EP0863704A1 - Stimulation of cell-mediated immune responses by targeted particulate genetic immunization - Google Patents
Stimulation of cell-mediated immune responses by targeted particulate genetic immunizationInfo
- Publication number
- EP0863704A1 EP0863704A1 EP96933987A EP96933987A EP0863704A1 EP 0863704 A1 EP0863704 A1 EP 0863704A1 EP 96933987 A EP96933987 A EP 96933987A EP 96933987 A EP96933987 A EP 96933987A EP 0863704 A1 EP0863704 A1 EP 0863704A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- antigen
- tumor rejection
- mammalian host
- cells
- antigenic protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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Definitions
- Genetic immunization for the purpose of stimulating antigen-specific immunity in a mammalian host, including a human host, is at the core of the present disclosure.
- This specification discloses delivery of particulate polynucleotides to the cytoplasm of host target cells, such as antigen presenting cells. These particulate polynucleotides encode an antigenic protein or antigenic protein fragment which accesses the cytoplasm of the target cell. Expression of the antigen gene results in antigen-specific immune responses, including but not limited to, the induction of antigen-specific cytotoxic T-lymphocytes (CTLs). Cytosolic access of the antigen allows membrane presentation of the antigenic pepude through the endogenous MHC class I pathway.
- CTLs cytotoxic T-lymphocytes
- Membrane presentation via the endogenous MHC class I pathway stimulates the induction of antigen-specific CTLs. Induced antigen-specific CTLs then target and destroy antigen-expressing affected host cells such as neoplastic cells or virally infected cells.
- Cytotoxic T-lymphocytes are a c ⁇ ttcal component of effective human immune responses to tumors or viral infecuons Cytotoxic T-lymphocytes destroy neoplasuc cells or virus infected cells through recognition of antigenic pepudes presented by MHC class I molecules on the surface of the affected target cells. These anugenic peptides are degradation products of foreign proteins present in the cytosol of the affected cell, which are processed and presented to CTLs through the endogenous MHC class I processing pathway.
- APCs can provide both the anugen-MHC class I ligand and the accessory signals required in the induction phase of CTL-mediated immunity.
- General properties of APCs include MHC class I and class II expression, expression of va ⁇ ous adhesion molecules important for APC-lymphocyte interaction, and expression of costimulatory molecules such as CD80 and CD86.
- Examples of APCs include macrophages and dendritic cells (including cutaneous epidermal Langerhans cells, dermal dend ⁇ tic cells, and dendritic cells resident in lymph nodes and spleen).
- Geneuc immunization possess several attractive features.
- Several in vivo gene transfer methods result in transgene expression, including retroviral or adenoviral mediated gene transfer, and direct injecuon of naked DNA (for a review, see K ⁇ shnaw, et al., 1995, Nature Med. 1: 521-522 and Pardoll, et al., 1995, Immunity 3: 165-169.) Williams, et al. (1991, Proc. Natl. Acad. Sci.
- biolistic biobalistic
- DNA induces antigen-specific CTL responses to viral proteins and protective immunity to viral challenge.
- the studies do not disclose targeting of genetic material to APCs for genetic immunization.
- Falo, et al. (1995, Nature Med. 1: 649-653) offer in vivo support for phagosome- to-cytosol pathway by showing that delivery of particulate protein antigen directly into animals results in antigen-specific CTL mediated tumor immunity in mice. They demonstrate that proteins injected directly into animals in vivo can specifically enter the phagosome-cytosol pathway of APCs if administered in particulate form. No details are forwarded regarding genetic immunization procedures. The capacity of in vivo administered genetic material to enter the cytosol of APCs, or other cell types, functionally intact through this pathway is not addressed. Pardoll and Beckerleg (1995, Immunity 3: 165-169) have recently reviewed the immunology of naked DNA vaccines.
- the present invention relates to therapeutic or prophylactic genetic immunization of a mammalian host which comprises delivery of a DNA fragment which encodes an antigenic protein to a target cell within the mammalian host, expression of the recombinant DNA fragment within the host cell, and subsequent presentation of the antigenic peptide or peptides by the host cell so as to stimulate cell-mediated immunity, humoral immunity, or both.
- the present invention further relates to therapeutic or prophylactic genetic immunization of a mammalian host which comprises delivery of a DNA sequence encoding a antigenic protein or biologically active fragment thereof to a specific target cell within the mammalian host.
- Antigenic peptides expressed from the DNA fragment are specific to an affected cell and subsequently stimulate antigen specific CTL production, thereby promoting destruction of affected cells such as neoplastic cells and virally infected cells.
- the present invention also relates to genetic immunization with particulate polynucleotides and inoculation of a mammalian host and subsequent delivery of these particulate-based transgenic polynucleotides to the cytosol of the target cell.
- the particulate polynucleotide expresses a protein or biologically active fragment thereof whereby an appropriate antigenic peptide fragment is generated and presented to the target cell membrane via the endogenous MHC class I pathway.
- an appropriate antigenic peptide fragment is generated and presented to the target cell membrane via the endogenous MHC class I pathway.
- Proper presentation of the antigenic peptide or peptides of interest through the MHC class I pathway stimulates CTL production and in turn promotes destruction of cells such as neoplastic cells or virally infected cells.
- the present invention also relates to in vivo methods of therapeutic or prophylactic genetic immunization of a mammalian host which comprises generating a DNA fragment which expresses an antigenic protein or antigenic protein fragment, distributing the DNA fragment on a particle surface which results in a particulate polynucleotide, inoculating the mammalian host with said particulate polynucleotide and delivering the particulate polynucleotide to the cytoplasm of a target cell of the mammalian host so that the expressed antigenic protein or antigenic protein fragment is presented to the membrane surface of said target cell through the MHC class I pathway.
- the mammalian host be a human.
- the DNA fragment of interest express 1) a tumor rejection antigen or an anugenic protein fragment or 2) a viral antigen or an antigenic protein fragment.
- human TRAs which may be utilized in the present invention include but are not limited to MAGE-1, MAGE 3, Melan-A, gplOO, p53, CEA and HER2/neu.
- viral antigens which may be utilized in the present invention include but are not limited to HIV gpl20, HIV gpl60, Influenza virus nucleoprotein and Hepat s B surface antigen.
- the preferred target cell in the present invention is an APC while the preferred localization or migrauon of the APC target cell is the lymphoid tissue of the human host.
- the mammalian host is immunized with the particulate polynucleotide by utilizing a mircoprojectile bombardment device.
- a mammalian host is immunized by inoculation with a pa iculate polynucleotide by a biolistic (biobalistic) procedure such that the paniculate polynucleotide enters the cytoplasm of at least an appropnate number of host cells.
- the transgenic polynucleotide is expressed at biologically effective levels such that antigenic pepude fragments are presented to the endogenous MHC class I pathway and displayed on the membrane surface of the host cells.
- Endogenous host cell membrane presentation of the introduced antigen promotes induction of antigen-specific CTLs, which in turn circulate throughout the mammalian host, preferably a human host, to destroy neoplastic cells or virally infected cells.
- a mammalian host preferably a human
- a mammalian host is immunized with a paniculate polynucleotide by microprojectile bombardment inoculation such that the paniculate polynucleotide enters the cytoplasm of at least an appropriate number of host cells, including APCs, in the path of the projectiles as a direct result of non-specific projectile bombardment.
- APCs of the skin include, but are not limited to epidermal Langerhans cells, keratinocytes, or dermal dendriuc cells.
- APCs of the lymphoid tissue which may be bombarded include, but are not limited to resident dendritic cells, macrophages, stromal cells, T-lymphocytes, or ⁇ -lymphocytes.
- a mammalian host is immunized with a particulate polynucleotide by direct injection, including but not limited to subcutaneous injection, epidermal injection, dermal injection, lymphatic injection and intra venous injection.
- the paniculate polynucleotide enters host cells and is expressed at biologically effective levels such that antigenic peptide fragments are presented to the endogenous MHC class I pathway and displayed on the membrane surface of the host cell. Endogenous target cell membrane presentation promotes the induction of antigen- specific CTLs, which in turn circulate throughout the mammalian host, preferably a human host, to destroy neoplastic cells or virally infected cells.
- the paniculate polynucleotide is delivered to a human host by subcutaneous injection and targeted to APCs through a phagosome-to- cytosol pathway and expressed at biologically effective levels by APCs.
- the present invention discloses that direct injection of the paniculate polynucleotide complex via a subcutaneous inoculation route results in targeted delivery to APCs and antigen expression in the lymphoid tissue.
- subcutaneous injection for direct targeting to an APC involves delivery of a paniculate polynucleotide encoding a tumor rejection antigen (TRA) or biologically active fragment thereof.
- TRA tumor rejection antigen
- Directed delivery of a TRA paniculate polynucleotide in this manner will maximize entry of tumor specific antigenic peptides into the class I pathway as well as avoiding substantial particulate translocation within non-APC cells.
- subcutaneous injection for direct targeting to an APC involves delivery of a paniculate polynucleotide encoding a viral antigen or biologically active fragment thereof.
- Directed delivery of a viral antigen encoding particulate polynucleotide in this manner will maximize entry of viral specific antigenic peptides into the class I pathway as well as avoiding substantial particulate translocation within non-APC cells.
- particulates composed of a variety of materials including but not limited to gold, iron, and synthetic plastics can access the phagosome-to-cytosol pathway used to target APCs in this invention.
- a mammalian host is immunized by injection, including but not limited to subcutaneous injection, epidermal injection, dermal injection, lymphatic injection and intra venous injection, with syngeneic APCs that have been antigen loaded in vitro by co-incubation with particulate polynucleotide.
- Particulate polynucleotides may, for example, enter the APC in vitro by either microprojectile bombardment or the phagosome-to-cytosol pathway.
- a mammalian host is immunized with particulate polynucleotide transfected APCs, such that the particulate polynucleotide specifically enters APCs in vitro and the APCs are injected into the host.
- the transgenic polynucleotide is expressed at biologically effective levels such that antigenic peptide fragments are processed and presented through the endogenous MHC class I pathway and displayed on the membrane surface of the APCs.
- endogenous APC cell membrane presentation of antigen promotes the induction of antigen-specific CTLs either at the site of injection, or in the lymphoid tissue.
- Induced antigen-specific CTLs in turn circulate throughout the mammalian host, preferably a human host, to destroy neoplastic cells or virally infected cells.
- a mammalian host is immunized by injection, including but not limited to subcutaneous injection, epidermal injection, dermal injection, lymphatic injection and intra venous injection, with syngeneic APCs that have been transfected in vitro with particulate polynucleotides.
- a mammalian host is immunized with particulate polynucleotide transfected APCs, such that the particulate polynucleotide enters APCs in vitro and the APCs are then injected into the host.
- Particulate polynucleotides may, for example, enter the APC in vitro by either microprojectile bombardment or the phagosome-to-cytosol pathway.
- APCs are transfected in vitro with antigen encoding polynucleotide and/or polynucleotides encoding a molecule or molecules which increases the efficiency of the antigen presenting function of the APC.
- Such molecules include but are not limited to cytokines and costimulatory molecules. Examples of cytokines include but are not limited to IL-12, IL-2, and IL-4. Examples of costimulatory molecules include but are not limited to CD80 and CD86.
- the transgenic antigen encoding polynucleotide is expressed at biologically effective levels such that antigenic peptide fragments are processed and presented through the endogenous MHC class I pathway and displayed on the membrane surface of the APCs.
- endogenous APC cell membrane presentation of antigen promotes the induction of antigen-specific CTLs either at the site of injection, or in the lymphoid tissue.
- Induced antigen-specific CTLs in turn circulate throughout the mammalian host, preferably a human host, to destroy neoplastic cells or virally infected cells.
- the transgenic cytokine and/or costimulatory encoding polynucleotide is expressed at biologically effective levels such that the antigen presenting function of the APC results in the induction of antigen specific immune responses either at the site of the injection, or in the lymphoid tissue.
- polynucleotides can be precipitated onto particulates composed of a variety of materials including but not limited to gold, iron, and synthetic plastics.
- APCs can be obtained from a variety of host tissue including, but not limited to bone ma ⁇ ow and peripheral blood, and that said APCs can be manipulated in vitro and then reintroduced into said host.
- Figure 1 shows functional presentation of ovalbumin by the transfected tumor cell lines M04 and EG7. Microcultures were prepared with the T-cell hybridoma RF33.70 (anti-OVA+ K b ) and the indicated number of transfected (squares) or untransfected
- Figure 2 shows that OVA expression by the B16 derived melanoma M04 does not significantly effect in vivo tumor growth or host survival following tumor challenge.
- Mice were challenged with M04 (circles) or B16 (squares) (5 x l /mouse, i.d., bilateral, mid- flanks).
- Tumor size ( Figure 2 A) was assessed 3x/week and is reported as the average tumor area in square millimeters until the first death occu ⁇ ed in each group.
- Survival Figure 2B is recorded as the percentage of surviving animals. All experiments included 5 mice/group and were repeated at least three times. Mice becoming moribund were sacrificed.
- Figure 3 shows that immunization by cutaneous delivery of OVA encoding DNA induces OVA-specific CTLs and antigen-specific, CTL mediated protection from lethal challenge with the OVA expressing melanoma M04.
- ln vitro restimulated splenocytes from OVA-immunized (genetically immunized as described in Example Section 7) mice were assayed for cytolytic function against the OVA-transfected lymphoma EG7 (closed squares) or the untransfected parent EL4 (open triangles) (A) .
- mice were genetically immunized with OVA (closed squares) or lacZ (open squares) and boosted 7 days later. Groups of immunized mice were challenged 7 days after the final immunization (day 0) with either the B16 melanoma (D), or the OVA-expressing subclone M04 (C).
- mice were divided into 2 groups, one of which was depleted of CD8 + lymphocytes by i.p. injection of anti-CD8 mAb 7 and 9 days after the last immunization.
- Intact (E) and CD8 + depleted (F) mice were then challenged 10 days after the final immunization (day 0) with M04. Survival was reported as the percentage of surviving animals (C-F). Animals surviving on day 60 had no sign of tumor growth. All experiments included 5 mice per group and were repeated at least 3 times. Mice that became moribund were sacrificed according to animal care guidelines.
- FIG. 4 shows that antigen presenting cells internalize and express particulate polynucleotides, and process and present the expressed antigen through the MHC class I restricted processing pathway.
- Dendritic cells were prepared by depleting bone ma ⁇ ow cells of lymphocytes and culturing ovemight in RPMI 1640 supplemented with 10%FCS, L-glutamine, antibiotics and 2-ME in 24 well plates at IO 6 cells/well. Cells were repleted on day 1 at 2.5 x 10 5 cells/well with GM-CSF (l ⁇ U/ml, Sigma, St. Louis, MO) and murine rIL-4 (lCPU/ml, Genzyme, Cambridge, MA) and loosely adherent cells were harvested on day 8.
- GM-CSF l ⁇ U/ml, Sigma, St. Louis, MO
- murine rIL-4 lCPU/ml, Genzyme, Cambridge, MA
- these dendritic cells expressed CD45, CD44, CDI lb (Mac-1), CD18, CD80, cD86 and class I and class II MHC antigens.
- Dendritic cells were pulsed 2hrs at 37°C with or without OVA peptide (20ng/ml) 4- ⁇ 2-microgl ⁇ bin (B2-M, lO ⁇ l/ml, human, Sigma) in reduced serum media (Optimen, Gibco, Grand Island, NY). Cells were then washed extensively, resuspended in PBS and irradiated (2000 rad) before injection into naive mice.
- the indicated number of bone ma ⁇ ow derived dendritic APCs were cocultured with OVA-encoding particulate polynucleotides prepared as described in Example Section 7 ( ⁇ O ⁇ l/ l/lO 4 cells of 7mg/ml particulates) using either Fe beads (closed squares) or gold beads (closed circles) as the particulate substrate or soluble OVA protein (2mg/ml) (open squares) for 24hrs., washed, and then the indicated number of APCs were co-cultured in microcultures with the T-cell hybridoma RF33.70 (anti-OVA+ K b ). After 18hrs incubation, supernatants were harvested and assayed for IL-2 using the indicator cell line HT2 (Rock, et al., 1990, J. Immunol. 145: 804-811).
- Figure 5 shows a comparison of particulate polynucleotide immunization administered by biolistic (biobalistic) or subcutaneous injection.
- biolistic biobalistic
- Subcutaneous injection Groups of 5 C57B1/6 mice were immunized, boosted on day 7, and then challenged by intradermal injection of 5 x IO 3 M05 tumor cells in each flank 7 days after boosting.
- Immunizations consisted of: (A) ⁇ -Gal encoding particulate polynucleotides prepared and delivered by biolistic (biobalistic) administration as described in Example Section 7; (B) OVA encoding particulate polynucleotides prepared and delivered by biolistic (biobalistic) administration as described in Example Section 7; (C) an excess quantity of OVA encoding polynucleotide without particulates delivered by subcutaneous injection; or, (D) an equivalent amount of OVA encoding particulate polynucleotides prepared as in Example Section 7 but administered by subcutaneous injection. Data is presented as % of animals tumor free in each group 50 days after tumor challenge.
- Figure 6 shows that immunization with APCs which had been co-incubated with particulate polynucleotide encoding OVA protects animals from challenge by OVA- expressing melanoma M05, Groups of 5 C57B1/6 mice were immunized subcutaneously as described in Figure 5 on one occasion and then challenged by intradermal injection of 1 x 10 s M05 tumor cells in each flank 10 days after immunization.
- Immunizations consisted of either: (open squares) paniculate polynucleotide encoding the i ⁇ elevant antigen ⁇ -galactosidase (lOO ⁇ l of 7mg/ml paniculate solution (particulate wt./vol PBS) per hind leg bilaterally; (open circles) salable pAc-neo-OVA (lOO ⁇ l per hind leg containing an approximately equivalent quantity of DN A/animal); or (closed squares) 5 x 10* bone ma ⁇ ow derived dendritic cells/lOO ⁇ l hind legs s.q. (prepared as described in Example 7). Survival was reported as the percentage of surviving animals. All experiments included 5 mice per group. Mice that became moribund were sacrificed according to animal care guidelines.
- mammalian host includes members of the animal kingdom, including but not limited to human beings.
- DNA fragment may include any nucleotide sequence, either DNA or RNA, which contains appropriate coding region and regulatory sequences to result in target cell expression of an antigenic protein or antigenic protein fragment for cell membrane presentation via the endogenous MHC Class I pathway.
- particle polynucleotide may refer to a particulate made from materials including but not limited to gold, iron, and synthetic plastics wherein the particle comprises a population of DNA fragments as defined in the preceding paragraph.
- the present invention relates to therapeutic or prophylactic genetic immunization of a mammalian host which comprises delivery of a DNA fragment to a target cell within the mammalian host, expression of the DNA fragment within the target cell, and subsequent presentation of a recombinant antigenic peptide(s) within the target cell so as to stimulate cell-mediated immunity, humoral immunity, or both.
- the present invention further relates to therapeutic or prophylactic genetic immunization of a mammalian host which comprises delivery of a DNA fragment encoding a protein or biologically active fragment thereof to a specific target cell within the mammalian host.
- Antigenic peptides expressed from the DNA sequence are specific to an affected cell such as a neoplasuc cell or virally infected cell.
- Antigen specific CTL production is stimulated, thereby promoting destruction of target cells such as neoplastic cells and virally infected cells.
- the present invention discloses genetic immunization methods for treatment or prevention of tumors or viral infections.
- Cytotoxic-T-cells are a significant component of the immune response to tumors and viral infections. Cytotoxic-T-cells kill neoplastic cells or virus-infected cells through the recognition of antigenic peptides presented by MHC class I molecules on the surface of the tumor target. These peptides are derived from tumor antigens that are synthesized by the affected cell and degraded in the cytosol. Attempts to induce tumor-specific CTL responses in vivo by immunization with killed tumor cells or component proteins have generally been unsuccessful, presumably because proteins in the extracellular fluids cannot enter the cytosol and access the MHC class I presentation pathway.
- a specific embodiment of the present invention relates to genetic immunization with particles containing the DNA fragment expressing the protein of interest.
- particles containing a DNA fragment are refe ⁇ ed to throughout this specification as
- In vivo cell delivery is best accomplished by administering the DNA fragment or protein of interest in paniculate form, such as coated beads or gold particles.
- the protein or biologically active fragment is expressed subsequent to cytosolic delivery within the target cell.
- the expressed protein or protein fragment specific to the affected cell provides a substrate for generating an antigenic peptide(s) for presentation to
- T-lymphocytes via the endogenous MHC class I pathway.
- Appropriate presentation of the antigenic peptide or peptides of interest through the MHC class I pathway stimulate CTL production and in turn promote destruction of the affected cell.
- the present invention is based on the premise that a DNA fragment expressing a tumor rejection antigen (TRA) or viral antigen or active fragment thereof may be targeted to a specific cell so as to promote the cascade of events culminating in CTL-mediated protective tumor immunity.
- TRA tumor rejection antigen
- the present invention discloses induction of CTL-mediated immunity by transfecting a target host cell with a DNA construct in particulate form which encodes an antigenic protein.
- the immunizing protein is produced intracellularly and hence has access to the MHC class I restricted presentation pathway.
- Naturally processed epitopes result, and the transfected cells may produce the immunizing protein for several days, potentially facilitating more intense immunogenic stimulation.
- a mammalian host is immunized with a particulate polynucleotide by a microprojectile bombardment device such that the particulate polynucleotide enters the cytoplasm of at least an appropriate number of host cells, preferably including but not necessarily limited to APCs, in the path of the projectiles as a direct result of non-specific projectile bombardment.
- APCs of the skin include, but are not limited to epidermal Langerhans cells, keratinocytes, or dermal dendritic cells.
- APCs of the lymphoid tissue which may be bombarded include, but are not limited to resident dendritic cells, macrophages, stromal cells, T-lymphocytes, or ⁇ -lymphocytes.
- the transgenic polynucleotide is expressed at biologically effective levels such that antigenic peptide fragments are processed and presented through the endogenous MHC class I pathway and displayed on the membrane surface of the APC cells.
- Endogenous APC cell membrane presentation of antigen promotes the induction of antigen-specific CTLs either at the site of bombardment, or after trafficking of bombarded cells to the lymphoid tissue.
- Induced antigen-specific CTLs in turn circulate throughout the mammalian host, preferably a human host, to destroy neoplastic cells or virally infected cells.
- a mammalian host may be immunized with a particulate polynucleotide by a biolistic (biobalistic) delivery procedure such that the particulate polynucleotide specifically enters host cells, including APCs, through the phagosome-to-cytosol pathway.
- Particulate polynucleotides enter host cells through the phagosome-to-cytosol pathway either at the bombarded site (skin or lymphoid tissue) or after trafficking to the lymphoid tissue.
- particulate polynucleotides may enter the lymphoid tissue either by direct trafficking of the particulates to the lymphoid tissue with subsequent uptake by cells in the lymphoid tissue, or by trafficking of host cells which have taken-up paniculate polynucleotides to the lymphoid tissue.
- the transgenic polynucleotide is expressed at biologically effective levels such that antigenic peptide fragments are processed and presented through the endogenous MHC class I pathway and displayed on the membrane surface of the host APCs.
- Endogenous APC cell membrane presentation of antigen promotes the induction of antigen-specific CTLs either at the site of bombardment, or in the lymphoid tissue.
- Induced antigen-specific CTLs in tum circulate throughout the mammalian host, preferably a human host, to destroy neoplastic cells or virally infected cells.
- a mammalian host is immunized with a particulate polynucleotide by direct injection, including but not limited to subcutaneous injection, epidermal injection, dermal injection, lymphatic injection and intra venous injection.
- the particulate polynucleotide enters host cells and is expressed at biologically effective levels such that antigenic peptide fragments are presented to the endogenous MHC class I pathway and displayed on the membrane surface of the host cell.
- Endogenous target cell membrane presentation promotes the induction of antigen- specific CTLs, which in turn circulate throughout the mammalian host, preferably a human host, to destroy neoplastic cells or virally infected cells.
- a mammalian host may be immunized with a particulate polynucleotide such that the particulate polynucleotide specifically enters host cells, including APCs, through the phagosome-to-cytosol pathway.
- Particulate polynucleotides enter host cells through the phagosome-to-cytosol pathway either at the injection site or after trafficking to the lymphoid tissue.
- particulate polynucleotides may enter the lymphoid tissue either by direct trafficking of the particulates to the lymphoid tissue with subsequent uptake by cells in the lymphoid tissue, or by trafficking of host cells which have taken-up particulate polynucleotides to the lymphoid tissue.
- the transgenic polynucleotide is expressed at biologically effective levels such that antigenic peptide fragments are processed and presented through the endogenous MHC class I pathway and displayed on the membrane surface of the host APCs.
- Endogenous APC cell membrane presentation of antigen promotes the induction of antigen-specific CTLs either at the site of injection, or in the lymphoid tissue.
- Induced antigen-specific CTLs in turn circulate throughout the mammalian host, preferably a human host, to destroy neoplastic cells or virally infected cells.
- a mammalian host is immunized with a particulate polynucleotide by direct injection, including but not limited to subcutaneous injection, epidermal injection, dermal injection, lymphatic injection and intra venous injection.
- the composition of the particulate polynucleotide is designed to include specific molecules which preferentially target APC phagocytic pathways
- Particulate polynucleotides enter host cells through the phagosome- to-cytosol pathway either at the injection site or after trafficking to the lymphoid tissue.
- particulate polynucleotides may enter the lymphoid tissue either by direct trafficking of the particulates to the lymphoid tissue with subsequent uptake by cells in the lymphoid tissue, or by trafficking of host cells which have taken-up particulate polynucleotides to the lymphoid tissue.
- the transgenic polynucleotide is expressed at biologically effective levels such that antigenic peptide fragments are processed and presented through the endogenous MHC class I pathway and displayed on the membrane surface of the host APCs. Endogenous APC cell membrane presentation of antigen promotes the induction of antigen-specific CTLs either at the site of injection, or in the lymphoid tissue.
- Induced antigen-specific CTLs in tum circulate throughout the mammalian host, preferably a human host, to destroy neoplastic cells or virally infected cells.
- the present invention is exemplified on several fronts using a murine melanoma model described in detail in Example Secuon 6.
- the primary limitation in studying antigen specific tumor immunity in a murine model is the lack of a defined tumor antigen recognized by MHC class I restricted CTLs. Since TRAs are not fundamentally different from any other protein synthesized by the cell, except that the host is not tolerant to them, a foreign protein synthesized by a tumor should function as a tumor antigen.
- Tumor immunization methods of the present invention are exemplified with a murine tumor model with a defined, endogenously synthesized TRA by transfecting the ovalbumin (OVA) gene into the C57B1/6 derived melanoma B16.
- OVA ovalbumin
- This system is attractive for several reasons: (1) the B16 melanoma is an extensively studied murine tumor, (2) in vivo growth characteristics and metastasis of this tumor line are well characterized, and (3) ovalbumin has a well defined structure.
- the intracellular processing and presentation of OVA in the C57B1/6 mouse is known.
- the structure of the processed peptide, presented in association with MHC class I K b is known.
- One embodiment of the present invention is immunization with paniculate polynucleotide of interest using a biolistic (biobalistic) device.
- a mammalian host is immunized with a particulate polynucleotide by a biolistic (biobalistic) procedure such that the particulate polynucleotide delivery prompts a series of biological events within the transfected cells so as to elicit protective immunity to lethal tumor challenge.
- the present invention is exemplified several fold by cutaneous antigen delivery using a biolistic (biobalistic) device.
- the target cell be a phagocytic APC, specifically an APC which is localized within, or can traffic to, the lymphoid tissue.
- a mammalian host is immunized with a particulate polynucleotide by direct injection, including but not limited to subcutaneous injection, epidermal injection, dermal injection, lymphatic injection and intra venous injection.
- the transgenic polynucleotide is expressed at biologically effective levels such that antigenic pepude fragments are processed and presented through the endogenous MHC class I pathway, and displayed on the membrane surface of the target cells.
- Endogenous APC cell membrane presentation promotes the induction of antigen-specific CTLs, which in turn circulate throughout the mammalian host, preferably a human host, to destroy neoplasuc cells or virally infected cells
- the target cell of a mammalian host immunized with a particulate polynucleotide by direct injection is a phagocytic APC.
- the transgenic polynucleotide is expressed at biologically effective levels such that antigenic peptide fragments are presented to the endogenous MHC class I pathway and displayed on the membrane surface of the APC cell APC cell membrane presentation promotes the induction of antigen specific CTLs, which in turn circulate throughout the mammalian host, preferably a human host, to destroy neoplastic cells or virally infected cells.
- the paniculate polynucleotide is delivered to a human host by subcutaneous injection.
- the present invention discloses that direct injection of the particulate polynucleotide complex results in targeted delivery to phagocytic APCs and gene expression in the lymphoid tissue It is this targeted delivery to phagocytic APCs and/or gene expression in the lymphoid tissue via subcutaneous administration which results in supenor induction of antigen-specific CTLs from naive precursors Therefore, central to the present invention are DNA fragments encoding a protein or biologically active fragment thereof that enter and are expressed by APCs.
- the antigen of interest is expressed within the APC cytoplasm and enters the MHC class I pathway, allowing the APCs to promote induction of antigen-specific CTLs.
- the data presented in this disclosure supports the novel premise that the most effective manner in which to induce such antigen-specific CTLs is by direct transfer of genetic material to the APCs in the lymphoid tissue or APCs capable of trafficking to the lymphoid tissue.
- a mammalian host is immunized by injection, including but not limited to subcutaneous injection, epidermal injection, dermal injection, lymphatic injection and intra venous injection, with syngeneic APCs that have been antigen loaded in vitro with particulate polynucleotide.
- a mammalian host is immunized with paniculate polynucleotide transfected APCs, such that the paniculate polynucleotide enters APCs in virro and the APCs are injected into the host.
- the transgenic polynucleotide is expressed a biologically effective levels such that antigenic peptide fragments are processed and presented through the endogenous MHC class I pathway and displayed on the membrane surface of the APCs.
- endogenous APC cell membrane presentation of antigen promotes the induction of antigen-specific CTLs either at the site of injection, or in the lymphoid tissue.
- Induced antigen-specific CTLs in turn circulate throughout the mammalian host, preferably a human host, to destroy cells or virally infected cells.
- a mammalian host is immunized by injection, including but not limited to subcutaneous injection, epidermal injection, dermal injection, lymphatic injection and intra venous injection, with syngeneic APCs that have been antigen loaded in vitro by co-incubation with particulate polynucleotide.
- syngeneic APCs are transfected in vitro by co-incubation with particulate polynucleotides such that the particulate polynucleotide enters the APCs through a phagosome-to-cytosol pathway.
- a mammalian host is immunized with particulate polynucleotide transfected APCs, such that the particulate polynucleotide specifically enters APCs through the phagosome-to-cytosol pathway and the APCs are then injected into the host. Subsequent to uptake by APCs the transgenic polynucleotide is expressed at biologically effective levels such that antigenic peptide fragments are processed and presented throughout the endogenous MHC class pathway and displaced on the membrane surface of the APCs. After injection of such APCs, endogenous APC cell membrane presentation of antigen promotes the induction of antigen-specific CTLs either at the site of injection, or in the lymphoid tissue. Induced antigen-specific CTLs in turn circulate throughout the mammalian host, preferably a human host, to destroy neoplastic cells or virally infected cells.
- a mammalian host is immunized by injection, including but not limited to subcutaneous injection, epidermal injection, dermal injection, lymphatic injection and intra venous injection, with syngeneic APCs that have been antigen loaded in vitro particulate polynucleotide.
- syngeneic APCs are transfened in vitro with microprojectile bombardment of APCs wiui particulate polynucleotides.
- the transgenic polynucleotide is expressed at biologically effective levels such that antigenic peptide fragments are processed and presented through the endogenous MHC class I pathway and displaced on the membrane surface of the APCs.
- APCs endogenous APC cell membrane presentation of antigen promotes the induction of antigen-specific CTLs either at the site of injection, or in the lymphoid tissue.
- Induced antigen-specific CTLs in tum circulate throughout the mammalian host, preferably a human host, to destroy neoplastic cells or virally infected cells.
- a mammalian host is immunized by injection, including but not limited to subcutaneous injection, epidermal injection, dermal injection, lymphatic injection and intra venous injection, with syngeneic APCs that have been transfected in vitro with particulate polynucleotide.
- a mammalian host is immunized with particulate polynucleotide transfected APCs, such that the particulate polynucleotide enters APCs in vitro and the APCs are injected into the host.
- Particulate polynucleotides enter APCs in vitro by either microprojectile bombardment or the phagosome-to-cytosol pathway.
- APCs are transfected in vitro with antigen encoding polynucleotides and/or polynucleotides encoding a molecule or molecules which increase the efficiency of the antigen presenting function of the APC, such as, but not limited to, cytokine molecules such as IL-12, IL-2, and/or IL-4 and/or costimulatory molecules such as CD80 and/or CD86.
- the transgenic antigen encoding polynucleotides is expressed at biologically effective levels such that antigenic peptide fragments are processed and presented through the endogenous MHC class I pathway and displayed on the membrane surface of the APCs.
- endogenous APC cell membrane presentation of antigen promotes the induction of antigen-specific CTLs either at the site of injection or in the lymphoid tissue.
- Induced antigen-specific CTLs in turn circulate throughout the mammalian host, preferably a human host, to destroy neoplastic cells or virally infected cells.
- the transgenic cytokine and/or costimulatory encoding polynucleotide is expressed at biologically effective levels such that the antigen presenting function of the APC results in the induction of antigen specific immune responses either at the site of injection, or in the lymphoid tissue.
- TRAs include, but are by no means limited to, MAGE-1 , MAGE-3, BAGE, GAGE-1 , GAGE-2, Tyrosinase, Melan-A(MART-l), gpl00(pmell7), gp75(TRPl), CEA (carcinoembryonic antigen) as well as viral derived tumor antigens from HPV, HBV, and EBV, as well as tumor associated oncogene/tumor suppressor gene mutation encoded antigens such as P53, P16, RAS, HER2/neu, C-ABL, and polymo ⁇ hic endothelial mucin antigens (as reviewed in Maeurer et.
- viral antigens include, but are by no means limited to, Influenza nucleoprotein (Donnelly et al., 1995, Nature Med. 1:583-587.), HIV gp 120, HIV gp 160, and Hepatitis B surface antigen (as reviewed in Pardoll and Beckerleg, Immunity, 1995, 3: 165-169).
- any eukaryotic promoter and/or enhancer sequence available which is known to up-regulate expression of a transgenic DNA sequence may be used in constructing a recombinant vector for combination with the particle of choice so as to generate the particulate polynucleotide of the present invention.
- promoter fragments include but are not limited to a cytomegalovirus (CMV) promoter, a Rous Sarcoma virus (RSV) promoter, a Murine Leukemia Virus
- a prefe ⁇ ed embodiment of the present invention is the use of paniculate bound DNA to deliver the DNA encoding a tumor or viral antigen specifically to the APCs in the lymphoid tissue, or APCs capable of trafficking to the lymphoid tissue, so as to promote MHC class I access of antigen, thus allowing APCs to stimulate induction of antigen-specific CTLs These CTLs will then circulate throughout the host and destroy neoplastic cells or virally infected cells.
- Tumor immunity based on the prefe ⁇ ed method of utilizing subcutaneous injection to promote a specific response in lymph node APCs is exemplified in Example Section 6, Example Section 7 and Example Section 8.
- Example Section 6 Tumor immunity based on the prefe ⁇ ed method of utilizing subcutaneous injection to promote a specific response in lymph node APCs is exemplified in Example Section 6, Example Section 7 and Example Section 8.
- the following examples are offered by way of illustration of the present invention, and not by way of limitation.
- OVA/B16 GENETIC MURINE TUMOR MODEL The OVA/B16 murine model was used to generate the data disclosed in Example Section 7 and 8 which exemplify the claimed invention.
- the OVA/B16 mu ⁇ ne system is attractive for several reasons: (1) the B16 melanoma is an extensively studied murine tumor, (2) in vivo growth characteristics and metastasis of this tumor line are well characterized, and (3) ovalbumin has a well defined structure.
- the intracellular processing and presentation of OVA in the C57B1/6 mouse is known.
- the structure of the processed peptide, presented in association with MHC class I K is known.
- mice and Cell Lines Female C57BL/6 mice, 5-8 weeks old were purchased from the Jackson Laboratories, Bar Harbor, ME.
- EL4 is a C57BL/6 T-lymphoma
- EG7 is a chicken egg ovalbumin (OVA)-transfected subclone of EL4 (Moore, et al. 1988, Cell 54: 777-785).
- the C57BL/6 derived murine melanoma B16 (Fidler, et al. , 1976, Cancer Res. 36: 3160-3165) was obtained from American Tissue Type Collection (ATCC).
- ATC American Tissue Type Collection
- M04 was constmcted by transfection of B16 with the pAc-Nco-OVA plasmid as described. (Falo, et al, 1995, Nature Med. 1 : 649-653, Moore, et al. 1988, Cell 54: 777-785)
- Monoclonal antibodies were prepared from the hybridomas GK1.5 (anti-CD4, ATCC TIB-207), 2.43 (anti-CD8 antibodies was raised in Balb/c nu/nu mice by i.p. injection of GKfl.5 cells (3 x IO 6 ) and IFA (0.5ml/mouse).
- DNA beads were identically prepared using an equivalent weight of Biomag iron oxide beads or gold beads as a particulate substrate.
- DNA coated beads were prepared as above and an equivalent quantity of DNA was injected, or the quantity specified in the figure descriptions.
- an excess quantity of free plasmid DNA was subcutaneously injected.
- Subcutaneous injections were administered in lOO ⁇ l volume of PBS in the hind legs, bilaterally.
- DNA beads were identically prepared as described above using an equivalent weight of Biomag iron oxide beads (Fe beads) or gold beads as paniculate substrate.
- Dendritic APCs were obtained from bone ma ⁇ ow as described in the Brief Description of the Drawings except that IL-4 was not used in tissue culture media with 25 ⁇ l of 7mg/ml particulate polynucleotide solution (particulate wt vol. PBS) for 18hrs at 37°C.
- Antigen-pulsed dendritic cells were prepared and injected as descnbed in the Bnef Desc ⁇ ption of the Drawings.
- dendritic cells were prepared by depleting bone ma ⁇ ow cells of lymphocytes and cultunng overnight in RPMI 1640 supplemented with 10%FCS, L-glutamine, antibiotics and 2-ME in 24 well plates at IO 6 cells/well. Cells were repleted on day 1 at 2.5 x 10 5 cells/well with GM-CSF (lOHJ/ml, Sigma, St. Louis, MO) and munne rIL-4 (10 3 U/ml, Genzyme, Cambridge, MA) and loosely adherent cells were harvested on day 8.
- GM-CSF lOHJ/ml, Sigma, St. Louis, MO
- munne rIL-4 10 3 U/ml, Genzyme, Cambridge, MA
- these dendntic cells expressed CD45, CD44, CDI lb (Mac-1), CD18, CD80, cD86 and class I and class II MHC antigens.
- Dendntic cells were pulsed 2hrs at 37°C with or without OVA peptide (20ng/ml) + ⁇ 2-m ⁇ croglob ⁇ n (B2-M, lO ⁇ l/ml, human, Sigma) in reduced serum media (Optimen, Gibco, Grand Island, NY). Cells were then washed extensively, resuspended in PBS and irradiated (2000 rad) before injection into naive mice.
- Cytotoxicity Assays Splenocytes from immunized animals were restimulated with minor modifications of previously described protocols. (Falo, et al., 1995, Nature Med. 1: 649-653). Briefly, 1 week after immunization splenocytes (30 x IO 6 ) were restimulated by coculture with irradiated (20,000 rad) EG7 cells (10 x IO 6 ). Effector cells were harvested five days later and cultured with 2xl0 4 5 'Cr labeled targets in round bottom microwells (200 ⁇ l) at the indicated effector target cell ratio. In some cases the effector cells were depleted of T-cell subsets using mAB plus complement before assay as described.
- OVA-immunized or tocZ-immunized animals were challenged by intradermal injection in the mid-flanks bilaterally with melanoma cells (2 x IO 4 ) at 2 times the dose lethal to 50% of the animals tested (LD JQ ) or the number of tumor cells indicated in section 4. Survival is recorded as the percentage of surviving animals.
- Melanoma cells for injection were washed three times in PBS. Injected cells were greater than 95% viable by trypan blue exclusion. All experiments included 5 mice per group and were repeated at least three times. Mice which became moribund were sacrificed according to animal care guidelines of the University of Pittsburgh Medical Center.
- mice were depleted of CD8 + cells. This was accomplished by i.p. injection of CD8 mAb (2.43) 7 and 9 days after immunization as described, followed by tumor challenge on day 10 (Falo, et al., 1995, Nature Med. 1: 649-653).
- mice Groups of mice that were immunized and boosted as described above were challenged 7 days later by i.d. injection of the M04 melanoma at a distant site to determine the ability of biolistic (biobalistic) immunization to induce protective tumor immunity.
- OVA-immunized mice were protected from lethal tumor challenge, while tumors in control mice (immunized similarly, but with the lacZ reporter gene) grew progressively and were lethal in 60% of the animals by day 40 (Figure 3C).
- OVA- immunized mice were not protected from challenge with the untransfected parent melanoma B16 ( Figure 3D), indicating that protective immunity was antigen specific, depending on OVA expression by the tumor target.
- Example Section 6 The materials and methods for inducing tumor immunity by subcutaneous administration of a particulate polynucleotide are as described throughout Example Section 6 and Example Section 7.
- This data demonstrates an ability to specifically target phagocytic APCs in the lymphoid tissue, or APCs capable of trafficking to the lymphoid tissue, by subcutaneous injection of particulate polynucleotides in vivo.
- APCs bone ma ⁇ ow derived dendritic cells co-cultured in vitro with particulate polynucleotides encoding ovalbumin (prepared as described in Example 7) (pAc-Neo-OVA) can stimulate the OVA (SIINFEKL* K b ) specific T cell hybridoma RF33.70 to produce IL-2 ( Figure 4).
- OVA SIINFEKL* K b
- Figure 4 demonstrate that these APCs functionally express the ovalbumin gene and generate the ovalbumin peptide-K b complex.
- the potency of stimulation is comparable to that observed with APCs pulsed with 2mg/ml of soluble OVA protein.
- Particulate polynucleotides are effective in this assay using either gold or Fe as the particulate substrate.
- the incubation of particulate polynucleotides with phagocytic APCs in vitro results in the endogenous production, processing, and presentation of the transfected antigen.
- antigen presenting cells in this case subcutaneously injected bone ma ⁇ ow derived dendritic cells, which have been incubated with particulate polynucleotides encoding the antigen OVA in vitro as described above, are capable of inducing protective immunity to tumor cells expressing the antigen gene.
- dendritic APCs which are potent simulators of antigen-specific CTLS and protective tumor immunity when peptide- loaded in vitro are at least similarly immunogenic which antigen-loaded by co-incubation with paniculate polynucleotides.
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US6110898A (en) * | 1996-05-24 | 2000-08-29 | University Of Maryland, Baltimore | DNA vaccines for eliciting a mucosal immune response |
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US6287569B1 (en) * | 1997-04-10 | 2001-09-11 | The Regents Of The University Of California | Vaccines with enhanced intracellular processing |
EP1056842A2 (en) * | 1998-02-11 | 2000-12-06 | Maxygen, Inc. | Genetic vaccine vector engineering |
AU2487300A (en) | 1998-12-31 | 2000-07-31 | Chiron Corporation | Polynucleotides encoding antigenic hiv type c polypeptides, polypeptides and uses thereof |
CA2370646A1 (en) * | 1999-04-21 | 2000-10-26 | Powderject Vaccines, Inc. | Nucleic acid immunization |
US6875612B2 (en) | 2001-03-30 | 2005-04-05 | Greenville Hospital System | Monocyte-specific particulate delivery vehicle |
US7211659B2 (en) | 2001-07-05 | 2007-05-01 | Chiron Corporation | Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof |
US11529414B2 (en) | 2020-06-23 | 2022-12-20 | Orbis Health Solutions, Llc | Viral vaccines for in vivo expression of a nucleic acid encoding an immunogenic peptide and methods of using the same |
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DATABASE MEDLINE [Online] US NATIONAL LIBRARY OF MEDICINE (NLM), BETHESDA, MD, US 4 September 1995 (1995-09-04) FOMSGAARD A: "ÄGenetic immunization--"the biological equivalent of cold fusion"?]" Database accession no. NLM7676526 XP002238693 & UGESKRIFT FOR LAEGER. DENMARK 4 SEP 1995, vol. 157, no. 36, 4 September 1995 (1995-09-04), pages 4932-4936, ISSN: 0041-5782 * |
DATABASE MEDLINE [Online] US NATIONAL LIBRARY OF MEDICINE (NLM), BETHESDA, MD, US15 April 1995 (1995-04-15) ZAROZINSKI C C ET AL: "Protective CTL-dependent immunity and enhanced immunopathology in mice immunized by particle bombardment with DNA encoding an internal virion protein." Database accession no. NLM7706740 XP002238694 & JOURNAL OF IMMUNOLOGY (BALTIMORE, MD.: 1950) UNITED STATES 15 APR 1995, vol. 154, no. 8, 15 April 1995 (1995-04-15), pages 4010-4017, ISSN: 0022-1767 * |
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