Douglas Hanlon

Enhanced and prolonged cross‐presentation following endosomal escape of exogenous antigens encapsulated in biodegradable nanoparticles

CD8+ T‐cell responses are critical in the immunological control of tumours and infectious diseases. To prime CD8+ T cells against these cell‐associated antigens, exogenous antigens must be cross‐presented by professional antigen‐presenting cells (APCs). While cross‐presentation of soluble antigens by dendritic cells is detectable in vivo, the efficiency is low, limiting the clinical utility of protein‐based vaccinations. To enhance the efficiency of presentation, we generated nanoparticles from a biodegradable polymer, poly(d,l‐lactide‐co‐glycolide) (PLGA), to deliver antigen into the major histocompatibility complex (MHC) class I antigen presentation pathway. In primary mouse bone marrow‐derived dendritic cells (BMDCs), the MHC class I presentation of PLGA‐encapsulated ovalbumin (OVA) stimulated T cell interleukin‐2 secretion at 1000‐fold lower concentration than soluble antigen and 10‐fold lower than antigen‐coated latex beads. The microparticles also served as an intracellular antigen reservoir, leading to sustained MHC class I presentation of OVA for 72 hr, decreasing by only 20% after 96 hr, a time at which the presentation of soluble and latex bead‐associated antigens was undetectable. Cytosol extraction demonstrated that antigen delivery via PLGA particles increased the amount of protein that escaped from endosomes into the cytoplasm, thereby increasing the access of exogenous antigen to the classic MHC class I loading pathway. These data indicate that the unique properties of PLGA particle‐mediated antigen delivery dramatically enhance and sustain exogenous antigen presentation by MHC class I, potentially facilitating the clinical use of these particles in vaccination.

Induction of human tumor-loaded dendritic cells

A preferred anti‐cancer vaccine would be tumor‐specific, simple to rapidly construct and safe to administer. It would permit immunization against a spectrum of the tumors distinctive antigens, without requiring their prior identification. Toward these goals, we describe a modification of standard extracorporeal photopheresis (ECP) which initiates, within a single day, both monocyte‐to‐dendritic cell (DC) differentiation and malignant cell apoptosis. The transition of monocytes to immature DCs was identified by the expression of cytoplasmic CD83 and membrane CD36 in the absence of membrane CD14 staining, as well as induction of membrane CD83 expression. Differentiating DCs were avidly phagocytic and engulfed apoptotic malignant T cells. Differentiating DCs were capable of stimulating significant proliferation of normal alloreactive lymphocyte responders, indicting increased expression of membrane MHC class II molecules. This approach provides a clinically practical means of developing tumor‐loaded cells that have initiated the transition to DCs without the requirement of exogenous cytokines, excessive cellular manipulation or isolation. Construction of DC vaccines using this methodology can be generalized to other diseases and may offer a novel approach for improved cancer immunotherapy.

Polymer nanoparticles containing tumor lysates as antigen delivery vehicles for dendritic cell–based antitumor immunotherapy

UNLABELLED Encapsulation of tumor-associated antigens in polymer nanoparticles (NP) is a promising approach to enhance efficiency of antigen delivery for anti-tumor vaccines. Head and neck squamous carcinoma (HNSCC) cell lines were initially used to generate tumor-associated antigens (TAA)-containing poly (lactic-co-glycolic acid) (PLGA) NP; encapsulation efficiency and release kinetics were profiled. Findings were adopted to entrap fresh tumor lysate from five patients with advanced HNSCC. To test the hypothesis that NP enhance antigen presentation, dendritic cell (DC) produced from patient blood monocyte precursors were loaded with either the un-encapsulated or NP-encapsulated versions of tumor lysates. These were used to stimulate freshly-isolated autologous CD8+ T cells. In four of five patients, anti-tumor CD8+ T cells showed significantly increased immunostimulatory IFN-γ (p=0.071) or decreased immmunoinhibitory IL-10 production (p=0.0004) associated with NP-mediated antigen delivery. The observations represent an enabling step in the production of clinically-translatable, inexpensive, highly-efficient, and personalized polymer-based immunotherapy for solid organ malignancies. FROM THE CLINICAL EDITOR Enhancing the antigen presentation may be a viable approach to increase the efficiency of tumor cell directed cytotoxicity via immune mechanisms. This study presents an example for this using head and neck cancer cell lines and nanotechnology-based encapsulated antigen presentation to dendritic cells. The observed CD8+ T-cell response was significantly enhanced. This method may pave the way to a highly efficient cancer cell elimination method with minimal to no toxicity.

Photoactivated 8-methoxypsoralen treatment causes a peptide-dependent increase in antigen display by transformed lymphocytes

Ex vivo exposure of malignant human T cells to photoactivated 8‐methoxypsoralen (8‐MOPa), followed by their i.v. return, appears to vaccinate patients against tumor‐associated antigens of cutaneous T cell lymphoma in a procedure termed photopheresis.The molecular basis of this Food and Drug Administration‐approved therapy, administered in 100 centers worldwide, is unclear. Most of the attention to the mechanism of action of the drug has focused on its capacity to form covalent cross‐links with pyrimidine bases of DNA, thereby inhibiting cellular proliferation. Because immunologic factors appear to be important in the clinical response and could potentially serve as a model for immunotherapy of other malignancies, we explored the possibility that 8‐MOP‐treated cells display increased quantities of antigenic peptides at their cell surface. In this work, human B‐lymphoblastoid tissue culture lines were exposed to 8‐MOPa and expression of cell surface class I major histocompatibility complex proteins assessed, since CD8 T cells recognize antigenic moieties in the context of class I molecules. A peak 200–300% increase in MHC class I expression in 8‐MOPa‐treated cells occurred at 20 hr. 8‐MOPa was far more effective in inducing this increase in class I MHC than other modalities, including mitomycin C, γ‐irradiation, ultraviolet B or heat or cold shock. This increase in surface class I MHC molecules appears to be driven by the degradation of cytoplasmic proteins into small peptides, followed by the transport of these peptides to MHC class I molecules in the endoplasmic reticulum. The data suggest that 8‐MOPa treatment may augment the immunogenicity of tumor and/or antigen‐presenting cells by enhancing processing and transport of class I MHC antigenic peptides. Int. J. Cancer 78:70–75, 1998.© 1998 Wiley‐Liss, Inc.

Targeting human dendritic cells via DEC-205 using PLGA nanoparticles leads to enhanced cross-presentation of a melanoma-associated antigen

Targeting antigen to dendritic cells (DCs) is a powerful and novel strategy for vaccination. Priming or loading DCs with antigen controls whether subsequent immunity will develop and hence whether effective vaccination can be achieved. The goal of our present work was to increase the potency of DC-based antitumor vaccines by overcoming inherent limitations associated with antigen stability and cross-presentation. Nanoparticles prepared from the biodegradable polymer poly(lactic-co-glycolic acid) have been extensively used in clinical settings for drug delivery and are currently the subject of intensive investigation as antigen delivery vehicles for vaccine applications. Here we describe a nanoparticulate delivery system with the ability to simultaneously carry a high density of protein-based antigen while displaying a DC targeting ligand on its surface. Utilizing a targeting motif specific for the DC-associated surface ligand DEC-205, we show that targeted nanoparticles encapsulating a MART-127–35 peptide are both internalized and cross-presented with significantly higher efficiency than isotype control-coated nanoparticles in human cells. In addition, the DEC-205-labeled nanoparticles rapidly escape from the DC endosomal compartment and do not colocalize with markers of early (EEA-1) or late endosome/lysosome (LAMP-1). This indicates that encapsulated antigens delivered by nanoparticles may have direct access to the class I cytoplasmic major histocompatibility complex loading machinery, overcoming the need for “classical” cross-presentation and facilitating heightened DC stimulation of anti-tumor CD8+ T-cells. These results indicate that this delivery system provides a flexible and versatile methodology to deliver melanoma-associated antigen to DCs, with both high efficiency and heightened potency.

A LYMPHOCYTE CELL SURFACE HEAT SHOCK PROTEIN HOMOLOGOUS TO THE ENDOPLASMIC RETICULUM CHAPERONE, IMMUNOGLOBULIN HEAVY CHAIN BINDING PROTEIN BIP

BE2 is a cell surface monomeric 78‐kDa protein (BE2‐78) expressed on the malignant lymphocytes of cutaneous T‐cell lymphoma and adult T‐cell leukemia, on some lymphocytes from patients with acquired immunodeficiency syndrome and on Epstein‐Barr virus‐transformed B cells. BE2‐78 positivity of cutaneous T‐cell lymphoma tumor cells is a useful diagnostic and prognostic determinant in evaluating patients with that disorder. The BE2‐78 protein was isolated from Epstein Barr virus‐transformed B cells, purified by 1‐ and 2‐dimensional electrophoresis and then sequenced. The sequence of 4 isolated peptide fragments was highly homologous with the 78‐kDa heat shock protein, BiP, an endoplasmic reticulum chaperone. The similarity between BiP and BE2‐78 was supported by the demonstration that BE2‐78, like BiP, avidly binds to ATP. However, polyclonal and monoclonal reagents that recognize cytoplasmic 70‐ and 78‐kDa heat shock proteins do not detect the BE2‐78 antigen on the cell surface of cutaneous T‐cell lymphoma or Epstein Barr virus‐transformed lymphocytes, and peptide mapping demonstrates sequence divergence, suggesting that either they are distinct or conformationally different molecules. Our results indicate that BE2‐78 is a cell surface heat shock protein. The possibility that malignant or transformed lymphocytes may express cell surface molecules with the capacity to bind a spectrum of exogenous or endogenous peptides has potential implications for tumor immunology. Int. J. Cancer 71: 1077‐1085, 1997.

The Clonotypic T Cell Receptor Is a Source of Tumor-associated Antigens in Cutaneous T Cell Lymphoma

Abstract: To develop cancer vaccines for the treatment of cutaneous T cell lymphoma (CTCL), immunogenic peptides were identified by two approaches. First, through the use of “reverse immunology” the peptide sequence of the idiotypic region of the β chain of the T cell receptor (TCR) was determined and a series of overlapping peptides synthesized and tested for CD8 T cell recognition. In two patients, the idiotypic CDR3 region provided immunogenic epitopes that were recognized in a class I‐restricted fashion by autologous CD8 T cell lines. In a second strategy, peptides were isolated directly from class I MHC molecules on the CTCL surface and sequenced. A peptide with partial homology to sequences contained in the conserved variable portion of the clonotypic TCR β chain was recognized as immunogenic by autologous CD8 T cells. Therefore, both approaches demonstrated that the clonotypic TCR in CTCL is a source of immunogenic tumor epitopes. To confirm that recognition of TCR‐derived sequences provides immunoprotection against tumor growth, a murine model of T cell lymphoma was studied. The immunogenicity of a thymoma, which lacks cell surface TCR expression, was enhanced by transfection of the β chain of the TCR. The studies reviewed in this paper demonstrate that the TCR can serve as one source for immunogenic tumor peptides in T cell lymphoma in vitro and in vivo. Presentation of TCR epitopes on dendritic cells that express high levels of MHC, costimulatory, and adhesion molecules may provide an effective means for immunization against T cell malignancy.

Enhanced Stimulation of Anti‐Ovarian Cancer CD8+ T Cells by Dendritic Cells Loaded with Nanoparticle Encapsulated Tumor Antigen

Citation 
Hanlon DJ, Aldo PB, Devine L, Alvero AB, Engberg AK, Edelson R, Mor G. Enhanced stimulation of anti‐ovarian cancer CD8+ T cells by dendritic cells loaded with nanoparticle encapsulated tumor antigen. Am J Reprod Immunol 2011; 65: 597–609

Efficient Tumor Antigen Loading of Dendritic Antigen Presenting Cells by Transimmunization

Extracorporeal photochemotherapy (ECP), or photopheresis, was originally introduced for the management of patients with cutaneous T cell lymphoma (CTCL). Today, ECP remains the only FDA approved tumor-targeting selective immunotherapy for the treatment of any cancer. The key cellular events permitting ECP-induced anti-tumor immunity against CTCL are the induction of apoptotis in the malignant T cells, and the induction of monocyte-to-dendritic cell (DC) differentiation. In standard ECP, leukocytes extracorporeally exposed to psoralen and ultraviolet A light (UVA) are circulated back to the patient. However, recent findings suggest that co-incubation of these cells prior to re-infusion allows for more efficient phagocytosis and processing of the apoptotic malignant T cells by the newly formed DCs. Moreover, such a co-incubation step permits the direct external manipulation of this system and the design of strategies to augment the production of tumor-loaded DCs. These considerations have led to the development of Transimmunization, so named because it causes transfer of tumor antigens to newly formed dendritic cells capable of initiating immunization against the tumor cells, as the replacement technology for ECP. We will review the scientific understanding of ECP and explain how this can lead a more efficienct, potentially broadly applicable, immunotherapy for cancer.

Configuration-dependent Presentation of Multivalent IL-15:IL-15Rα Enhances the Antigen-specific T Cell Response and Anti-tumor Immunity.

Here we report a “configuration-dependent” mechanism of action for IL-15:IL-15Rα (heterodimeric IL-15 or hetIL-15) where the manner by which IL-15:IL-15Rα molecules are presented to target cells significantly affects its function as a vaccine adjuvant. Although the cellular mechanism of IL-15 trans-presentation via IL-15Rα and its importance for IL-15 function have been described, the full effect of the IL-15:IL-15Rα configuration on responding cells is not yet known. We found that trans-presenting IL-15:IL-15Rα in a multivalent fashion on the surface of antigen-encapsulating nanoparticles enhanced the ability of nanoparticle-treated dendritic cells (DCs) to stimulate antigen-specific CD8+ T cell responses. Localization of multivalent IL-15:IL-15Rα and encapsulated antigen to the same DC led to maximal T cell responses. Strikingly, DCs incubated with IL-15:IL-15Rα-coated nanoparticles displayed higher levels of functional IL-15 on the cell surface, implicating a mechanism for nanoparticle-mediated transfer of IL-15 to the DC surface. Using artificial antigen-presenting cells to highlight the effect of IL-15 configuration on DCs, we showed that artificial antigen-presenting cells presenting IL-15:IL-15Rα increased the sensitivity and magnitude of the T cell response, whereas IL-2 enhanced the T cell response only when delivered in a paracrine fashion. Therefore, the mode of cytokine presentation (configuration) is important for optimal immune responses. We tested the effect of configuration dependence in an aggressive model of murine melanoma and demonstrated significantly delayed tumor progression induced by IL-15:IL-15Rα-coated nanoparticles in comparison with monovalent IL-15:IL-15Rα. The novel mechanism of IL-15 transfer to the surface of antigen-processing DCs may explain the enhanced potency of IL-15:IL-15Rα-coated nanoparticles for antigen delivery.