The present invention provides an effective method for the transfection of dendritic cells by non-viral methods. The present invention provides this benefit by incubating dendritic cells and a specified transfection agent. The transfection agent comprises a polynucleotide and microparticles, with the microparticles being comprised of biodegradable polymer and cationic detergent. The dendritic cells and transfection agent are incubated for a time sufficient to transfect the dendritic cells with the polynucleotide.

Description of the Invention

FIELD OF THE INVENTION

The present invention relates to compositions and methods of providing dendritic cells for immunotherapy in connection with, for example, viruses or tumors. In particular, the invention relates to methods for generating antigen presenting dendritic cells by transfection, allowing for, e.g., the activation and expansion of large numbers of viral- or tumor-antigen-specific T cells for use in adoptive cellular immunotherapy against viruses and tumors.

BACKGROUND OF THE INVENTION

The generation of an immune response involves the sensitization of helper (CD4+) (T.sub.H) and cytotoxic (CD8+) (CTL) T cell subsets through their interaction with antigen presenting cells. Antigen presenting cells express major histocompatibility (MHC)-class I or class II molecules associated with antigenic fragments (i.e., specific amino acid sequences derived from an antigen which bind to MHC I and MHC II for presentation on the cell surface). The MHC in humans is also referred to as the HLA (human leukocyte antigen) complex. The sensitized CD4+ T cells produce lymphokines that participate in the activation of B cells as well as various T cell subsets. The sensitized CD8+ T cells increase in numbers in response to lymphokines and act to destroy cells that express the specific antigenic fragments associated with matching MHC-encoded class I molecules. In the course of a tumor or viral infection, cytotoxic T cells eradicate cells expressing tumor or virus associated antigens.

Dendritic cells (DCs) are thought to be the most potent antigen presenting cells of the immune system (reviewed in Steinman, R. M. 1991. The dendritic cells system and its role in immunogenicity. Ann. Rev. Immunol. 9:271; Banchereau, J. B. and R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature. 392:245). Given their broad spectrum of roles in initiating the immune response by internalizing and processing antigens, migrating to lymphoid organs, secreting cytokines, and expressing co-stimulatory molecules required for lymphocyte signaling, it is no surprise that dendritic cells are logical targets for clinical use (Banchereau, J. B. and R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature. 392:245). By targeting antigens into dendritic cells in vivo or exposing dendritic cells to antigen ex vivo, it may be possible to enhance the immunogenicity of vaccines by eliciting helper and cytotoxic T cells, antibodies, and IL-12 for prophylactic applications, or induce T cell mediated anti-tumor responses for cancer immunotherapy. Akbari, et al. have suggested that transfection and activation of dendritic cells are key events for immunity following DNA vaccination by scarification of the ear skin in mouse models (O. Akbari, N. P., S. Garcia, R. Tascon, D. Lowrie, and B. Stockinger. 1999. DNA vaccination: transfection and activation of dendritic cells as key events for immunity. J. Exp. Med. 189:169). Anti-tumor CTL activity and protection against lethal tumor challenge in mouse models have been demonstrated using cytokine-driven bone-marrow-derived dendritic cells (BMDCs) pulsed with tumor-associated peptides (J. I. Mayordomo, T. Z., W. J. Storkus. 1995. Bone marrow-derived dendritic cells pulsed with synthetic tumour peptides elicit protective and therapeutic antitumour immunity. Nature Med. 1:1297), and whole tumor lysates (R. C. Fields, K. S., and J. J. Mule'. 1998. Murine dendritic cells pulsed with whole tumor lysates mediate potent antitumor immune responses in vitro and in vivo. Proc. Natl. Acad. Sci. USA 95:9482) transferred by the subcutaneous route.

In vitro generation of dendritic cells has been optimized sufficiently so that genetic immunotherapy based on passive transfer of dendritic cells has become an attractive target for development (N. Romani, S. G., D. Brang. 1994. Proliferating dendritic cell progenitors in human blood. J. Exp. Med. 180:83). However, in vitro transfection efficiency of dendritic cells by non-viral methods has been extremely poor (J. F. Arthur, L. H. B., M. D. Roth, L. A. Bui, S. M. Kiertscher, R. Lau, S. Dubinett, J. Glaspy, W. H. McBride, and J. S. Economou. 1997. A comparison of gene transfer methods in human dendritic cells. Cancer Gene Ther. 4:17) and has limited progress toward effective dendritic-cell-based immunotherapy. While progress has been made by the use of electroporation, the efficiency of transfection is extremely low and results in substantial loss of cell viability (V. F. I. Van Tendeloo, H.-W. S., F. Lardon, GLEE Vanham, G. Nijs, M. Lenjou, L. Hendriks, C. Van Broeckhoven, A. Moulijn, I. Rodrigus, P. Verdonk, D. R. Van Bockstaele, and Z. N. Berneman. 1988. Nonviral transfection of distinct types of human dendritic cells: high efficiency gene transfer by electroporation into hematopoietic progenitor- but not monocyte-derived dendritic cells. Gene Ther. 5:700). To date, no purely chemical method has been shown to be effective.

Particulate carriers have been used in order to achieve controlled, parenteral delivery of therapeutic compounds. Such carriers are designed to maintain the active agent in the delivery system for an extended period of time. Examples of particulate carriers include those derived from polymethyl methacrylate polymers, as well as microparticles derived from poly(lactides) (see, e.g., U.S. Pat. No. 3,773,919), poly(lactide-co-glycolides), known as PLG (see, e.g., U.S. Pat. No. 4,767,628) and polyethylene glycol, known as PEG (see, e.g., U.S. Pat. No. 5,648,095). Polymethyl methacrylate polymers are nondegradable while PLG particles biodegrade by random nonenzymatic hydrolysis of ester bonds to lactic and glycolic acids, which are excreted along normal metabolic pathways.

For example, U.S. Pat. No. 5,648,095 describes the use of microspheres with encapsulated pharmaceuticals as drug delivery systems for nasal, oral, pulmonary and oral delivery. Slow-release formulations containing various polypeptide growth factors have also been described. See, e.g., International Publication No. WO 94/12158, U.S. Pat. No. 5,134,122 and International Publication No. WO 96/37216.

Particulate carriers have also been used with adsorbed or entrapped antigens in attempts to elicit adequate immune responses. Such carriers present multiple copies of a selected antigen to the immune system and promote trapping and retention of antigens in local lymph nodes. The particles can be phagocytosed by macrophages and can enhance antigen presentation through cytokine release. For example, commonly owned, co-pending application Ser. No. 09/015,652, filed Jan. 29, 1998, describes the use of antigen-adsorbed and antigen-encapsulated microparticles to stimulate cell-mediated immunological responses, as well as methods of making the microparticles.

In commonly owned provisional Patent Application 60/036,316, for example, a method of forming microparticles is disclosed which comprises combining a polymer with an organic solvent, then adding an emulsion stabilizer, such as polyvinyl alcohol (PVA), then evaporating the organic solvent, thereby forming microparticles. The surface of the microparticles comprises the polymer and the stabilizer. Polynucleotides such as DNA, polypeptides, and antigens may then be adsorbed on those surfaces. See also PCT US99/17308.

Commonly owned Provisional Application No. 60/146,391 discloses a method of forming microparticles with adsorbent surfaces that are capable of adsorbing a variety of macromolecules including polynucleotides. In one embodiment, the microparticles are comprised of both a polymer and a detergent. The microparticles are derived from a polymer, such as a poly(.alpha.-hydroxy acid), preferably, a poly(D,L-lactide-co-glycolide), a polyhydroxy butyric acid, a polycaprolactone, a polyorthoester, a polyanhydride, a polycyanoacrylate, and the like, and are formed with detergents, such as cationic, anionic, or nonionic detergents, which detergents may be used in combination. Cationic detergents disclosed are cetrimide (CTAB), benzalkonium chloride, DDA (dimethyl dioctodecyl ammonium bromide), DOTAP, and the like. It is noted that these microparticles yield improved adsorption of viral antigens, and provide for superior immune responses, as compared to microparticles formed by a process using only PVA.

Dendritic cells can capture antigen at peripheral sites via macropinocytosis using membrane ruffling, or may also internalize antigen by receptor-mediated processes involving Fc.gamma.III, the mannose receptor, or the C-type lectin DEC-205 (reviewed in Lanzavecchia, A. 1996. Mechanisms of antigen uptake for presentation. Curr. Op. Immunol. 8:348). Thus, dendritic cells may be targeted by the capture of larger (>250 nm) particulate antigens by phagocytosis. Biodegradable polymer microspheres such as poly-lactide-co-glycolide (PLG) are readily internalized by phagocytic cells up to a diameter of 5 .mu.m (Ikada, Y. T. et al. 1990. Phagocytosis of polymer microspheres by macrophages. Adv. Polymer. Sci. 94:107) and have been utilized as carriers for drug delivery systems.

Recently, Newman, et al. reported cytoplasmic delivery of Texas red labeled dextran encapsulated in PLGA microspheres following phagocytosis in mouse peritoneal macrophages (K. D. Newman, G. K., J. Miller, V. Chlumecky, J. Samuel. 1999. Cytoplasmic delivery of a fluorescent probe by poly(D,L lactic-co-glycolic acid) microspheres. In 1999 AAPS Annual Meeting Abstracts Online, vol. 1).

The application of synthetic biopolymers for nucleic acid delivery has proven advantageous by protecting DNA against nuclease degradation and increasing cellular uptake (C. Chavany, T. S.-B., T. Le Doan, F. Puisieux, P. Couvreur, and C. Helene. 1994. Adsorption of oligonucleotides onto polyisohexylcyanoacrylate nanoparticles protects them against nucleases and increases their cellular uptake. Pharm. Res. 11:1370).

Evidence for direct transfection of non professional antigen presenting cells mediated by PLG was recently reported by Ciftci and Su who found PLG microparticles containing a DNA:polycation complex provided controlled release of DNA and surfactant-enhanced uptake and gene expression in 293 and MCF-7 cells (K. Ciftci, J. S. 1999. DNA-PLGA microparticles: a promising delivery system for cancer gene therapy. In 1999 AAPS Annual Meeting Abstracts Online, vol. 1).

While polyalkylcyanoacrylate nanoparticles have been used to bind CTAB-oligonucleotide complexes to deliver antisense oligonucleotides to macrophage cell lines in vitro (C. Chavany, T. S.-B., T. Le Doan, F. Puisieux, P. Couvreur, and C. Helene. 1994. Adsorption of oligonucleotides onto polyisohexylcyanoacrylate nanoparticles protects them against nucleases and increases their cellular uptake. Pharm. Res. 11:1370; E. Fattal, C. V., I. Aynie, Y. Nakada, G. Lambert, C. Malvy, and P. Couvreur. 1998. Biodegradable polyalkylcyanoacrylate nanoparticles for the delivery of oligonucleotides. J. Controlled Release 53:137), these vehicles have not been shown to transfect dendritic cells with plasmids carrying recombinant genes.

Hence, there is a need in the art for an effective non-viral technique for the transfection of dendritic cells. While microparticle technology has been heretofore used for introduction of polynucleotides into cells, applicants are aware of no such technology having been used for the transfection of dendritic cells, which are notoriously resistant to transfection.

SUMMARY OF THE INVENTION

The present invention provides an effective method for the transfection of dendritic cells by non-viral methods. The present invention provides this benefit by incubating dendritic cells and a specified transfection agent. The transfection agent comprises polynucleotide and microparticles, with the microparticles being comprised of a biodegradable polymer and a cationic detergent. The dendritic cells and transfection agent are incubated for a time sufficient to transfect the dendritic cells with the polynucleotide.

For the transfecting agent, the cationic detergent preferably comprises CTAB or cetrimide, while the polymer preferable is a poly(.alpha.-hydroxy acid), for example, a poly(lactide), a copolymer of D,L-lactide and caprolactone, or a copolymer of D,L-lactide and glycolide or glycolic acid, such as poly(D,L-lactide-co-glycolide). In a further preferred embodiment, the polynucleotide is provided in the form of a plasmid. In still further preferred embodiments, the polynucleotide encodes an antigen associated with a virus, such as HIV, meningitis A, meningitis B or meningitis C, or a tumor.

The dendritic cells can originate from any available source, for example, the bone marrow or blood of a vertebrate subject, preferably a human subject. Dendritic cells can be cultured, for example, for about 5 to about 10 days prior to transfection, in the presence of appropriate growth factors, for example, GM-CSF.

The dendritic cells and transfecting agent are preferably incubated for about 24 hours under appropriate conditions.

In some embodiments of the present invention, an effective amount of the transfected dendritic cells of the present invention are administered to a vertebrate subject in need thereof. In other embodiments, T cells are first activated by the dendritic cells of the present invention and then administered to a vertebrate subject in need thereof. The dendritic cells and/or T cells may originate, for example, from the vertebrate subject or a healthy vertebrate subject MHC-matched to the vertebrate subject. The dendritic cells and or T cells may be administered parenterally to the vertebrate subject.

One advantage of the present invention is that polynucleotides can be efficiently internalized by dendritic cells.

Another advantage of the present invention is that gene expression can be effected within dendritic cells.

Yet another advantage of the present invention is that antigen can be processed and presented in connection with MHC molecules on the surface of dendritic cells.

Another advantage of the present invention is that polynucleotides can be rapidly internalized and expressed, with antigen presentation.

Still another advantage of the present invention is that the methods of the invention can be used, for example, in genetic immunotherapy or vaccination with relative safely. For instance, both cationic detergents, such as CTAB, and biodegradable polymers, such as PLG, have been utilized in biomedical applications. Moreover, the obvious safety concerns with the use of live viral vectors can be avoided (reviewed in Rock, S. R. et al. 1998. Fully mobilizing host defense: building better vaccines. Nature Biotech. 16:1025).

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, polymer chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.); Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Handbook of Surface and Colloidal Chemistry (Birdi, K. S., ed, CRC Press, 1997) and Seymour/Carraher's Polymer Chemistry (4th edition, Marcel Dekker Inc., 1996).

Formation of Microparticles

In the present invention, a polynucleotide comprising an antigen of interest is adsorbed upon microparticles formed from a polymer and a cationic detergent.

The adsorption of polynucleotides to the surface of the adsorbent microparticles occurs via any bonding-interaction mechanism, including, but not limited to, ionic bonding, hydrogen bonding, covalent bonding, Van der Waals bonding, and bonding through hydrophilic/hydrophobic interactions. Those of ordinary skill in the art may readily select cationic detergents appropriate for the invention. As noted above, known cationic detergents include, but are not limited to, cetyl trimethyl ammonium bromide (CTAB), cetrimide (a mixture consisting chiefly of tetradecyltrimethylammonium bromide, together with smaller amounts of dodecyltrimethylammonium bromide and CTAB), benzalkonium chloride, DDA (dimethyl dioctodecyl ammonium bromide), DOTAP, and the like. CTAB is particularly preferred. Microparticles manufactured with cationic detergents, such as CTAB, e.g., CTAB-PLG microparticles, readily adsorb negatively charged polynucleotides.

Biodegradable polymers for manufacturing microparticles for use with the present invention are readily commercially available from, e.g., Boehringer Ingelheim, Germany and Birmingham Polymers, Inc., Birmingham, Ala. For example, useful polymers for forming the microparticles herein include those derived from polyhydroxybutyric acid; polycaprolactone; polyorthoester; polyanhydride; as well as a poly(.alpha.-hydroxy acid), such as poly(L-lactide), poly(D,L-lactide) (both known as APLA" herein), poly(hydroxybutyrate), copolymers of D,L-lactide and glycolide, such as poly(D,L-lactide-co-glycolide) (designated as "PLG" or "PLGA" herein) or a copolymer of D,L-lactide and caprolactone. Particularly preferred polymers for use herein are PLA and PLG polymers. These polymers are available in a variety of molecular weights, and the appropriate molecular weight for a given use is readily determined by one of skill in the art. Thus, e.g., for PLA, a suitable molecular weight will be on the order of about 2000 to 5000. For PLG, suitable molecular weights will generally range from about 10,000 to about 200,000, preferably about 15,000 to about 150,000, and most preferably about 50,000 to about 100,000.

If a copolymer such as PLG is used to form the microparticles, a variety of lactide:glycolide ratios will find use herein. PLG copolymers with varying lactide:glycolide ratios and molecular weights are readily available commercially from a number of sources including from Boehringer Ingelheim, Germany and Birmingham Polymers, Inc., Birmingham, Ala. These polymers can also be synthesized by simple polycondensation of the lactic acid component using techniques well known in the art, such as described in Tabata et al., J. Biomed. Mater. Res. (1988) 22:837-858.

The polynucleotide/microparticles are prepared using any of several methods well known in the art. For example, double emulsion/solvent evaporation techniques, such as those described in U.S. Pat. No. 3,523,907 and Ogawa et al., Chem. Pharm. Bull. (1988) 36:1095-1103, can be used herein to make the microparticles.

A water-in-oil-in-water (w/o/w) solvent evaporation system can be used to form the microparticles, as described by O'Hagan et al., Vaccine (1993) 11:965-969 and Jeffery et al., Pharm. Res. (1993) 10:362. In this technique, the particular polymer is combined with an organic solvent, such as ethyl acetate, dimethylchloride (also called methylene chloride and dichloromethane), acetonitrile, acetone, chloroform, and the like. The polymer will be provided in about a 1-30%, preferably about a 2-15%, more preferably about a 3-10% and most preferably, about a 4% solution, in organic solvent. The polymer solution is emulsified using, e.g., a homogenizer. The emulsion is then optionally combined with a larger volume of an aqueous solution of an emulsion stabilizer such as polyvinyl alcohol (PVA), polyvinyl pyrrolidone, and a detergent, specifically a cationic detergent. The emulsion may be combined with more than one emulsion stabilizer and/or detergent, e.g., a combination of PVA and a cationic detergent. Certain polynucleotides may adsorb more readily to microparticles having a combination of stabilizers and/or detergents. Where an emulsion stabilizer is used, it is typically provided in about a 2-15% solution, more typically about a 4-10% solution. Generally, a weight-to-weight detergent to polymer ratio in the range of from about 0.00001:1 to about 0.1:1 will be used, more preferably from about 0.0001:1 to about 0.01:1, more preferably from about 0.001:1 to about 0.01:1, and even more preferably from about 0.005:1 to about 0.01:1. The mixture is then homogenized to produce a stable w/o/w double emulsion. Organic solvents are then evaporated.

The formulation parameters can be manipulated to allow the preparation of small microparticles on the order of 0.05 .mu.m (50 nm) to larger microparticles 50 .mu.m or even larger. See, e.g., Jeffery et al., Pharm. Res. (1993) 10:362-368; McGee et al., J. Microencap. (1996). For example, reduced agitation results in larger microparticles, as does an increase in internal phase volume. Small particles are produced by low aqueous phase volumes with high concentrations of emulsion stabilizers.

Microparticles can also be formed using spray-drying and coacervation as described in, e.g., Thomasin et al., J. Controlled Release (1996) 41:131; U.S. Pat. No. 2,800,457; Masters, K. (1976) Spray Drying 2nd Ed. Wiley, New York; air-suspension coating techniques, such as pan coating and Wurster coating, as described by Hall et al., (1980) The "Wurster Process" in Controlled Release Technologies: Methods, Theory, and Applications (A. F. Kydonieus, ed.), Vol. 2, pp. 133-154 CRC Press, Boca Raton, Fla. and Deasy, P. B., Crit. Rev. Ther. Drug Carrier Syst. (1988) S(2):99-139; and ionic gelation as described by, e.g., Lim et al., Science (1980) 210:908-910.

Particle size can be determined by, e.g., laser light scattering, using for example, a spectrometer incorporating a helium-neon laser. Generally, particle size is determined at room temperature and involves multiple analyses of the sample in question (e.g., 5-10 times) to yield an average value for the particle diameter. Particle size is also readily determined using scanning electron microscopy (SEM).

Following preparation, microparticles can be stored as is or freeze-dried for future use.

C. Isolation of Dendritic Cells

Dendritic cells are obtained from any tissue where they reside including non-lymphoid tissues such as the epidermis of the skin (Langerhans cells) and lymphoid tissues such as the spleen, bone marrow, lymph nodes and thymus as well as the circulatory system including blood (blood dendritic cells), for example peripheral blood and cord blood, and lymph (veiled cells).

For example, explants of mouse (Larsen et al., J. Exp. Med. 172:1483-1493 (1990)) or human skin (Richters et al., J. Invest. Dermatol. (1994)) placed in organ culture permit selective migration of dendritic cells into the medium surrounding the explant.

Recent studies have described methods for the isolation and expansion of human dendritic cells, including, from human peripheral blood. (Macatonia et al., 1991, Immunol. 74: 399-406; O'Doherty et al., 1993, J. Exp. Med. 178: 1067-1078 (isolation); and Markowicz et al., 1990, J. Clin. Invest. 85: 955-961; Romani et al., 1994, J. Exp. Med. 180: 83-93; Sallusto et al., 1994, J. Exp. Med. 179: 1109-1118; Berhard et al., 1995, J. Exp. Med. 55: 1099-1104 (expansion)).

Van Tendeloo et al., 1998, Gene Ther. 5: 700-707, discloses techniques for deriving dendritic cells (including Langerhans' cells) from CD34+ progenitor cells obtained from bone marrow and cord blood and from mononuclear cells from peripheral blood.

Dendritic cells may also be treated to induce maturation or activation, e.g., by culturing, preferably in the presence of a specific growth or stimulatory factor or factors. In the examples below, dendritic cells are modified by culturing with GM-CSF.

Additional techniques relating to the preparation of dendritic cells can be found, for example, in U.S. Pat. Nos. 5,788,963, 5,962,318, and 5,851,756, the disclosures of which are herein incorporated by reference.

According to a preferred embodiment of the invention, dendritic cells are obtained from a patient to be treated. The dendritic cells are used to activate T cells of the patient, either in vitro or in vivo, for immunotherapy

According to an alternate embodiment, dendritic cells are obtained from a healthy individual. The relevant HLA antigens (both class I and II, e.g., HLA-A, B, C and DR), for example, on the individual's peripheral blood mononuclear cells (PBMC's), are identified and dendritic cells that match the patient, in terms of HLA antigens, are isolated and expanded as described above. For example, in certain instances, a late stage cancer patient who has been treated with radiation and/or chemotherapy agents is not able to provide sufficient or efficient dendritic cells. Thus, dendritic cells from healthy HLA-matched individuals, such as siblings, can be obtained and expanded using any of the methods described above.

D. Antigens

Selected antigens that may be expressed include one or more selected antigens of a vertebrate infectious agent or cancer and can correspond to either structural or non-structural proteins. The invention herein described can provide for association of such antigens with MHC molecules at the surface of dendritic cells such that an immune response to the antigen of interest can be mounted.

For example, the present invention is useful for stimulating an immune response against a wide variety of antigens from the herpes virus family, including proteins derived from herpes simplex virus (HSV) types 1 and 2, such as HSV-1 and HSV-2 glycoproteins gB, gD and gH; antigens derived from varicella zoster virus (VZV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) including CMV gB and gH; and antigens derived from other human herpesviruses such as HHV6 and HHV7. (See, e.g. Chee et al., Cytomegaloviruses (J. K. McDougall, ed., Springer-Verlag 1990) pp. 125-169, for a review of the protein coding content of cytomegalovirus; McGeoch et al., J. Gen. Virol. (1988) 69:1531-1574, for a discussion of the various HSV-1 encoded proteins; U.S. Pat. No. 5,171,568 for a discussion of HSV-1 and HSV-2 gB and gD proteins and the genes encoding therefor; Baer et al., Nature (1984) 310:207-211, for the identification of protein coding sequences in an EBV genome; and Davison and Scott, J. Gen. Virol. (1986) 67:1759-1816, for a review of VZV.)

Antigens from the hepatitis family of viruses, including hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), the delta hepatitis virus (HDV), hepatitis E virus (HEV) and hepatitis G virus (HGV), can also be conveniently used in the techniques described herein. By way of example, the viral genomic sequence of HCV is known, as are methods for obtaining the sequence. See, e.g., International Publication Nos. WO 89/04669; WO 90/11089; and WO 90/14436. The HCV genome encodes several viral proteins, including E1 (also known as E) and E2 (also known as E2/NSI) and an N-terminal nucleocapsid protein (termed "core") (see, Houghton et al., Hepatology (1991) 14:381-388, for a discussion of HCV proteins, including E1 and E2). Each of these proteins, as well as antigenic fragments thereof, will find use in the present composition and methods.

Similarly, the sequence for the .delta.-antigen from HDV is known (see, e.g., U.S. Pat. No. 5,378,814) and this antigen can also be conveniently used in the present composition and methods. Additionally, antigens derived from HBV, such as the core antigen, the surface antigen, sAg, as well as the presurface sequences, pre-S1 and pre-S2 (formerly called pre-S), as well as combinations of the above, such as sAg/pre-S1, sAg/pre-S2, sAg/pre-S1/pre-S2, and pre-S1/pre-S2, will find use herein. See, e.g., "HBV Vaccines--from the laboratory to license: a case study" in Mackett, M. and Williamson, J. D., Human Vaccines and Vaccination, pp. 159-176, for a discussion of HBV structure; and U.S. Pat. Nos. 4,722,840, 5,098,704, 5,324,513, incorporated herein by reference in their entireties; Beames et al., J. Virol. (1995) 69:6833-6838, Birnbaum et al., J. Virol. (1990) 64:3319-3330; and Zhou et al., J. Virol. (1991) 65:5457-5464.

Antigens derived from other viruses will also find use in the claimed compositions and methods, such as without limitation, proteins from members of the families Picornaviridae (e.g., polioviruses, etc.); Caliciviridae; Togaviridae (e.g., rubella virus, dengue virus, etc.); Flaviviridae; Coronaviridae; Reoviridae; Birnaviridae; Rhabodoviridae (e.g., rabies virus, etc.); Filoviridae; Paramyxoviridae (e.g., mumps virus, measles virus, respiratory syncytial virus, etc.); Orthomyxoviridae (e.g., influenza virus types A, B and C, etc.); Bunyaviridae; Arenaviridae; Retroviradae (e.g., HTLV-I; HTLV-II; HIV-1 (also known as HTLV-III, LAV, ARV, hTLR, etc.)), including but not limited to antigens from the isolates HIV.sub.IIIb, HIV.sub.SF2, HIV.sub.LAV, HIV.sub.LAI, HIV.sub.MN); HIV-1.sub.CM235, HIV-1.sub.US4; HIV-2; simian immunodeficiency virus (SIV) among others. Additionally, antigens may also be derived from human papillomavirus (HPV) and the tick-borne encephalitis viruses. See, e.g. Virology, 3rd Edition (W. K. Joklik ed. 1988); Fundamental Virology, 2nd Edition (B. N. Fields and D. M. Knipe, eds. 1991), for a description of these and other viruses.

More particularly, the gp120 envelope proteins from any of the above HIV isolates, including members of the various genetic subtypes of HIV, are known and reported (see, e.g., Myers et al., Los Alamos Database, Los Alamos National Laboratory, Los Alamos, N. Mex. (1992); Myers et al., Human Retroviruses and Aids, 1990, Los Alamos, N. Mex.: Los Alamos National Laboratory; and Modrow et al., J. Virol. (1987) 61:570-578, for a comparison of the envelope sequences of a variety of HIV isolates) and antigens derived from any of these isolates will find use in the present methods. Furthermore, the invention is equally applicable to other immunogenic proteins derived from any of the various HIV isolates, including any of the various envelope proteins such as gp160 and gp41, gag antigens such as p24gag and p55gag, as well as proteins derived from the pol region.

Influenza virus is another example of a virus for which the present invention will be particularly useful. Specifically, the envelope glycoproteins HA and NA of influenza A are of particular interest for generating an immune response. Numerous HA subtypes of influenza A have been identified (Kawaoka et al., Virology (1990) 179:759-767; Webster et al., "Antigenic variation among type A influenza viruses," p. 127-168. In: P. Palese and D. W. Kingsbury (ed.), Genetics of influenza viruses. Springer-Verlag, New York). Thus, proteins derived from any of these isolates can also be used in the compositions and methods described herein.

Antigens derived from meningitis A, meningitis B, meningitis C, and other related viruses will also find use in the compositions and methods of the present invention. For examples of meningitis B antigens see, for example, PCT 99/00695 filed Apr. 7, 1999; PCT IB98/01665 filed Oct. 9, 1998 and PCT US99/09346 filed Apr. 30, 1999.

Non-viral organisms that are controlled by T cell immune responses include: pathogenic protozoa (e.g. Pneumocystis carinii, Trypanosoma, Leishmania, Plasmodia, and Toxoplasma gondii); bacteria (e.g., Mycobacteria, and Legioniella) and fungi (e.g. Histoplasma capsulatum and Cocidioides immitus). Hence, antigens derived from these organisms are also useful in connection with the present invention.

Tumor antigens for use in the invention include, but are not limited to, melanoma tumor antigens (Kawakami et al., Proc. Natl. Acad. Sci. USA 91:3515-3519 (1994); Kawakami et al., J. Exp. Med., 180:347-352 (1994); Kawakami et al. Cancer Res. 54:3124-3126 (1994), including MART-1 (Coulie et al., J. Exp. Med. 180:35-42 (1991), gp100 (Wick et al., J. Cutan. Pathol. 4:201-207 (1988) and MAGE antigen, MAGE-1, MAGE-2 and MAGE-3 (Van der Bruggen et al., Science, 254:1643-1647 (1991)); CEA, TRP-1, P-15 and tyrosinase (Brichard et al., J. Exp. Med. 178:489 (1993)); HER-2/neu gene product (U.S. Pat. No. 4,968,603); estrogen receptor, milk fat globulin, p53 tumor suppressor protein (Levine, Ann. Rev. Biochem. 62:623 (1993)); mucin antigens (Taylor-Papdimitriou, International Pub. No. WO90/05142)); telomerases; nuclear matrix proteins; prostatic acid phosphatase; papilloma virus antigens; and antigens associated with the following cancers: melanomas, metastases, adenocarcinoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, colon cancer, non-Hodgkins lymphoma, Hodgkins lymphoma, leukemias, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer and others (e.g., Rosenberg, Ann. Rev. Med. 47:481-91 (1996).

E. Polynucleotides

In accordance with the invention, one or more polynucleotides are inserted ex vivo into dendritic cells, such that one or more selected antigens are presented in effective amounts on the surface of the dendritic cells. By "effective amount" is meant that presentation is sufficient to enable the dendritic cells to provoke an immune response.

Techniques for nucleic acid manipulation are well known. Reagents useful in applying such techniques, such as restriction enzymes and the like, are widely known in the art and commercially available from a number of vendors.

Large amounts of polynucleotide sequences encoding the selected antigens for expression in the dendritic cells of the invention may be obtained using known procedures for molecular cloning and replication of a vector carrying the sequences in a suitable host cell. The nucleic acid sequences for use in the present invention may also be produced in part or in total by chemical synthesis, and may be performed on commercial automated oligonucleotide synthesizers.

Polynucleotides encoding the desired antigens for presentation in the dendritic cells are preferably recombinant expression vectors in which high levels of expression may occur, and which contain appropriate regulatory sequences for transcription and translation of the inserted nucleic acid sequence. The vectors may also contain polynucleotide sequences encoding selected class I and class II MHC molecules, costimulation and other immunoregulatory molecules, ABC transporter proteins, including the TAP1 and TAP2 proteins. Thus, various combinations of polynucleotide sequences may be inserted in a suitable expression vector or vectors. The vector may contain additional elements needed for subsequent replication, such as an origin of replication. The vectors may also contain at least one positive marker that enables the selection of dendritic cells carrying the inserted nucleic acids.

Preferred recombinant expression vectors for the invention include plasmid vectors. Preferred plasmid expression vectors include pCMV (see, for example, U.S. Pat. No. 5,688,688, the entire disclosure of which is hereby incorporated by reference).

Polynucleotides encoding the desired antigen or antigens are introduced into dendritic cells using the transfection methods of the present invention discussed below.

F. Association of Microparticles with Polynucleotides

In order to associate a polynucleotide of interest with a microparticle of interest, microparticles are simply mixed with polynucleotides, for example, in an appropriate buffer solution. The resulting formulation can be lyophilized prior to use. Generally, polynucleotides are added to the microparticles to yield microparticles with adsorbed polynucleotides having a weight-to-weight ratio of from about 0.0001:1 to 0.25:1 polynucleotides to microparticles, preferably, 0.001:1 to 0.1, more preferably 0.01 to 0.05. Polynucleotide content of the microparticles can be determined using standard techniques.

The microparticles of the present invention may have polynucleotides entrapped or encapsulated within them, as well as having polynucleotides adsorbed thereon.

The association of the microparticle with the polynucleotide is referred to alternatively herein as "polynucleotide/microparticles", "transfecting agent" and "transfection agent".

G. Transfection of Dendritic Cells

Once the dendritic cells and polynucleotide/microparticles are prepared, they are incubated in solution for a time and at a temperature sufficient for transfection to occur. According to a preferred embodiment, dendritic cells and polynucleotide/microparticles are incubated for 24 hours at 37.degree. C. in humidified CO.sub.2 incubator.

Expression of the polynucleotide of interest after transfection into dendritic cells may be confirmed by immunoassays or biological assays. For example, expression of introduced polynucleotides into cells may be confirmed by detecting the binding to the cells of labeled antibodies specific for the antigens of interest using assays well known in the art such as FACS (Fluorescent Activated Cell Sorting) or ELISA (enzyme-linked immunoabsorbent assay) or by simply by staining (e.g., with (.beta.-gal) and determining cell counts.

T cell activation may be detected by various known methods, including measuring changes in the proliferation of T cells, killing of target cells and secretion of certain regulatory factors, such as lymphokines, expression of mRNA of certain immunoregulatory molecules, or a combination of these.

H. Use of Dendritic Cells to Present Antigen In Vitro and In Vivo

According to an embodiment of the invention, dendritic cells transfected by polynucleotide/microparticles using any of the methods described herein are used to activate T cells in vitro. T cells or a subset of T cells can be obtained from various lymphoid tissues. Such tissues include but are not limited to spleens, lymph nodes, and peripheral blood.

The cells can be co-cultured with transfected dendritic cells as a mixed T cell population or as a purified T cell subset. For instance, it may be desired to culture purified CD8+ T cells with antigen transfected dendritic cells, as early elimination of CD4+ T cells may prevent the overgrowth of CD4+ cells in a mixed culture of both CD8+ and CD4+ T cells. T cell purification may be achieved by positive or negative selection, including but not limited to, the use of antibodies directed to CD2, CD3, CD4, CD5, and CD8. On the other hand, it may be desired to use a mixed population of CD4+ and CD8+ T cells to elicit a specific response encompassing both a cytotoxic and T.sub.H immune response.

After activation in vitro, the T cells are administered to a patient in a dose sufficient to induce or enhance an immune response to the selected antigen expressed by the dendritic cells of the invention.

T cells, as well as dendritic cells as described below, may be introduced into the subject to be treated by using one of a number of methods of administration of therapeutics known in the art. For example, the cells may be administered (with or without adjuvant) parenterally (including, for example, intravenous, intraperitoneal, intramuscular, intradermal, and subcutaneous administration). Alternatively, the cells may be administered locally by direct injection into a tumor or infected tissue. Adjuvants include any known pharmaceutically acceptable carrier. Parenteral vehicles for use as pharmaceutical carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, and lactated Ringer's. Other adjuvants may be added as desired such as antimicrobials.

As an example, T cells may be administered, by intravenous infusion, at doses of about 10.sup.8 to 10.sup.9 cells/m.sup.2 of body surface area (see, Ridell et al., 1992, Science 257: 238-241) Infusion can be repeated at desired intervals, for example, monthly. Recipients are monitored during and after T cell infusions for any evidence of adverse effects.

According to a preferred embodiment, the T cells are obtained from the same patient from whom the dendritic cells were obtained.

According to another embodiment, the T cells are obtained from a patient and the dendritic cells, which are used to stimulate the T cells, are obtained from an HLA-matched healthy donor (e.g., a sibling), or vice versa.

According to yet another embodiment, both the T cells and the dendritic cells are obtained from an HLA-matched healthy donor. This embodiment may be particularly advantageous, for example, when the patient is a late stage cancer patient who has been treated with radiation and/or chemotherapy agents and may not be able to provide sufficient or efficient dendritic or T cells.

According to another embodiment of the invention, dendritic cells isolated from a patient are cultured, transfected in vitro and administered back to the patient to stimulate an immune response, including T cell activation. As such, the dendritic cells constitute a vaccine and/or immunotherapeutic agent. As an example, dendritic cells presenting antigen are administered, via intravenous infusion, at a dose of, for example, about 10.sup.6 to 10.sup.8 cells. The immune response of the patient can be monitored. Infusion can be repeated at desired intervals based upon the patient's immune response.
 

Claim 1 of 5 Claims

1. A method for producing an immune response in a vertebrate subject in need thereof comprising: activating T cells by subjecting them to dendritic cells produced by a method that comprises incubating the dendritic cells and a transfection agent for a time sufficient to transfect the dendritic cells with the polynucleotide, said transfection agent comprising a polynucleotide that encodes an antigen associated with a virus or a tumor adsorbed on microparticles comprised of a biodegradable polymer and a cationic detergent, wherein polynucleotide is not entrapped within said microparticles; administering said activated T cells to said subject.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.