Characterization of a Sustained-Release Delivery System for Combined CytokinefPeptide Vaccination Using a Poly- N-Acetyl Glucosamine-based Polymer Matrix1

Identification of tumor-associated antigens (TAAs) and their class I MHC-restricted epitopes now allows for the rational design of peptide-based cancer vaccines. A biocompatible system capable of sustained release of biologically relevant levels of cytokine and TAA peptide could provide a more effective microenvironment for antigen presentation, Our goal was to test a sustained-release cytokine/TAA peptide-based formulation using a highly purified polysaccharide [poly-N-acetyl glucosamine (p-G1cNAc)] polymer. Granulocyte-macrophage colony-stimulating factor (GMCSF; 100 Lg) and MART-1(27_35) peptide (128 ig in DMSO) were formulated into p-GlcNAc. Peptide release was assayed in vitro using interleukin 2 production from previously characterized MART-1(27_35)-specific Jurkat T cells (JRT22). GM-CSF release was assayed via ELISA and proliferation of M-07e (GM-CSF-dependent) cells, Local bioavailability of MART-1(2735) peptide for uptake and presentation by antigen-presenting cells was demonstrated for up to 6 days (>0.5 Wml). More than 1.0 Wml GM-CSF was concomitantly released over the same period. Biocompatibility and local tissue response to p-GlcNAc releasing murine GM-CSF was determined in C57BL/6 mice via s.c. injection using murine GM-CSF (0.2 iWml) in 200 p.1 of a 2.5% polymer gel. Significant lymphocytic and eosinophilic infiltration was Received 8/5/96; revised 1/29/97: accepted 2/20/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I Supported by Marine Polymer Technologies (Danvers, MA) and The Center for Molecular and Structural Biology and Department of Surgery. Medical University of South Carolina. 2 To whom requests for reprints should be addressed. at Medical University of South Carolina. Department of Surgery. Room 420 CSB, 171 Ashley Avenue, Charleston. SC 29425. Phone: (803) 792-3276: Fax: (803) 792-2048. observed 2-7 days after injection with polymer containing murine GM-CSF. The results of our studies show that this biocompatible system is capable of a sustained concomitant release of biologically active peptide and cytokine into the local microenvironment. These findings support further studies to validate a p-GlcNAc delivery system vehicle for a cytokinelTAA peptide-based cancer vaccine. INTRODUCTION CTLs that specifically recognize and respond to both autologous and allogenic tumor cells in a MHC-restricted manner can be isolated in vitro ( 1-3). These T cells are capable of mediating antitumor responses in select patients (4-6) and provide evidence that manipulation of the human immune systern can result in the regression ofcancer. Recent progress in our knowledge of antigen processing/presentation and techniques for the isolation of peptides presented in a MHC-restricted fashion has led to the identification of TAAs3 recognized by T lymphocytes (7-13). Several of the genes encoding for TAAs have been cloned, their class I MHC-restricted epitopes described, and in some cases the functional specificity of T-cell receptor heterodimer recognition characterized ( 14-I 7). These findings support the concept that CTL-mediated tumor regression iii t’lt’() can be explained by T-cell recognition of specific 9or 10-amino acid peptides bound to MHC class I molecules presented on the surface of cancer cells and have been a major step forward in cancer vaccine development (18). Recent work. performed in murine models using TAA epitopes. provides in vito evidence that a cancer therapy approach based on relevant TAA peptides may be effective and justify development and design of peptide-based cancer saccines ( I 9 -2 1 ). An attractive aspect of this approach is the ability to present biologically relevant levels of TAA epitopes away from the potentially immunosuppressive influences that may exist at the tumor site. Additionally. several cytokines including GM-CSF, IL-l2, and IFN-’y have been documented to elicit an antitumor effect when present at the local tumor site (22-26). In the context of TAA peptide vaccine, these cytokines could be administered concomitantly with peptide to create a local microenvironment which would provide a potentially more effective milieu for antigen presentation. Our objective, therefore, has been to develop a vaccine which uses TAA-derived peptides in combination with cyto3 The abbreviations used are: TAA. tumor-associated antigen: GM-CSF. granulocyte-macrophage colony-stimulating factor: IL. interleukin: APC, antigen-presenting cell: p-GIcNAc. poly-N-acetyl glucosamine: mGM-CSF, niurine GM-CSF: HPLC. high-performance liquid chroii,atography: TFA, trifluoroacetic acid: rhGM-CSF, recombinant human GM-CSF. on May 3, 2017. © 1997 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from 868 Characterization of a Sustained-Release Delivery System kines svhich are potentially capable of enhancing in i’ivo APC uptake and presentation so as to achieve a more efiective tumorspecific CTL response. Optimal CTL activation may be dependent on mode of presentation. antigen localization and concentration, and overcoming anergizing mechanisms by the tumor ( 19. 21. 27). As such. both the duration and availability of peptide for uptake by locally recruited APCs are significant variables in this approach. Sustained levels of biologically available peptide in an enhanced antigen presentation environment are potentially desirable for an augmentation in immune response ( 19, 28. 29). The vehicle. therefore, used for peptide vaccine administration may be of critical importance. Several possible modalities for peptide vaccination have been described, including Freund’s adjuvant (19). encapsulation of peptide in liposomes (30), or modification as lipopeptides (3 1 ). A newer approach. however, is now possible with the recent availability of a highly purified. biocompatible polysaccharide matrix: p-GlcNAc. The goal of this study was to characterize the p-G1cNAc-based matrix as a sustained-release yehide for a cytokine/TAA peptide-based vaccine. MATERIALS AND METHODS p-GlcNAc Polymer. p-GlcNAc was provided as either a sterile fully deacetylated lyophilized disc able to fit in the bottom of a standard 24-well plate or as a hydrated 2.5% sterile gel for injection. p-GIcNAc is a highly purified polysaccharide polymer produced by a fermentation process and isolated from controlled aseptic cultures grown on a defined culture medium under conditions that are consistent with Good Laboratory Practice (GLP) guidelines (Marine Polymer Technologies. Danvers. MA). It has passed the Food and Drug Administration biocompatibility testing including USP class VI sensitization assay. irritation test, systemic toxicity, cytotoxicity. mutagenicity. subchronic toxicity. and pyrogenicity. For in vitro testing. rhGMCSF was reconstituted in HBSS at a concentration of 200 p g/ml. Sterile p-GIcNAc polymer was provided in a fully deacetylated form (to minimize hydrophobicity) with 0.5 ml of reconstituted GM-CSF dissolved (for a total of 100 ig ofGM-CSF) into 1.5 ml of the p-GlcNAc polymer prior to lyophilization. MART127 55) peptide (0.1 ml of peptide/DMSO solution at 1280 p.g/ml for a total of’ I 28 ig of MARTI peptide) was solubilized into postlyophilization porous matrix. In vito studies used mGM-CSF (Genzyme Diagnostics, Cambridge, MA: 200 1ii delivered per animal dissolved into 0.5 ml of a 2.5% hydrated p-GlcNAc gel for injection (with a final concentration of 0.2 jig/mI GM-CSF). Cell Lines. Jurkat 22 (JRT22) cells were maintained in culture with RPMI 1640 and 10% FBS at 37#{176}C. These cells have been previously characterized for class I MHC-restricted recognition of MART-l(27 55) peptide ( l7. T2 cells ( 17) were maintamed in RPMI l64() with 10% FCS. M-07e GM-CSF-dependent cells (32) were maintained in RPMI 1640 and 10% FCS with 1(8) units/mI GM-CSF. Peptide Synthesis. Peptides (kindly provided by Dr. Y. Kawakami, NIH, Bethesda. MD) were synthesized with a solidphase method using a multiple peptide synthesizer (Gilson Co., Inc., Worthington. OH) and purified by HPLC on a C4 column (VYDAC, Hesperia, CA) with 0.05% TFA/water-acetonitrile. The MART-l(27 5) peptide (AAGIGILTV) is located in a hydrophobic putative transmembrane domain in MARTI ( 14). Detection of MART-i Peptide by HPLC. HPLC-rnass spectrometry data were obtained using the Medical University of South Carolina Mass Spectrometry Research Resource Facility (courtesy of Drs. Kuruppu Dharmasiri and Daniel R. Knapp). Briefly, supernatants from one set of 24-h peptide release assay samples stored at -4#{176}Cwere thawed at room temperature and analyzed by HPLC-electrospray tiiass spectrometry using a Finnigan LCQ instrument. RPM! 1640 with I .0 jig/mI MART-I peptide and RPMI 1640 alone were used as positive and negative controls, respectively. HPLC was performed using a 2 mm X 10-cm C 5 column with a gradient of acetonitrile/0.05% TFA in 0.05% aqueous TFA at a flow rate of 200 il/min. Peptide Release Assay. Peptide or peptide/mGM-CSF containing lyophilized p-GIcNAc matrices were incubated in I .0 ml ofRPMI 1640 in 24-well plates at 37#{176}C, with the p-G1cNAc supernatant changed to fresh media every 24 h. Twenty-four-h samples were stored at -4#{176}Cuntil further testing. T2 cells were then preincubated for 2 h with either peptide ( I p.g/ml in RPMI 1640) or p-GlcNAc supernatant. After incubation, the cells were washed twice with PBS and then added to effector JRT22 cells at a 1:1 ratio for a total of I X l0 ’ cells/mI in a 24-well plate. The ability of the peptide-pulsed T2 cells to stimulate IL-2 release from JRT22 cells was then assessed using the ELISA (Endogen. Inc., Cambridge. MA). Internal controls were performed for each assay using ELISA standards for IL-2 detection, and positive controls containing RPMI 1640 with 10% FCS and either 1 rig, 10 ng, 101) pg. or 1 pg of MART-I peptide/mI. A standard curve was then generated correlating units of IL-2 released by the JRT22 cells to the amount of MART-l peptide present. The correlation of IL-2 produced in response to MART-I peptide was approximately lO(X)-1200 pg/mI of IL-2 per 1 ig/ml MART-I peptide present in the supernatant. This assay was modified to assess local bioavailability of peptide release from the matrix by either placing T2 cells directly onto the matrix in I ml of RPM! 1640 with 10% FCS in a 24-well plate for 12 h or coincubated T2 cells with the supernatantcontaining peptide released from the matrix into culture supernatant at 37#{176}C. The cells were then harvested, washed twice, and then added to the effector cells as described previously. GM-CSF Release Assays. GM-CSF or peptide/GMCSF containing lyophilized p-G1cNAc matrices were incubated in 1.0 ml of RPMI 1640 in 24-well plates at 37#{176}C.with the p-G1cNAc supernatant changed to fresh media every 24 h. Twenty-four-h samples were stored at 30#{176}C until further testing. The presence of rhGM-CSF was then assessed by proliferation of M-07e cells in response to GM-CSF and was verified using the ELISA (Endogen. Inc.). Briefly, for the proliferation assay, 7.5 x l0 M-07e cells [which are derived from a human megakaryoblastic leukemia cell line and are dependent on either human GM-CSF or human IL-3 for growth and survival (32)] were seeded per well in a 24-well plate and grown for 3 days in 1.5 ml of common medium containing either 0. 1, 10, or 50 units/ml of human recombinant GM-CSF (Genetics Institute, Cambridge. MA) or the pGlcNAc test supernatants. At day 3, M-07e cells were counted and, by comparing these cell numbers with those of the standard curve. equivalent units of GM-CSF produced in 24 h were derived. Comparable data were obtained on May 3, 2017. © 1997 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Days Clinical Cancer Research 869