because the immune response to another non-cross-reactive antigen (ovalbumin) was not altered. These results are consistent with the notion that FVIII-PS is tolerogenic and suggest that immunization with this tolerogenic form of the protein could be a useful treatment option to minimize immunogenicity of FVIII and other protein-based therapeutics.

Nonlinear pharmacokinetics of factor VIII and its phosphatidylinositol lipidic complex in hemophilia A mice.

Factor VIII (FVIII) is an important cofactor in the blood coagulation cascade and its deficiency or dysfunction causes hemophilia A (HA), a bleeding disorder. Replacement with recombinant FVIII is limited by a short half-life and the development of inhibitory antibodies. A phosphatidylinositol (PI) containing lipid nanoparticle was developed that, when associated with FVIII, reduces immunogenicity and prolongs circulation of the therapeutic protein in HA mice. A multiple dose level pharmacokinetic (PK) study of human free FVIII and its FVIII-PI complex over a clinically relevant range of doses (20, 40 and 200 IU/kg) was conducted in HA mice to investigate linearity of the PK and to determine if the reduced catabolism of FVIII following association with PI particles, previously only observed in the terminal phase following 400 IU/kg, could be extendable over a range of doses. The findings suggest that the disposition of FVIII is best characterized by a two-compartment model with saturable Michaelis-Menten elimination. Spontaneous complexation of FVIII with PI particles significantly increases plasma survival of the protein at 20 and 40 IU/kg doses. Human simulations at 40 IU/kg project an increase in the terminal half-life and the time to reach a minimum therapeutic threshold of 0.01 IU/ml of 5.4 h and 40 h, respectively, compared with free FVIII. Formulation with PI containing lipid particles may represent a viable delivery strategy for improving FVIII therapy.

Changes in ovarian tumor cell number, tumor vasculature, and T cell function monitored in vivo using a novel xenograft model.

Despite an initial response to chemotherapy, most patients with ovarian cancer eventually progress and succumb to their disease. Understanding why effector T cells that are known to infiltrate the tumor do not eradicate the disease after cytoreduction is critically important to the development of novel therapeutic strategies to augment tumor immunity and improve patient outcomes. Such studies have been hampered by the lack of a suitable in vivo model. We report here a simple and reliable model system in which ovarian tumor cell aggregates implanted intraperitoneally into severely immunodeficient NSG mice establish tumor microenvironments within the omentum. The rapid establishment of tumor xenografts within this small anatomically well-defined site enables the recovery, characterization, and quantification of tumor and tumor-associated T cells. We validate here the ability of the omental tumor xenograft (OTX) model to quantify changes in tumor cell number in response to therapy, to quantify changes in the tumor vasculature, and to demonstrate and study the immunosuppressive effects of the tumor microenvironment. Using the OTX model, we show that the tumor-associated T cells originally present within the tumor tissues are anergic and that fully functional autologous T cells injected into tumor-bearing mice localize within the tumor xenograft. The transferred T cells remain functional for up to 3 days within the tumor microenvironment but become unresponsive to activation after 7 days. The OTX model provides for the first time the opportunity to study in vivo the cellular and molecular events contributing to the arrest in T cell function in human ovarian tumors.

A mechanistic marker-based screening tool to predict clinical immunogenicity of biologics.

BACKGROUND: The efficacy and safety of therapeutic proteins are undermined by immunogenicity driven by anti-drug antibodies. Immunogenicity risk assessment is critically necessary during drug development, but current methods lack predictive power and mechanistic insight into antigen uptake and processing leading to immune response. A key mechanistic step in T-cell-dependent immune responses is the migration of mature dendritic cells to T-cell areas of lymphoid compartments, and this phenomenon is most pronounced in the immune response toward subcutaneously delivered proteins. METHODS: The migratory potential of monocyte-derived dendritic cells is proposed to be a mechanistic marker for immunogenicity screening. Following exposure to therapeutic protein in vitro, dendritic cells are analyzed for changes in activation markers (CD40 and IL-12) in combination with levels of the chemokine receptor CXCR4 to represent migratory potential. Then a transwell assay captures the intensity of dendritic cell migration in the presence of a gradient of therapeutic protein and chemokine ligands. RESULTS: Here, we show that an increased ability of the therapeutic protein to induce dendritic cell migration along a gradient of chemokine CCL21 and CXCL12 predicts higher immunogenic potential. Expression of the chemokine receptor CXCR4 on human monocyte-derived dendritic cells, in combination with activation markers CD40 and IL-12, strongly correlates with clinical anti-drug antibody incidence. CONCLUSIONS: Mechanistic understanding of processes driving immunogenicity led to the development of a predictive tool for immunogenicity risk assessment of therapeutic proteins. These predictive markers could be adapted for immunogenicity screening of other biological modalities.

Protein drugs have a structure that is very similar to proteins in the human body, however there are slight differences. This can result in a person's immune system having a negative reaction to the drug. Unfortunately, during the early stages of drug development, it is currently difficult to predict which of these potential new drugs might cause this problem. We think that there is a higher chance for undesired immune responses to protein drugs when they cause an increase in the movement of the body's immune cells from the drug injection site to the lymph nodes. The more immune cells that arrive in the lymph node, the stronger the immune reaction. We developed a method that can predict how likely it is that a protein drug will trigger the unwanted activation and movement of these immune cells. This tool has the potential to improve our ability to predict, and thus avoid, unwanted immune system reactions toward protein drugs. This could shorten the time and cost required to develop new drugs.

Immunogenicity of Therapeutic Biological Modalities - Lessons from Hemophilia A Therapies.

The introduction and development of biologics such as therapeutic proteins, gene-, and cell-based therapy have revolutionized the scope of treatment for many diseases. However, a significant portion of the patients develop unwanted immune reactions against these novel biological modalities, referred to as immunogenicity, and no longer benefit from the treatments. In the current review, using Hemophilia A (HA) therapy as an example, we will discuss the immunogenicity issue of multiple biological modalities. Currently, the number of therapeutic modalities that are approved or recently explored to treat HA, a hereditary bleeding disorder, is increasing rapidly. These include, but are not limited to, recombinant factor VIII proteins, PEGylated FVIII, FVIII Fc fusion protein, bispecific monoclonal antibodies, gene replacement therapy, gene editing therapy, and cell-based therapy. They offer the patients a broader range of more advanced and effective treatment options, yet immunogenicity remains the most critical complication in the management of this disorder. Recent advances in strategies to manage and mitigate immunogenicity will also be reviewed.

Understanding preclinical and clinical immunogenicity risks in novel biotherapeutics development.

Immunogenicity continues to pose a challenge in the development of biotherapeutics like conventional therapeutic-proteins and monoclonal antibodies as well as emerging modalities such as gene-therapy components, gene editing, and CAR T cells. The approval of any therapeutic is based on a benefit-risk evaluation. Most biotherapeutics address serious medical conditions where the standard of care has a poor outcome. Consequently, even if immunogenicity limits the utility of the therapeutic in a sub-set of patients, the benefit-risk assessment skews in favor of approval. Some cases resulted in the discontinuation of biotherapeutics due to immunogenicity during drug development processes, This special issue presents a platform for review articles offering a critical assessment of accumulated knowledge as well as novel findings related to nonclinical risks that extend our understanding of the immunogenicity of biotherapeutics. Some of the studies in this collection leveraged assays and methodologies refined over decades to support more clinically relevant biological samples. Others have applied rapidly advancing methodologies in pathway-specific analyses to immunogenicity. Similarly, the reviews address urgent issues such as the rapidly emerging cell and gene therapies which hold immense promise but could have limited reach as a significant number of the patient population could potentially not benefit due to immunogenicity. In addition to summarizing the work presented in this special issue we have endeavored to identify areas where additional studies are required to understand the risks of immunogenicity and develop appropriate mitigation strategies.

Subcutaneous absorption of monoclonal antibodies: role of dose, site of injection, and injection volume on rituximab pharmacokinetics in rats.

PURPOSE: To determine the effect of dose, the anatomical site of injection, and the injection volume on subcutaneous absorption of rituximab in rats and to explore absorption mechanisms using pharmacokinetic modeling. METHODS: Rituximab serum concentrations were measured following intravenous and subcutaneous administration at the back, abdomen, and foot of rats. Several pharmacokinetic models were developed that included linear and saturable absorption, and degradation and/or protective binding at the injection site. RESULTS: Rituximab exhibited linear kinetics following intravenous administration; however, bioavailability following subcutaneous injection was inversely related to the dose level. For the 1 mg/kg dose, bioavailability was approximately 70% at all tested injection sites, with faster absorption from the foot (T(max) = 12 h for foot vs. 4.6 days for back). Bioavailability for the 10 mg/kg dose was 44 and 31% for the abdomen and back sites and 18% for 40 mg/kg injected at the back. A pharmacokinetic model that included binding as part of the absorption mechanism successfully captured the nonlinearities in rituximab absorption. CONCLUSION: The anatomical site of subcutaneous injection influences the rate of absorption and bioavailability of rituximab in rats. Saturable binding may be a major determinant of the nonlinear absorptive transport of monoclonal antibodies.

Biophysical Characterization of Tolerogenic Lipid-Based Nanoparticles Containing Phosphatidylcholine and Lysophosphatidylserine.

Autoimmune conditions, allergies, and immunogenicity against therapeutic proteins are initiated by the unwanted immune response against self and non-self proteins. The development of tolerance induction approaches can offer an effective treatment modality for these clinical conditions. We recently showed that oral administration of lipidic nanoparticles containing phosphatidylcholine (PC) and lysophosphatidylserine (Lyso-PS) converted an immunogen to a tolerogen and induced immunological tolerance towards several antigens. While the biophysical properties such as lamellar characteristics of this binary lipid system are critical for stability, therapeutic delivery, and mechanism of tolerance induction, such information has not been thoroughly investigated. In the current study, we evaluated the lamellar phase properties of PC/Lyso-PS system using orthogonal biophysical methods such as fluorescence (steady-state, anisotropy, PSvue, and Laurdan), dynamic light scattering, and differential scanning calorimetry. The results showed that Lyso-PS partitioned into the PC bilayers and led to changes in the particles' lamellar phase properties, lipid-packing, and lipid-water dynamics. Additionally, the biophysical characteristics of PC/Lyso-PS system are different from the well-studied PC/double-chain phosphatidylserine (PS) system. Notably, the incorporation of Lyso-PS significantly reduced the hydrodynamic diameter of PC particles. Results from the in vivo uptake study and intestinal loop assay utilizing flow cytometry analysis also indicated that the uptake of Lyso-PS-containing nanoparticles by immune cells in the gut and Peyer's patches is significantly higher than that of double-chain PS due to the differential transport through microfold cells. It was also found that the acyl chain mismatch between PC and Lyso-PS is critical for the miscibility and particle stability. Collectively, the results suggest that these biophysical characteristics likely influence the in vivo behaviors and contribute to the oral tolerance property of PC/Lyso-PS system.

Phosphatidylserine reduces immune response against human recombinant Factor VIII in Hemophilia A mice by regulation of dendritic cell function.

A major clinical complication in the treatment of Hemophilia A using exogenously administered recombinant Factor VIII (FVIII) is the development of neutralizing antibodies. It has been shown previously that FVIII complexed with phosphatidylserine (PS) reduces the development of total and neutralizing antibody titers in hemophilic mice. The effect of complexation of FVIII with PS upon dendritic cell (DC) uptake, maturation and processing, T-cell proliferation and cytokine secretion profiles was investigated. Flow cytometric studies of DC showed that PS inhibited the up-regulation of cell surface co-stimulatory markers (CD86 and CD40). PS reduced T-cell proliferation and significantly increased levels of TGF-ß and IL-10 but reduced secretion of IL-6 and IL-17 compared to controls. The data suggest that PS reduces immunogenicity of FVIII by regulating dendritic cell maturation and subsequent T-lymphocyte activity through modulation of cytokine secretion. A possible mechanism for PS-mediated induction of FVIII tolerance is discussed.

Immunogenicity Challenges Associated with Subcutaneous Delivery of Therapeutic Proteins.

The subcutaneous route of administration has provided convenient and non-inferior delivery of therapeutic proteins compared to intravenous infusion, but there is potential for enhanced immunogenicity toward subcutaneously administered proteins in a subset of patients. Unwanted anti-drug antibody response toward proteins or monoclonal antibodies upon repeated administration is shown to impact the pharmacokinetics and efficacy of multiple biologics. Unique immunogenicity challenges of the subcutaneous route have been realized through various preclinical and clinical examples, although subcutaneous delivery has often demonstrated comparable immunogenicity to intravenous administration. Beyond route of administration as a treatment-related factor of immunogenicity, certain product-related risk factors are particularly relevant to subcutaneously administered proteins. This review attempts to provide an overview of the mechanism of immune response toward proteins administered subcutaneously (subcutaneous proteins) and comments on product-related risk factors related to protein structure and stability, dosage form, and aggregation. A two-wave mechanism of antigen presentation in the immune response toward subcutaneous proteins is described, and interaction with dynamic antigen-presenting cells possessing high antigen processing efficiency and migratory activity may drive immunogenicity. Mitigation strategies for immunogenicity are discussed, including those in general use clinically and those currently in development. Mechanistic insights along with consideration of risk factors involved inspire theoretical strategies to provide antigen-specific, long-lasting effects for maintaining the safety and efficacy of therapeutic proteins.

Tolerogenic form of Factor VIII to prevent inhibitor development in the treatment of Hemophilia A.

BACKGROUND: The development of antidrug antibodies, also termed inhibitors, against administered factor VIII (FVIII) is one of the major complications in the clinical management of hemophilia A. Once formed, these inhibitory antibodies abrogate the activity of FVIII, resulting in loss of hemostatic efficacy and patients are subjected to increased risk of bleeding tendencies. Current treatment options after inhibitor development are expensive and ineffective in some cases. Therefore, treatment strategies that can prevent inhibitor formation is an effective approach in the management of hemophilia A. OBJECTIVES: We aimed to evaluate and discuss the use of a tolerogenic form of FVIII as an immunotherapy strategy to prevent inhibitor risk. METHODS: FVIII was associated with nanoparticles containing lysophosphatidylserine (Lyso-PS) and administered to hemophilia A mice via intravenous route. These animals then received weekly rechallenge injections with free FVIII, and plasma was collected at the end of the study to evaluate for inhibitor development. To investigate whether Lyso-PS nanoparticles influence the plasma survival of FVIII, a pharmacokinetic study following a single intravenous administration of FVIII in the presence and absence of Lyso-PS nanoparticles was performed. For dosing convenience, the tolerogenic effect of Lyso-PS nanoparticles following oral administration was also examined. RESULTS AND CONCLUSIONS: The results demonstrated that FVIII associated with Lyso-PS nanoparticles significantly reduced inhibitor development while improving plasma survival of FVIII following intravenous administration, suggesting a multifunctional FVIII form to improve clinical utility. Additionally, reduction in inhibitor formation can also be achieved using Lyso-PS nanoparticles through the user-friendly oral route of administration.

Rational design of a nanoparticle platform for oral prophylactic immunotherapy to prevent immunogenicity of therapeutic proteins.

The safety and efficacy of several life-saving therapeutic proteins are compromised due to their immunogenicity. Once a sustained immune response against a protein-based therapy is established, clinical options that are safe and cost-effective become limited. Prevention of immunogenicity of therapeutic proteins prior to their initial use is critical as it is often difficult to reverse an established immune response. Here, we discuss a rational design and testing of a phosphatidylserine-containing nanoparticle platform for novel oral prophylactic reverse vaccination approach, i.e., pre-treatment of a therapeutic protein in the presence of nanoparticles to prevent immunogenicity of protein therapies.

Preclinical evaluation of cancer immune therapy using patient-derived tumor antigen-specific T cells in a novel xenograft platform.

OBJECTIVES: With a rapidly growing list of candidate immune-based cancer therapeutics, there is a critical need to generate highly reliable animal models to preclinically evaluate the efficacy of emerging immune-based therapies, facilitating successful clinical translation. Our aim was to design and validate a novel in vivo model (called Xenomimetic or 'X' mouse) that allows monitoring of the ability of human tumor-specific T cells to suppress tumor growth following their entry into the tumor. METHODS: Tumor xenografts are established rapidly in the greater omentum of globally immunodeficient NOD-scid IL2Rγnull (NSG) mice following an intraperitoneal injection of melanoma target cells expressing tumor neoantigen peptides, as well as green fluorescent protein and/or luciferase. Changes in tumor burden, as well as in the number and phenotype of adoptively transferred patient-derived tumor neoantigen-specific T cells in response to immunotherapy, are measured by imaging to detect fluorescence/luminescence and flow cytometry, respectively. RESULTS: The tumors progress rapidly and disseminate in the mice unless patient-derived tumor-specific T cells are introduced. An initial T cell-mediated tumor arrest is later followed by a tumor escape, which correlates with the upregulation of the checkpoint molecules programmed cell death-1 (PD-1) and lymphocyte-activation gene 3 (LAG3) on T cells. Treatment with immune-based therapies that target these checkpoints, such as anti-PD-1 antibody (nivolumab) or interleukin-12 (IL-12), prevented or delayed the tumor escape. Furthermore, IL-12 treatment suppressed PD-1 and LAG3 upregulation on T cells. CONCLUSION: Together, these results validate the X-mouse model and establish its potential to preclinically evaluate the therapeutic efficacy of immune-based therapies.

Novel phosphatidylserine-binding molecule enhances antitumor T-cell responses by targeting immunosuppressive exosomes in human tumor microenvironments.

BACKGROUND: The human tumor microenvironment (TME) is a complex and dynamic milieu of diverse acellular and cellular components, creating an immunosuppressive environment, which contributes to tumor progression. We have previously shown that phosphatidylserine (PS) expressed on the surface of exosomes isolated from human TMEs is causally linked to T-cell immunosuppression, representing a potential immunotherapeutic target. In this study, we investigated the effect of ExoBlock, a novel PS-binding molecule, on T-cell responses in the TME. METHODS: We designed and synthesized a new compound, (ZnDPA)6-DP-15K, a multivalent PS binder named ExoBlock. The PS-binding avidity of ExoBlock was tested using an in vitro competition assay. The ability of this molecule to reverse exosome-mediated immunosuppression in vitro was tested using human T-cell activation assays. The in vivo therapeutic efficacy of ExoBlock was then tested in two different human tumor xenograft models, the melanoma-based xenomimetic (X-)mouse model, and the ovarian tumor-based omental tumor xenograft (OTX) model. RESULTS: ExoBlock was able to bind PS with high avidity and was found to consistently and significantly block the immunosuppressive activity of human ovarian tumor and melanoma-associated exosomes in vitro. ExoBlock was also able to significantly enhance T cell-mediated tumor suppression in vivo in both the X-mouse and the OTX model. In the X-mouse model, ExoBlock suppressed tumor recurrence in a T cell-dependent manner. In the OTX model, ExoBlock treatment resulted in an increase in the number as well as function of CD4 and CD8 T cells in the TME, which was associated with a reduction in tumor burden and metastasis, as well as in the number of circulating PS+ exosomes in tumor-bearing mice. CONCLUSION: Our results establish that targeting exosomal PS in TMEs with ExoBlock represents a promising strategy to enhance antitumor T-cell responses.