Glucan-Based Macrophage-Targeted Adjuvant - American Chemical

Chapter 6

A New β-Glucan-Based Macrophage-Targeted Adjuvant G. R. Ostroff, D. D. Easson, Jr., and S. Jamas

Downloaded by UNIV OF PITTSBURGH on July 1, 2013 | http://pubs.acs.org Publication Date: August 15, 1991 | doi: 10.1021/bk-1991-0469.ch006

Alpha-Beta Technology, Inc., Two Biotech Park, Worcester, MA 01605

Through polysaccharide engineering, the composition, structure and porosity of novel glucan carbohydrate microcapsules (Adjuvax) can be controlled to yield sophisticated targeted antigen or drug delivery vehicles. The unique β(1-3)/β(1-6) - linked glucan structure of Adjuvax allows the targeting of antigens to macrophages or neutrophils through interactions with the β-glucan receptor found uniquely on the surface of these cell types. Adjuvax- ligand complexes are formed by physically entrapping or crosslinking the ligand within the β-glucan microcapsule. Sustained release rate is dependent upon microcapsule porosity, ligand molecular weight, degree of Adjuvax - ligand crosslinking, and the rate of carbohydrate biodegradation. Microcapsule porosity is controlled by varying the ratio of the β(1-3)/β(1-6) linkages in the carbohydrate molecule through genetic and process manipulations. The in vitro sustained release rate of entrapped proteins ranged from 1 hour for a 12 kD protein to 6 hours for a 150 kD protein. Adjuvax - ligand crosslinking increased the sustained release rate of the 12 kD protein to greater than 24 hours. Adjuvax coadministered with bovine serum albumin or crosslinked to a series of peptide antigens increased mouse antibody titers, 1,000 fold over antigen only controls. Adjuvax stimulation of antibody titer was equivalent to Complete Freund's Adjuvant (CFA) without the toxicity and histopathology associated with use of CFA. These results suggest that the use of this safe, non– antigenic, newly defined carbohydrate adjuvant may prove useful in new generation vaccines.

Adjuvax is a non-antigenic, polysaccharide vaccine adjuvant and drug delivery vehicle designed to deliver a broad spectrum of antigens or drugs to the macrophage cell surface. The active component of Adjuvax is a branched β-glucan polymer of 0097-6156/91/0469-0052$06.00/0 © 1991 American Chemical Society

In Polymeric Drugs and Drug Delivery Systems; Dunn, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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defined structure, known as P G G glucan, which has been engineered for optimal biologic activity (1). The P G G glucan molecule is formulated as a highly purified carbohydrate microcapsule combining three key adjuvant properties: • macrophage targeting of antigens • macrophage activation • sustained release of antigens The P G G glucan molecules bind to specific β-glucan receptors on macrophages and neutrophilic phagocytes (2,3,4,) and activate a cascade of immunologic responses (2 J,6,7,) which promote the production of higher titers of serum antibodies to specific antigens (8,9,10). This report demonstrates the feasibility for developing Adjuvax as a vaccine adjuvant. EXPERIMENTAL Materials. Microspherical P G G glucan (Adjuvax, Alpha-Beta Technology, Worcester, M A ) was prepared from Saccharomyces cereviseae strain R4 cells (11). Zymosan, cytochrome c (cyt c), bovine serum albumin ( B S A ) , yeast alcohol dehydrogenase (ADH), Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA) were purchased from Sigma Chemical Co. (St. Louis, MO). Measurement of Adjuvax Phagocytosis. Adjuvax microcapsules were assayed at three doses for their relative capacity to trigger phagocytosis after 30 minutes of exposure to adherent human monocytes (2). Adjuvax concentrations of SxKfi/tnL to 6 x l 0 / m L corresponding to particle-to-cell ratios of approximately 5 to 50 were used. The number of Adjuvax particles ingested by at least 300 monocytes was determined by direct visual observation with a 1000X light microscope. The results are expressed as the percentage of monocytes ingesting >3 particles. 7

Sustained Release Studies. The ligand proteins B S A , cyt c, and A D H dissolved at 10 mg/mL in water, were loaded into Adjuvax microcapsules by hydration (0.07 mL protein/mg Adjuvax) for four hours at room temperature. A control, run in parallel contained no Adjuvax. The loaded microcapsules were dried by lyophilization and ground to a fine powder. Protein release was monitored by hydrating the powder with saline and agitating at 37 C. Samples were removed at regular intervals, centrifuged to remove the Adjuvax microcapsules, and soluble supernatants containing the released protein were assayed (absorbance, 280 nm). Percent protein retained was calculated by dividing the absorbance (280 nm) of each time point by the absorbance (280 nm) of the protein only control. e

Adjuvax - Ligand Crosslinking. Whole glucan particles containing crosslinked cyt c were prepared by first reacting 5 mg cyt c with 2.5 mg of the heterobifunctional crosslinking reagent sulfosuccinimidyl 6- (4 -azido-2 -nitrophenylamino-hexanoate (sulfoSANPAH, Pierce Chemical Co., Rockford, IL) in 1 m L of 10 m M sodium phosphate buffer p H 7.4 for 16 hours at 25°C in the dark. One m L of the sulfoSANPAH-cyt c conjugate was swelled into the P G G glucan microcapsule by ,

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mixing with 150 mg of dry Adjuvax, followed by incubation at 25C for 2 hours in the dark. The sulfoSANPAH-cyt c conjugate was crosslinked to the Adjuvax by exposure to bright light. The unre acted sulfoS A N P A H , cyt c and sulfoS ANPAH-cyt c conjugate were removed by washing with saline. The crosslinked Adjuvax sulfoS ANPAH-cyt c conjugate was dried by lyophilization and stored at 4°C. In vivo Adjuvant Characterization of Adjuvax. The adjuvant effect of Adjuvax in mice was determined by measuring the antibody production to B S A and a 55 amino acid (P55) antigen coupled to B S A in two different Adjuvax formulations. One formulation was prepared by the physical entrapment of the antigen within the Adjuvax microcapsules. The second formulation contained the antigen crosslinked to the Adjuvax microcapsules. A standard adjuvant control contained antigen mixed with C F A (primary immunization) or IFA (secondary immunization). For each antigen, three groups of ten mice were immunized subcutaneously on day 1 with 0.2 m L of the entrapped formulation, 0.2 m L of the crosslinked formulation or 0.2 mL of a C F A - antigen emulsion. A secondary immunization was administered on day 14 with 0.2 mL of the same Adjuvax formulations or 0.2 mL of an IFA-antigen emulsion. On day 27, serum was collected and analyzed for antiP55 antibody or anti-BS A antibody by ELISA. RESULTS A d j u v a x A c t i v a t i o n of Phagocytosis by H u m a n M o n o c y t e s . The P G G carbohydrate structure of Adjuvax was optimized using in vitro assays for its ability to interact with the β-glucan receptor of human monocytes and neutrophils. This receptor is identified with the phagocytic receptor for particulate activators of the alternate complement pathway (2). Figure 1 compares the engineered Adjuvax microcapsule to zymosan, the typical particulate activator of phagocytosis. These results demonstrate the increased potency of Adjuvax over standard glucans (zymosan) to be targeted to, bound and phagocytosed by macrophages. Sustained Antigen and D r u g Release from Adjuvax. Physical entrapment of certain ligands (antigen or drug) within the hollow Adjuvax microcapsules represents the most basic delivery mode. The ligand can be trapped within the microcapsules by a variety of techniques tailored specifically to the physical properties of the molecule. The sustained release of ligand from Adjuvax microcapsules under physiologic conditions is monitored in vitro and is dependent upon natural diffusion. In vivo release is additionally dependent on biodégradation of the Adjuvax P G G matrix. Both these mechanisms are controlled by the primary structure of the engineered P G G molecules and are a function of the molecular weight of the entrapped ligand. The release rate of proteins that have been physically entrapped within Adjuvax microcapsules can be used as a preliminary indication of the sustained release potential of this system. Three proteins of different molecular weight (cyt c = 14 k D ; B S A = 67kD and A D H = 150 kD) were entrapped within Adjuvax


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microcapsules. Figure 2 shows the effect of antigen molecular weight on sustained release rate. The time to release 50% of the protein ( T ) from the Adjuvax microcapsules increased as the molecular weight of the antigen increased (31 minutes for cyt c; 80 minutes for BSA; and 200 minutes for ADH). These sustained release results show that entrapped low molecular weight ligands are rapidly released from Adjuvax microcapsules. To extend the application of Adjuvax microcapsules to low molecular weight molecules, chemical attachment techniques have been developed which do not interfere with microcapsule targeting or ligand functionality. Proteins and peptides were covalently crosslinked to Adjuvax microcapsules through amino groups using the heterobifunctional crosslinking agent sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamine) hexonate. Figure 3 compares the release rates of ligand from a crosslinked Adjuvax χ cyt-c conjugate and a physically entrapped Adjuvax:cyt-c formulation. Crosslinking significantly increased the retention time of cyt-c from 31 minutes to greater than 144 hours. Release of crosslinked ligands was dependent upon ligand or polysaccharide degradation.


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In Vivo Adjuvant Effect of Adjuvax in Mice. Adjuvax was compared to C F A for its ability to stimulate antibody production to protein (BSA) and peptide (a 55 amino acid residue oligopeptide) antigens in two different Adjuvax formulations. One formulation was prepared by physical entrapment of the antigens within the Adjuvax microcapsules, the second formulation contained the antigens crosslinked to the Adjuvax microcapsules. The B S A formulation contained an Adjuvax/antigen ratio of 100 mg Adjuvax/10 mg antigen per dose (0.2mL), the peptide formulation contained an Adjuvax/antigen ratio of 100 mg Adjuvax/50 mg antigen per dose (0.2mL). Controls containing the same amounts of antigen were mixed with C F A , a standard adjuvant. Figures 4 and 5 summarize the antibody titer results. These results show that the covalently crosslinked Adjuvax formulations were superior to the physically entrapped Adjuvax formulations. In addition, the Adjuvax crosslinked formulations were as effective in stimulating antibody titers as Freund's Adjuvant. Furthermore, animals immunized with Adjuvax did not experience local inflammatory reactions or granuloma formation which was observed with all CFA/IFA immunized animals. DISCUSSION We have established that Adjuvax is effectively targeted to the macrophage via the β-glucan receptor. Adjuvax has a novel, engineered glucan structure (PGG glucan) that has a higher avidity for the β-glucan receptor and is thus more potent than previousl described glucans. In addition, Adjuvax has been recently shown to effectively activate monocytes and neutrophils resulting in enhanced cytokine (ILl a , EL-Ιβ, IL-6 and TNF ) and colony stimulating factor production (GM-CSF) (72). Thus, the strong adjuvant properties of Adjuvax can be attributed to the unique targeted antigen delivery and macrophage activation features of the P G G glucan matrix.


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Figure 1. Stimulation of phagocytic capacity in human monocytes with Adjuvax.

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Time (minutes) Figure 2. Effect of ligand molecular weight on the sustained release rate from Adjuvax.



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Figure 4. Comparison of Freund's Adjuvant to Adjuvax formulations in stimulating antibody response to P55 oligopeptide antigen. Relative antibody titers between adjuvant groups at day 27 were determined by measuring the absorbance at 450 nm of a 1:500 dilution of anti-P55 immune sera by ELIS A .


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Figure 5. Comparison of Freund's Adjuvant to Adjuvax formulations in stimulating antibody response to B S A . Relative antibody titers between adjuvant groups at day 27 were determined by measuring the absorbance at 450 nm of a 1:500 dilution of anti-BSA immune sera by ELIS A.

In addition, Adjuvax protects an antigen from degradation and rapid clearance from circulation through entrapment and sustained release from P G G glucan microcapsules. Diffusional release of entrapped proteins and peptides from the Adjuvax microcapsule was shown to be dependent upon molecular branching within the capsule matrix and protein/peptide molecular weight. It was also shown that covalent crosslinking of peptides or proteins to the Adjuvax decreases the release rate to the extent that release becomes dependent upon in vivo biodégradation of the crosslinking bonds rather than diffusion. In vivo adjuvant studies with peptide and protein loaded Adjuvax, crosslinked Adjuvax-antigen conjugates, or the standard C F A adjuvant demonstrate that the three formulations stimulate comparable antibody responses in mice. The trends observed showed that Adjuvax-antigen loaded preparations yielded weaker antibody responses than C F A , while Adjuvax-antigen crosslinked preparations yielded stronger antibody responses than C F A , although there were no statistically significant differences between the C F A and Adjuvax groups. These results support the importance of the targeted antigen delivery features of Adjuvax which require the prolonged association between the antigen and the Adjuvax microcapsule to obtain the greatest stimulation of antibody titers. It was also shown that, unlike C F A and many other adjuvants, Adjuvax itself is nonantigenic (data not shown) allowing repeated use of Adjuvax-based drug or vaccine formulations with minimal side-effects. In addition, unlike many other oil or detergent based adjuvants, the saline based Adjuvax does not cause local


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pathologies,such as granulomas at the injection s i t e ,and the effectiveness of Adjuvax to stimulate antibody titers also has been established in a wide variety of animal species ranging from mice to horses (data not shown). These combined efficacy and safety features of Adjuvax support its further evaluation and development for use as an adjuvant or drug delivery vehicle for human and veterinary applications. Literature Cited


1. Jamas S,Easson D D ,Ostroff G R ,Onderdonk AB. 1990. ACS Preprint. 2. Czop,J. K.; Austen,K. F. J. Immunol. 1985, 134, 2588. 3. Czop,J.; Fearon D. T.; Austen,K.F. J. Immunol. 1978, 120, 1132. 4. Czop, J.K.; Puglisi, A.V.; Miorandi, D.M.; Austen, K.F. J. Immunol. 1988, 141, 3170. 5. Williams,J.D.; Czop,J.K.; Austen,K.F. J. Immunol. 1984, 132, 3034. 6. Czop,J.K.; Austen,K.F. Proc. Natl. Acad. Sci. USA 1985, 82, 2751. 7. Janusz,M.J.; Austen,K.F.; Czop,J.K. J. Immunol. 1987, 138,3897. 8. Benach,J.L.; Habicht,G.S.; Holbrook,T.W.; Cook,J.A. Infect. Immun. 1982, 35, 947. 9. Cook,J.A.; Holbrook,T.W.; Dougherty,W.J. Infect. Immun. 1982, 37, 1261. 10. Holbrook,T.W.; Cook,J.A.; Parker,B.W. Infec. Immun. 1981, 32, 542. 11. Ostroff, G.R.; Easson,D.D.; Jamas,S. 198th ACS National Meeting. September 1989. 12. Symposium: The Beta-Glucan Receptor and Response to PGG,International Congress for Infectious Diseases,Montreal Canada,July 1990. Received March 19, 1991


Glucan-Based Macrophage-Targeted Adjuvant - American Chemical

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