Hyaluronidase Enhances Targeting of Hydrogel-Encapsulated Anti-CTLA-4 to Tumor Draining Lymph Nodes and Improves Anti-Tumor Efficacy.

Immunotherapy targeting checkpoint inhibitors, such as CTLA-4 and/or PD-1, has emerged as a leading cancer therapy. While their combination produces superior efficacy compared to monotherapy, it also magnifies inflammatory and autoimmune toxicity that limits clinical utility. We previously reported that a peri-tumor injection of low-dose hydrogel-encapsulated anti-CTLA-4 produced anti-tumor responses that were equal to, or better than, systemic dosing despite a >80% reduction in total dose. Injection of hydrogel-encapsulated anti-CTLA-4 was associated with low serum exposure and limited autoimmune toxicity, but still synergized with anti-PD-1. In this report, we employ live and ex vivo imaging to examine whether peri-tumor administration specifically targets anti-CTLA-4 to tumor-draining lymph nodes (TDLN) and whether the incorporation of hyaluronidase enhances this effect. Tumor-free survival analysis was also used to measure the impact of hyaluronidase on tumor response. Compared to systemic dosing, peri-tumor injection of hydrogel-encapsulated anti-CTLA-4/DyLight 800 resulted in preferential labeling of TDLN. Incorporating hyaluronidase within the hydrogel improved the rapidity, intensity, and duration of TDLN labeling and significantly improved tumor-free survival. We conclude that hydrogel-encapsulated anti-CTLA acts as a localized antibody reservoir and that inclusion of hyaluronidase optimizes the blockade of CTLA-4 in TDLN and thereby imparts superior anti-tumor immunity.

Development of a blocker of the universal phosphatidylserine- and phosphatidylethanolamine-dependent viral entry pathways.

Envelope phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtr) have been shown to mediate binding of enveloped viruses. However, commonly used PtdSer binding molecules such as Annexin V cannot block PtdSer-mediated viral infection. Lack of reagents that can conceal envelope PtdSer and PtdEtr and subsequently inhibit infection hinders elucidation of the roles of the envelope phospholipids in viral infection. Here, we developed sTIM1dMLDR801, a reagent capable of blocking PtdSer- and PtdEtr-dependent infection of enveloped viruses. Using sTIM1dMLDR801, we found that envelope PtdSer and/or PtdEtr can support ZIKV infection of not only human but also mosquito cells. In a mouse model for ZIKV infection, sTIM1dMLDR801 reduced ZIKV load in serum and the spleen, indicating envelope PtdSer and/or PtdEtr support in viral infection in vivo. sTIM1dMLDR801 will enable elucidation of the roles of envelope PtdSer and PtdEtr in infection of various virus species, thereby facilitating identification of their receptors and transmission mechanisms.

Peri-tumor administration of controlled release anti-CTLA-4 synergizes with systemic anti-PD-1 to induce systemic antitumor immunity while sparing autoimmune toxicity.

Combination immunotherapy targeting the PD-1 and CTLA-4 checkpoint inhibitor pathways provides substantial clinical benefit in patients with advanced-stage cancer but at the risk of dose-limiting inflammatory and autoimmune toxicity. The delicate balance that exists between unleashing tumor killing and promoting systemic autoimmune toxicity represents a major clinical challenge. We hypothesized that targeting anti-CTLA-4 so that it perfuses tumor-draining lymph nodes would provide a significant therapeutic advantage and developed an injectable hydrogel with controlled antibody release characteristics for this purpose. Injection of hydrogel-encapsulated anti-CTLA-4 at a peri-tumor location (MC-38 tumor model) produced dose-dependent antitumor responses and survival that exceeded those by anti-CTLA-4 alone (p < 0.05). Responses to 100 µg of targeted anti-CTLA-4 also equaled or exceeded those observed with a series of systemic injections delivering 600 µg (p < 0.05). While preserving antitumor activity, this approach resulted in serum anti-CTLA-4 exposure (area under the curve) that averaged only 1/16th of that measured with systemic therapy. Consistent with the marked differences in systemic exposure, systemic anti-CTLA-4 stimulated the onset of autoimmune thyroiditis in iodide-exposed NOD.H-2h4 mice, as measured by anti-thyroglobulin antibody titer, while hydrogel-encapsulated anti-CTLA-4 had a minimal effect (p ≤ 0.01). At the same time, this targeted low-dose anti-CTLA-4 approach synergized well with systemic anti-PD-1 to control tumor growth and resulted in a high frequency of complete responders that were immune to tumor re-challenge at a distant site. We conclude that targeted and controlled delivery of low-dose anti-CTLA-4 has the potential to improve the benefit-risk ratio associated with combination checkpoint inhibitor therapy.

Regulation of Cell Surface CB2 Receptor during Human B Cell Activation and Differentiation.

Cannabinoid receptor type 2 (CB2) is the primary receptor pathway mediating the immunologic consequences of cannabinoids. We recently reported that human peripheral blood B cells express CB2 on both the extracellular membrane and at intracellular sites, where-as monocytes and T cells only express intracellular CB2. To better understand the pattern of CB2 expression by human B cells, we examined CD20+ B cells from three tissue sources. Both surface and intracellular expression were present and uniform in cord blood B cells, where all cells exhibited a naïve mature phenotype (IgD+/CD38Dim). While naïve mature and quiescent memory B cells (IgD-/CD38-) from tonsils and peripheral blood exhibited a similar pattern, tonsillar activated B cells (IgD-/CD38+) expressed little to no surface CB2. We hypothesized that regulation of the surface CB2 receptor may occur during B cell activation. Consistent with this, a B cell lymphoma cell line known to exhibit an activated phenotype (SUDHL-4) was found to lack cell surface CB2 but express intracellular CB2. Furthermore, in vitro activation of human cord blood resulted in a down-regulation of surface CB2 on those B cells acquiring the activated phenotype but not on those retaining IgD expression. Using a CB2 expressing cell line (293 T/CB2-GFP), confocal microscopy confirmed the presence of both cell surface expression and multifocal intracellular expression, the latter of which co-localized with endoplasmic reticulum but not with mitochondria, lysosomes, or nucleus. Our findings suggest a dynamic multi-compartment expression pattern for CB2 in B cells that is specifically modulated during the course of B cell activation.

Human tumor infiltrating lymphocytes cooperatively regulate prostate tumor growth in a humanized mouse model.

BACKGROUND: The complex interactions that occur between human tumors, tumor infiltrating lymphocytes (TIL) and the systemic immune system are likely to define critical factors in the host response to cancer. While conventional animal models have identified an array of potential anti-tumor therapies, mouse models often fail to translate into effective human treatments. Our goal is to establish a humanized tumor model as a more effective pre-clinical platform for understanding and manipulating TIL. METHODS: The immune system in NOD/SCID/IL-2Rγnull (NSG) mice was reconstituted by the co-administration of human peripheral blood lymphocytes (PBL) or subsets (CD4+ or CD8+) and autologous human dendritic cells (DC), and animals simultaneously challenged by implanting human prostate cancer cells (PC3 line). Tumor growth was evaluated over time and the phenotype of recovered splenocytes and TIL characterized by flow cytometry and immunohistochemistry (IHC). Serum levels of circulating cytokines and chemokines were also assessed. RESULTS: A tumor-bearing huPBL-NSG model was established in which human leukocytes reconstituted secondary lymphoid organs and promoted the accumulation of TIL. These TIL exhibited a unique phenotype when compared to splenocytes with a predominance of CD8+ T cells that exhibited increased expression of CD69, CD56, and an effector memory phenotype. TIL from huPBL-NSG animals closely matched the features of TIL recovered from primary human prostate cancers. Human cytokines were readily detectible in the serum and exhibited a different profile in animals implanted with PBL alone, tumor alone, and those reconstituted with both. Immune reconstitution slowed but could not eliminate tumor growth and this effect required the presence of CD4+ T cell help. CONCLUSIONS: Simultaneous implantation of human PBL, DC and tumor results in a huPBL-NSG model that recapitulates the development of human TIL and allows an assessment of tumor and immune system interaction that cannot be carried out in humans. Furthermore, the capacity to manipulate individual features and cell populations provides an opportunity for hypothesis testing and outcome monitoring in a humanized system that may be more relevant than conventional mouse models.

Differential expression of intracellular and extracellular CB(2) cannabinoid receptor protein by human peripheral blood leukocytes.

mRNA encoding for the CB(2) cannabinoid receptor is expressed by many subsets of human peripheral blood leukocytes (PBL), but little is known about the resulting protein expression and function. Employing clones from the A549 and 293T cell lines that were constructed to express both full-length human CB(2) and GFP, we developed a flow cytometry assay for characterizing CB(2) protein expression. A monoclonal antibody directed against human CB(2) selectively stained the surface of transduced but not parental cell lines. When cells were fixed and permeabilized, imaging flow cytometry identified large stores of intracellular protein. Total cellular staining for CB(2) corresponded closely with the level of GFP expression. When exposed to Δ(9)-tetrahydrocannabinol, CB(2)-expressing cells internalized cell surface CB(2) receptors in a time- and dose-dependent manner. Applying these approaches to human PBL, CB(2) protein was identified on the surface of human B cells but not on T cells or monocytes. In contrast, when PBL were fixed and permeabilized, intracellular CB(2) expression was readily detected in all three subsets by both conventional and imaging flow cytometry. Similar to the protein expression pattern observed in fixed and permeabilized PBL, purified B cells, T cells, and monocytes expressed relatively equal levels of CB(2) mRNA by quantitative real-time RT-PCR. Our findings confirm that human PBL express CB(2) protein but that its distribution is predominantly intracellular with only B cells expressing CB(2) protein at the extracellular membrane. The differential role of intracellular and extracellular CB(2) receptors in mediating ligand signaling and immune function remains to be determined.

Reconstitution of huPBL-NSG mice with donor-matched dendritic cells enables antigen-specific T-cell activation.

Humanized mouse models provide a unique opportunity to study human immune cells in vivo, but traditional models have been limited to the evaluation of non-specific T-cell interactions due to the absence of antigen-presenting cells. In this study, immunodeficient NOD/SCID/IL2r-γ(null) (NSG) mice were engrafted with human peripheral blood lymphocytes alone or in combination with donor-matched monocyte-derived dendritic cells (DC) to determine whether antigen-specific T-cell activation could be reconstituted. Over a period of 3 weeks, transferred peripheral blood lymphocytes reconstituted the spleen and peripheral blood of recipient mice with predominantly human CD45-positive lymphocytes. Animals exhibited a relatively normal CD4/CD8 ratio (average 1.63:1) as well as reconstitution of CD3/CD56 (averaging 17.8%) and CD20 subsets (averaging 4.0%). Animals reconstituted with donor-matched CD11c+ DC also demonstrated a CD11c+ population within their spleen, representing 0.27% to 0.43% of the recovered human cells with concurrent expression of HLA-DR, CD40, and CD86. When immunized with adenovirus, either as free replication-incompetent vector (AdV) or as vector-transduced DC (DC/AdV), there was activation and expansion of AdV-specific T-cells, an increase in Th1 cytokines in serum, and skewing of T-cells toward an effector/memory phenotype. T-cells recovered from animals challenged with AdV in vivo proliferated and secreted a Th1-profile of cytokines in response to DC/AdV challenge in vitro. Our results suggest that engrafting NSG mice with a combination of lymphocytes and donor-matched DC can reconstitute antigen responsiveness and allow the in vivo assessment of human immune response to viruses, vaccines, and other immune challenges.

Redirecting lentiviral vectors pseudotyped with Sindbis virus-derived envelope proteins to DC-SIGN by modification of N-linked glycans of envelope proteins.

Redirecting the tropism of viral vectors enables specific transduction of selected cells by direct administration of vectors. We previously developed targeting lentiviral vectors by pseudotyping with modified Sindbis virus envelope proteins. These modified Sindbis virus envelope proteins have mutations in their original receptor-binding regions to eliminate their natural tropisms, and they are conjugated with targeting proteins, including antibodies and peptides, to confer their tropisms on target cells. We investigated whether our targeting vectors interact with DC-SIGN, which traps many types of viruses and gene therapy vectors by binding to the N-glycans of their envelope proteins. We found that these vectors do not interact with DC-SIGN. When these vectors were produced in the presence of deoxymannojirimycin, which alters the structures of N-glycans from complex to high mannose, these vectors used DC-SIGN as their receptor. Genetic analysis demonstrated that the N-glycans at E2 amino acid (aa) 196 and E1 aa 139 mediate binding to DC-SIGN, which supports the results of a previous report of cryoelectron microscopy analysis. In addition, we investigated whether modification of the N-glycan structures could activate serum complement activity, possibly by the lectin pathway of complement activation. DC-SIGN-targeted transduction occurs in the presence of human serum complement, demonstrating that high-mannose structure N-glycans of the envelope proteins do not activate human serum complement. These results indicate that the strategy of redirecting viral vectors according to alterations of their N-glycan structures would enable the vectors to target specific cells types expressing particular types of lectins.

Clarifying CB2 receptor-dependent and independent effects of THC on human lung epithelial cells.

Marijuana smoking is associated with a number of abnormal findings in the lungs of habitual smokers. Previous studies revealed that Delta(9)-tetrahydrocannabinol (THC) caused mitochondrial injury in primary lung epithelial cells and in the cell line, A549 [Sarafian, T. A., Kouyoumjian, S., Khoshaghideh, F., Tashkin, D. P., and Roth, M. D. (2003). Delta 9-tetrahydrocannabinol disrupts mitochondrial function and cell energetics. Am J Physiol Lung Cell Mol Physiol 284, L298-306; Sarafian, T., Habib, N., Mao, J. T., Tsu, I. H., Yamamoto, M. L., Hsu, E., Tashkin, D. P., and Roth, M. D. (2005). Gene expression changes in human small airway epithelial cells exposed to Delta9-tetrahydrocannabinol. Toxicol Lett 158, 95-107]. The role of cannabinoid receptors in this injury was unclear, as was the potential impact on cell function. In order to investigate these questions, A549 cells were engineered to over-express the type 2 cannabinoid receptor (CB2R) using a self-inactivating lentiviral vector. This transduction resulted in a 60-fold increase in CB2R mRNA relative to cells transduced with a control vector. Transduced cell lines were used to study the effects of THC on chemotactic activity and mitochondrial function. Chemotaxis in response to a 10% serum gradient was suppressed in a concentration-dependent manner by exposure to THC. CB2R-transduced cells exhibited less intrinsic chemotactic activity (p<0.05) and were 80- to 100-fold more sensitive to the inhibitory effects of THC. Studies using SR144528, a selective CB2R antagonist, verified that these effects were mediated by the CB2R. Marijuana smoke extract, but not smoke extracts from tobacco or placebo marijuana cigarettes, reproduced these effects (p<0.05). THC decreased ATP level and mitochondrial membrane potential (Psi(m)) in both control and CB2R-transduced cells. However, these decreases did not play a significant role in chemotaxis inhibition since cyclosporine A, which protected against ATP loss, did not increase cell migration. Moreover, CB2R-transduced cells displayed higher Psi(m) than did control cells. Since both Psi(m) and chemotaxis are regulated by intracellular signaling, we investigated the effects of THC on the activation of multiple signaling pathways. Serum exposure activated several signaling events of which phosphorylation of IkappaB-alpha and JNK was regulated in a CB2R- and THC-dependent manner. We conclude that airway epithelial cells are sensitive to both CB2R-dependent and independent effects mediated by THC.

Centrifugation enhances integrin-mediated transduction of dendritic cells by conventional and RGD-modified adenoviral vectors.

The level of antigen loading can impact on the capacity for dendritic cells (DC) to activate T cell responses. Several different approaches to adenoviral (Ad)-based transduction were therefore assessed for their effect on both transgene expression and T cell activation. While a conventional E1(-)/E3Delta Ad vector (Ad/GFP) produced a concentration-dependent expression of GFP, a modified vector expressing Arginine-Glycine-Aspartic Acid (RGD) sequence on its fiber knob (Ad-RGD/GFP) enhanced transgene expression by 9-20-fold at each MOI. The addition of centrifugal force (2000xg) during DC transduction with Ad/GFP also increased expression up to 20-fold. However, combining centrifugation with the Ad-RGD/GFP vector produced no effect on transduction rate and only a 1.5- to 2-fold increase in GFP expression, suggesting overlapping mechanisms of action. Consistent with this, exogenous RGD peptide blocked transduction regardless of the vector used, or the addition of centrifugal force, and transduction was primarily limited to DC expressing the CD51 integrin receptor. Ad vectors expressing ovalbumin (OVA) were used to assess transduced DC for their capacity to activate OVA-specific T cells. We observed a significant relationship between transgene expression and the capacity for T cell activation regardless of whether transgene expression was increased by using a higher MOI, an RGD-modified vector, or by employing centrifugal force. Furthermore, combining these approaches produced synergistic effects on T cell activation. We conclude that RGD-modified vectors and centrifugation both enhance DC transduction by increasing entry via integrin receptors and that the capacity for T cell activation can be optimized by combining approaches to achieve the highest possible level of transgene expression.

Adenoviral-encoded antigens are presented efficiently by a subset of dendritic cells expressing high levels of alpha(v)beta3 integrins.

Dendritic cells (DC) play a central role in antigen presentation and are often targeted by adenoviral (Ad)-based gene therapy. However, DC lack the coxsackie-Ad receptor, and little is known about the process by which they acquire and present Ad-encoded antigens. We examined the expression of alpha(v)beta3 integrins (CD51/CD61) on mouse bone marrow-derived DC (BM-DC) and their susceptibility to transduction by Ad vectors. Less than 10% of BM-DC precursors expressed CD51, but expression increased over time in culture with granulocyte macrophage-colony stimulating factor (GM-CSF)/interleukin (IL)-4. After 7 days, 28 +/- 1.7% of CD11c+ DC expressed high levels of CD51 (CD51(hi)), and the remaining DC expressed low levels of CD51 (CD51(lo)). CD51(hi) CD express higher major histocompatibility complex type 1 (MHC I); however, both of the DC subsets expressed similar levels of MHC II and costimulatory molecules. When exposed to a first-generation Ad vector, transgene expression was restricted to the CD51(hi) DC subset and blocked by soluble peptides expressing an arginine, glycine, aspartic acid (RGD) sequence, confirming the role of integrins in viral entry. Consistent with this, a modified Ad expressing an RGD-binding sequence in its fiber knob (Ad-RGD) transduced the CD51(hi) DC subset with significantly higher efficiency. When BM-DC were transduced with an Ad-expressing ovalbumin (Ad-OVA), the CD51(hi) subset proved superior in activating OT-I (T cell receptor-OVA) T cells. Similar to in vitro effects, systemic administration of GM-CSF/IL-4 increased the expression of CD51 on splenic DC and rendered these cells susceptible to Ad transduction. These results suggest that a limited subset of DC expressing high levels of alpha(v)beta3 integrins is preferentially transduced by Ad vectors and activates CD8+ T cell responses against Ad-encoded antigens.

Modifying adenoviral vectors for use as gene-based cancer vaccines.

The past decade has produced significant advances in our understanding of antigen-presenting cells, tumor antigens, and other components of the immune response to cancer. Gene-based vaccination is emerging as one of the more promising approaches for loading dendritic cells (DC) with tumor-associated antigens. In this respect, it is proposed that adenoviral (AdV) vectors can deliver high antigen concentrations, promote effective processing and MHC expression, and stimulate potent cell-mediated immunity. While AdV vectors have performed well in pre-clinical vaccine models, their application to patient care has limitations. The in vivo administration of AdV vectors is associated with both innate and adaptive host responses that result in tissue inflammation and injury, viral neutralization, and premature clearance of AdV-transduced cells. A variety of strategies have been developed to address these limitations. The ideal vaccine would avoid vector-related immune responses, have relative specificity for transducing DC, and induce high levels of transgene expression. This review describes the range of host responses to AdV vaccines, identifies strategies to reduce viral recognition and enhance transgene antigen expression, and suggests future approaches to vector development and administration. There is every reason to believe that safer and more effective forms of AdV-based vaccines can be developed and applied to patient therapy.

Helper-dependent adenoviral vectors efficiently express transgenes in human dendritic cells but still stimulate antiviral immune responses.

Adenoviral (AdV) vectors can be used to transduce a wide range of human cells and tissues. However, pre-existing immunity to AdV, and enhancement of this immunity after repeated administration, limits their clinical application. This may be especially relevant when vectors are loaded into APCs. Helper-dependent AdV (Hd-AdV), in which viral coding regions are replaced by human stuffer DNA, offers a new approach for limiting antiviral responses. To evaluate their immunogenicity, human dendritic cells (DCs) were infected with either an Hd-AdV or a conventional replication-deficient E1-deleted AdV (E1-AdV) and were evaluated for their capacity to stimulate antiviral T cell responses. Hd-AdV proved to be 50- to 275-fold more effective than E1-AdV at expressing the lacZ transgene in human DCs. PCR demonstrated similar transduction efficiencies, but RT-PCR revealed much higher expression of transgene mRNA after transduction with Hd-AdV. Functionally, DCs transduced with Hd-AdV stimulated the proliferation of autologous T cells to the same level as DCs transduced with E1-AdV. Identical viral-specific T cell responder frequencies were observed and T cells stimulated with either type of AdV-transduced DC lysed viral-infected target cells. Disrupting transcription of vector-based genes had no effect on T cell activation, suggesting that responses against both vectors were directed against preformed components of the viral capsid. We conclude that Hd-AdV vectors can be used to obtain higher transgene expression in human DCs but that they still evoke a vector-related immune response similar to that generated by E1-AdV.

Increased dendritic cell number and function following continuous in vivo infusion of granulocyte macrophage-colony-stimulating factor and interleukin-4.

Dendritic cells (DCs) are rare antigen-presenting cells that play a central role in stimulating immune responses. The combination of recombinant granulocyte macrophage-colony-stimulating factor (rGM-CSF) and recombinant interleukin-4 (rIL-4) provides an important stimulus for generating DCs from murine bone marrow precursors in vitro. Using miniature osmotic pumps, we now demonstrate that continuous infusion of these cytokines for 7 days had a similar effect in vivo, increasing the number and function of splenic DCs. Administration of rGM-CSF/rIL-4 (10 microg/d each) increased the concentration of CD11(+) DCs by 2.7-fold and the absolute number of splenic DCs by an average of 5.7-fold. DC number also increased in peripheral blood and lymph nodes. The resultant DCs exhibited a different phenotype and function than those in control mice or mice treated with rGM-CSF alone. rGM-CSF/IL-4 increased both the myeloid (CD11c(+)/CD11b(+)) and the lymphoid (CD11c(+)/CD8alpha(+)) subpopulations, whereas rGM-CSF increased only myeloid DCs. DCs were highly concentrated in the T-cell areas of white pulp after rGM-CSF/IL-4 administration, whereas they were diffusely distributed throughout white pulp, marginal zones, and red pulp in mice treated with rGM-CSF alone. rGM-CSF/rIL-4 also significantly increased the expression of major histocompatibility complex (MHC) class I and MHC class II on CD11c(+) cells and increased their capacity to take up antigens by macropinocytosis and receptor-mediated endocytosis. Splenic DCs generated in response to rGM-CSF/rIL-4 were functionally immature in terms of allostimulatory activity, but this activity increased after short-term in vitro culture. Systemic treatment with rGM-CSF/rIL-4 enhanced the response to an adenoviral-based vaccine and led to antigen-specific retardation in the growth of established tumor. We conclude that systemic therapy with the combination of rGM-CSF/rIL-4 provides a new approach for generating DCs in vivo.