CZ92998A3 - Process of stimulating cellularly mediated immunity response by making use of immunization through gene material bound on particles - Google Patents

Abstract

The present invention relates to various methods of genetic immunization for the purpose of providing antigen-specific immunity in a mammalian host, including a human host. The invention is based on the ability to direct particulate polynucleotides which express an antigenic protein or protein fragment to the cytoplasm of host target cells, such as antigen presenting cells. A directed delivery of such particulate polynucleotides to the cytoplasm of antigen presenting cells will stimulate antigen-specific CTL production, thus promoting destruction of affected cells such as neoplastic cells and virally infected cells.

Description

The invention describes gene immunization suitable for stimulating an antigen-specific immune response in mammalian hosts, including humans. The invention describes the administration of particle-bound polynucleotides into the cytoplasm of target host cells, which are, for example, antigen-presenting cells. Particles carrying polynucleotides encode an antigenic protein called antigenic protein, which accumulates in the cytoplasm of target cells. Expression of a gene encoding an antigen elicits a specific immune response to that antigen, including, but not limited to, the induction of antigen-specific T-lymphocytes (CTL). The accumulation of antigen in the cytosol allows the presentation of antigenic peptides on the membrane by means of endogenous MHC class I proteins (major histocompatibility complex proteins). The presentation of antigens by MHC class I proteins stimulates the induction of antigen-specific cytotoxic T-lymphocytes (CTL). Specific CTLs induced by the antigen then target and destroy host cells that express the antigen, such as tumor cells or virus-infected cells.

Current state of the art

Cytotoxic T-lymphocytes (CTL) are a critical component of an effective immune response to tumors or viral infections in humans. Cytotoxic T-lymphocytes destroy neoplastic cells or virus-infected cells by recognizing antigenic peptides presented on MHC class I proteins ·· 9 • 9 ·· ·

on the surface of affected target cells. These antigenic peptides are degradation products of foreign proteins present in the cytosol of affected cells, which are processed and presented to cytotoxic T-lymphocytes using the endogenous MHC class I pathway.

Although recognition of a foreign protein presented by MHC class I molecules may be sufficient for * recognition and destruction of the affected cell by cytotoxic

T-lymphocytes, yet the induction of antigen-specific CTLs from T-lymphocyte precursors requires additional signals.

Specialized antigen-presenting cells (APCs) can provide both ligands for the antigen-MHC class I complex and additional signals for the elicitation phase of cyto-mediated immunity. ----------General features of APC include expression of MHC proteins

I. and II. classes as well as the expression of stimulatory molecules such as CD80 and CD86. Examples of APCs include macrophages and dendritic cells (including skin epidermal Lagerhans cells, dermal dendritic cells, and dendritic cells found in the lymph nodes and spleen).

Attempts to induce an in vivo antigen-specific CTL response by immunizing the host with killed tumor cells, killed virus-infected cells, or protein fragments thereof have mostly been unsuccessful,

- primarily because -proteins., from extracellular fluids. they could not enter the cytosol and thus achieve their presentation by MHC class I antigens.

Gene immunization has several advantageous features. There are several in vivo gene transfer methods used leading to transgene expression. These include, for example, gene transfer mediated by retroviruses and adenoviruses and direct • · • · · • ·

-3injection of bare DNA (for a review see Krishnaw, et al., 1995, Nature Med. 1: pages 521 to 522 and Pardoll et al., 1995, Immunity 3: pages 165 to 169.)

Williams et al (1991, Proc. Nati. Acad. Sci. USA 88: pages 2726-2730) reported expression of luciferase protein in intact epidermal cells following biolistic (bioballistic) introduction of the firefly luciferase gene. CTLs were not included in this study, nor was attention focused on directing host cells to the process of generating a cell-mediated immune response.

Tang et al. (1992, Nature 356: pages 152 to 154) —used a bioballistic device to generate a humoral response to—foreign—because—into—epidermal_ tissue of mice a gene encoding hGH was introduced under the control of either CMV promoter or β-actin promoter. Antibodies against hGH were demonstrated in mice subjected to this immunization process. However, Tang et al do not describe gene immunization targeting a cell-mediated immune response. Tang et al also do not describe or propose the direct use of APC cells for gene immunization.

Fynan et al. (1993, Proc. Nati. Acad. Sci. USA 90: pp. 11478-11482) confirmed the results of Tang et al. when they used a plasmid containing a DNA construct encoding the influenza virus hemagglutinin glycoprotein. Fynan et al ' compared the humoral response induced by introducing DNA on coated gold particles into the epidermis using a gene gun with other mechanisms and found that the use of a biolistic (bioballistic) device resulted in:

1. the emergence of 95% protection against the lethal consequences of influenza exposure, ·♦

2. it has been found to be the most effective route for DNA immunization, showing to be more effective than mucosal, intramuscular or intravenous antigen administration immunization procedures and that

3. immunization requires 250 to 2500 times less DNA than standard saline inoculations.

However, the direct use of APC cells for gene immunization was neither described nor considered in the work of Fynan et al., nor was CTL cell-mediated immunity observed.

Liu and colleagues (Montgomery et al. 1993, DNA Cell Biol. 12: page 777 to 783, Ulmer et al. 1993, Science. 259: page 1745 to 1749, Donelly et al. 1995, -Nature—Medidne__1_:__page 583 to 587.) for caries and Γΐ; that non-targeted, non-specific intramuscular injection of free DNA (naked DNA) induces an antigen-specific response to viral proteins in cytotoxic T-lymphocytes as well as protection of immunity against viral infections. However, these studies do not describe the targeted use of genetic material for gene immunization of antigen-presenting cells.

Sun et al. (1995, Proc. Nat. Acad. Sci. USA 92: pages 2889-2893) use a biolistic (bioballistic) device to induce an anti-tumor response in mice. The authors inserted a plasmid construct expressing IL-6 directly into the tumor site in mice. Expression of IL-6. induced a form of cytokine gene therapy non-specifically targeting the tumor. The work does not claim to induce specific immunity to the tumor. Also, the targeted use of APC cells for gene immunization is not described or proposed by Sun and others.

Kundig et al. (1995, Science. 268: pages 1343-1346) have shown that the localization of protein antigen in lymphatic organs is critical for antigen induction.

specific responses in vivo. Gene immunization is not described in the thesis.

Kovascovcs-Bankowski and Rock (1995, Science 267: pages 243-246) demonstrated that a protein antigen not normally presented by the endogenous MHC class 1 pathway can be taken up into the cytosol via the phagosome. The authors came up with the idea that a protein bound to particles and internalized inside the phagosome is virtually capable of entering the cytosol and being used in the cytosolic pathway of presentation on MHC class 1 antigens. However, the capacity of functionally intact genetic material to enter the cytosol via phagosomes is not mentioned in the thesis.

_______Falo et al. (1995, Nature Med. 1: pages 649 to 653) offers in—vxvo—support—for—the antigen's passage from the phagosome to the cytosol by showing that the entry of a particle-bound protein antigen directly into animals induces antigen-specific CTL-mediated tumor immunity in mice. They demonstrated that proteins injected directly into animals in vivo can specifically enter the cytosol of antigen-presenting cells via phagosome formation when administered bound to particles. However, no details regarding the method of gene immunization are recommended. The capacity of in vivo administered genetic material to enter the cytosol of antigen-presenting cells or other types of functionally intact cells by this route is not stated.

Pardoll and Backerleg (1995, Immunity 3: pages 165 to 169) provided a review on the immunology of vaccines containing free DNA. They emphasized the importance of further research to elucidate and define the hitherto unknown mechanism of DNA immunization. In particular, they underline the conclusion that it will be highly important to dissect the mechanism by which free DNA activates the immune response. Only such a study can • ·

-6 demonstrate that the introduction of reasonable modifications could maximize both qualitatively and quantitatively the limited possibilities of this immunization.

Despite the achievements mentioned in the previous literature review, there is still a need to develop a procedure for gene immunization that will specifically target such a type of cell that will stimulate an antigen-specific immune response in the host, mediated by cytotoxic T-lymphocytes and thus allow direct specific destruction of neoplastic or virally infected cells in the host's body. The invention describes a solution to both of these requirements.

The essence of the invention-------The invention relates to therapeutic or prophylactic gene immunization of mammalian hosts, which includes insertion of DNA fragments encoding an antigenic protein into the target cells of the mammalian host, expression of recombinant DNA fragments in the host cell and subsequent presentation of the antigenic peptide or peptides by the host cell by stimulating cell-mediated immunity, humoral immunity, or both.

The invention further describes therapeutic or prophylactic gene immunization of a mammalian host, which includes insertion

A DNA sequence encoding an antigenic protein or a biologically active fragment of this protein to a specific target cell of a mammalian host. Antigenic peptides, expressed from DNA fragments, stimulate and support, and are specific to the affected cell, the production of antigen-specific destruction of the affected cell, such as and subsequently

CTL, whereby the cell is a neoplastic or virus-infected cell.

• *

The invention further describes gene immunization using polynucleotides bound to particles, inoculation of mammalian hosts and subsequent penetration of these particles with transgenic polynucleotides into the cytosol of target cells. After being absorbed by this target cell, the polynucleotides attached to the particles express a protein or a biologically active fragment of this protein, thereby creating a self-antigenic peptide fragment that is presented on the membrane of the target cell using the endogenous MHC class I pathway. Self-presentation of the antigenic peptide or peptides that are the subject of our interest using the MHC class I presentation pathway stimulates the production of cytotoxic T-lymphocytes and thus stimulates the destruction of cells such as neoplastic cells or

-Virus-infected cells.

Furthermore, the invention describes a method of in vfvo therapeutic or prophylactic gene immunization of mammalian hosts, which includes the creation of DNA fragments that express an antigenic fragment surface-bound to an antigenic protein, a particle, which to the particle and the so-called DNA binding of this to a protein or fragment to a polynucleotide particle of an immobilized mammalian cell host, polynucleotide where delivery to the cytoplasm of the target expressed antigenic protein or a fragment of the antigenic protein is created on the membrane surface of the mentioned target cell using the MHC class I antigen presentation pathway.

In a particularly preferred embodiment of the invention, the mammalian host cells are of human origin. ''' , „ ₌

In other advantageous embodiments of the invention, the DNA fragment used expresses:

1. antigen causing tumor rejection (Tumor Rejection Antigen, TRA) or antigenic protein fragment or

2. viral antigen or antigenic protein fragment.

-8• · · *

Examples of human TRA antigens that can be used in the method of the invention include, but are not limited to, MAGE-1, MÁGE 3, Melan-A, gp100, p53, CEA, and HER2/neu. Examples of viral antigens that may be used in the invention include, but are not limited to, HIV gp 120, HIV gp160, influenza virus nucleoprotein, and hepatitis B virus surface antigen.

The invention preferably uses antigen presenting cells (APC) as target cells. The localization of these target cells in the lymphoid tissue or the ability to migrate to this tissue of the human host is advantageous.

In another advantageous arrangement of the invention, the mammalian host is immunized with a polynucleotide on particles using a projectile-bombardment device.

In a particular embodiment, the mammalian host is immunized by inoculation with a particle-bound polynucleotide using a biolistic (bioballistic) process. In a particular embodiment of the invention, a human host is immunized with a particle-bound polynucleotide using a bioballistic procedure such that the polynucleotide enters the cytoplasm of at least a sufficient number of host cells. Transgenic polynucleotides are concentrated in such a way that they are presented using expressed in biologically effective antigenic peptide fragments are endogenous presentation pathways of MHC class I antigens and are presented on the membrane surface of host cells. .Presentation of inserted antigens on cell endogenous host membranes increases the induction of antigen-specific cytotoxic T-lymphocytes, which further circulate in the mammalian organism, preferably in the human host, and thus destroy neoplastic cells or virus-infected cells.

In a special embodiment of the invention, the mammalian host, preferably a human, is immunized with a polynucleotide bound to particles using a microprojectile

bombardment, so a sufficient number of particles penetrates the polynucleotide immobilized on using projectiles into the cytoplasm of host cells, including antigen-presenting cells. In skin bombardment, antigen presenting cells (ACPs) may include, but are not limited to, epidermal Lagerhans cells, keratinocytes, or dermal dendritic cells. When lymphoid tissue is bombarded, antigen presenting cells (APCs) of lymphoid tissue that may be affected include, but are not limited to: resident ^lymphoid cells; macrophagesT—cells—st-roma-tu-,—T_- lymphocytes. or β-lymphocytes.

In another preferred embodiment of the invention, the mammalian host is immunized with particles with a bound polynucleotide, which is administered by direct injection, including, but not limited to, dermal injection, lymphatic injection, and intravenous injection. Particles with attached nucleotides enter the host cells and are expressed in a biologically effective amount so that the antigenic peptide fragments are presented using the endogenous presentation pathway of MHC class I antigens and are presented on the membrane surface of the host cell. Presentation on the endogenous membranes of target cells increases the induction of antigen-specific CTLs, which further circulate in the mammalian organism, preferably in the human host, and thus destroy neoplastic cells or virus-infected cells.

In a particularly preferred embodiment of the invention, the bound polynucleotide particles are delivered to the human host by subcutaneous injection and are modified to facilitate phagosome-mediated transfer to the cytosol of the APC, where they are expressed at a biologically effective concentration. The invention demonstrates

-10Λ» that direct injection of particles with bound polynucleotides (polynucleotide complex) via subcutaneous inoculation leads to penetration of this complex into the target antigen-presenting cells and the antigen is then expressed in the lymphoid tissue.

In another particularly advantageous embodiment of the invention, the subcutaneous injection used to direct the inoculum to the APC contains a particle with a bound polynucleotide encoding an antigen (TRA) or a biologically active fragment thereof. Specifically directed introduction of particles with bound polynucleotide encoding TRA in this way allows to increase the entry of specific antigenic peptides into processing in the presentation pathway of MHC class I antigens and thus prevents to a greater extent tra-n-location of immunization particles in other than in antigen presenting cells (APCj~ cells.

In another particularly advantageous embodiment of the invention, the subcutaneous injection used to direct the inoculum to the APC contains a particle with a bound polynucleotide encoding a viral antigen or a biologically active fragment thereof. The specifically directed introduction of particles with a bound polynucleotide encoding a raft antigen in this way allows to increase the entry of specific antigenic peptides into the processing in the presentation pathway of MHC class I antigens and thus prevents to a greater extent the translocation of immunization * particles in cells other than antigen presenting cells (APC). ’ —.= =

It is also known to those skilled in the art that particles made of various materials, including but not limited to gold, iron, and synthetic materials, can enter the cytosol of antigen-presenting cells via the phagosome. This delivery method is used in the invention to specifically target APC.

-11• that

In the following preferred embodiment of the invention, mammalian host cells are immunized by injection, including, but not limited to, subcutaneous injection, epidermal injection, dermal injection, lymphatic injection, and intravenous injection of syngeneic antigen-presenting cells (APCs), which present on their surface the antigen that has been injected into them introduced in vitro by incubation with particles with bound polynucleotide.

For example, polynucleotide-bearing particles can enter antigen-presenting cells in vitro either by microprojectile bombardment or via phagosomes. Specifically, mammalian host cells are immunized with polynucleotide-bearing particles in vitro by transfection of arrťi“gene~pre-z-en-t-ujú-G-í-eh—cell__(APC), which are then injected into the host organism! Subsequently—upon antigen uptake by antigen-presenting cells, transgenic polynucleotides are expressed in a biologically effective amount so that antigenic peptide fragments are processed and presented by the endogenous pathway of MHC class I antigens and are presented on the membrane surface of antigen-presenting cells.

After the injection of antigen-presenting cells prepared in this way, the antigen is presented on their endogenous cell membranes, which starts the induction of antigen-specific cytotoxic T-lymphocytes either at the injection site or in lymphoid tissues. Antigen-induced specific cytotoxic T-lymphocytes (CTL) further circulate in the mammalian organism, preferably in the human host, where they destroy neoplastic cells or virally infected cells.

In another preferred embodiment of the invention, the mammalian host is immunized by injection, including but not limited to subcutaneous injection, epidermal injection, dermal injection, lymphatic injection, and intravenous injection containing syngeneic antigen presenting cells that have been subjected to in vitro transfection with polynucleotide-carrying particles. The mammalian host is immunized with antigen-presenting cells into which polynucleotide-carrying particles have been transfected, in such a way that the particles with the polynucleotide enter the APC in vitro and the antigen-presenting cells (APC) modified in this way are then injected into the host organism.

Polynucleotide-carrying particles can, for example, enter antigen-presenting cells in vitro either by microprojectile bombardment or by trafficking into the cytosol of the cell via a phagosome. Antigen-presenting cells are subjected to in vitro transfection with an antigen that is -encoded—poly-yn.-k-1-eo-tide—and/or polynucleotides encoding a molecule or molecules that increase the effectiveness of pre-z-en- tational—--------function of antigen presenting cells. Such molecules include, but are not limited to, cytokines and stimulatory molecules. Examples of cytokines include, but are not limited to, IL12, IL-2, and IL-4. Examples of stimulatory molecules include, but are not limited to, CD80 and CD86 antigens.

Following entry into antigen-presenting cells, the transgenic antigen encoded by the polynucleotide is expressed in a biologically effective concentration so that antigenic peptide fragments are processed and presented using the endogenous presentation pathway of MHC class I antigens and are thus presented on the membrane surface of antigen-presenting cells (APCs). . ^: ₌ . _ , . _

After injection of such APCs, the presentation of antigens on the endogenous cell membrane of that cell increases the induction of specific cytotoxic T-lymphocytes. This induction then occurs either directly at the injection site or in the lymphoid tissue of the host. Specific cytotoxic T-lymphocytes induced by this antigen circulate in the mammalian organism,

preferably in the human body, thus destroying neoplastic cells or virus-infected cells.

Following entry into the APC, either the transgenic cytokine or the costimulatory molecule, encoded by the polynucleotide, is expressed at a biologically effective concentration to induce the antigen presentation function of the APC, which further induces the induction of an antigen-specific response, either directly at the site of injection or in lymphoid tissue.

It is also well known to those skilled in the art that polynucleotides can be attached to particles composed of various materials including, but not limited to, gold, iron and plastics.

It is well known to those skilled in the art that various recombinant vectors can be used to apply the generated transgenic sequences in the desired form. A preferred vector, which is preferred because it is very easy to work with, is a DNA plasmid.

It is further well known to those skilled in the art that antigen-presenting cells can be obtained from various host tissues, including, but not limited to, bone marrow and peripheral blood, and that said antigen-presenting cells can be returned to the host after in vitro manipulation.

The aim of the invention is therapeutic or prophylactic gene immunization against neoplastic cells. ___ __

Another object of the invention is therapeutic and prophylactic gene immunization against viral infections.

Another object of the invention is gene immunization of mammalian hosts, preferably humans, by specific targeting of particles carrying polynucleotides that encode genes for TRA (tumor rejection antigens) to the host's

-14antigen-presenting cells located in the lymphoid tissue of the host, or able to migrate to this tissue and thus participate in inducing a CTL response.

Another object of the invention is gene immunization of mammalian hosts, preferably humans, by specific targeting of particles carrying polynucleotides that encode viral antigen genes to the host's immune cells, which are involved in eliciting a CTL response.

Another aim of the invention is gene immunization of mammalian hosts, preferably humans, by specific targeting of particles carrying polynucleotides encoding viral antigens to antigen-presenting cells located in the lymphoid tissue of the host, or able to migrate to this tissue and thus to -induce—CTL—about dresponse ----- ---Brief description of images

Figure 1 shows the functional presentation of ovalbumin by transfected M04 and EG7 tumor cells. Microcultures were prepared from hybridoma RF33.70 T-lymphocytes (anti-OVA + K ^b ) and the indicated amount of transfected (squares) and non-transfected (circles) tumor cells in the presence (open symbols) or absence (filled symbols) of delivered exogenous ... ..ovalbumin peptide SIINFEKL (10 ng/ml) see Rock et al. 1990, J. Immunol. 45, pp. 804-811. After an 18-hour incubation, supernatants were collected and assayed for IL-2 using the indicator cell line HT2 (see Rock et al. 1990, J. Immunol. 45, pp. 804-811). Figure A shows cell line B16 and ovalbumin-transfected subclone MO4, Figure B shows cell line EL4 and ovalbumin-transfected subclone EG7. Presentation of ovalbumin (OVA) ·· ·

-15 peptides by transfected tumor cells was not significantly increased in the presence of exogenous peptide SIINFEKL in the tested cultures. The numbers of APC are shown on the horizontal axes and CPMxlO' ³ on the vertical axes.

Figure 2 shows that expression of ovalbumin OVA by B16-derived MO4 melanoma cells does not affect in vivo tumor growth or host survival after tumor cell administration. Mice were intradermally injected with M04 (circles) or B16 (squares) tumor cells at a dose of 5×10 ⁴ per mouse into the center of both groins. Tumor size (Figure 2A) was determined 3 times per week and is expressed as the average tumor area in square millimeters (vertical axis) until the first death in each group. Survival (Figure “2B') bvTo~za-z narne navá-no-^-ake—or^> cent o—pr-eží vaj. icich animals and___________ is shown on the vertical axis. In both—-------cases, the days since the last immunization ^are shown on the horizontal axis. In all experiments, experimental groups consisted of five animals and were repeated three times. Mortally ill mice were sacrificed.

Figure 3 shows that immunization after cutaneous administration of OVA-encoding DNA induces OVA-specific cytotoxic T-lymphocytes and antigen-specific, CTL-mediated protection against an otherwise lethal M04 melanoma that expresses OVA on its surface. Splenic cells restimulated in vitro after collection from OVA-immunized mice (gene-immunized mice see Example 2) were tested for their cytolytic function against OVA-transfected EG7 lymphoma (filled squares) or against non-transfected parental EL4 lymphoma (open triangles) (A) . The effector cell population was incubated with complement alone (open squares) or with monoclonal antibodies against CD4 ⁺ (filled triangles), CD8 ⁺ (open triangles), or Thyl.2 ⁺ (filled circles)

-16 lymphocytes and with complement, and then their cytolytic activity against the target cells of the EG7 tumor line was determined. The ratio of E/T cells is shown on the horizontal axis, % specific ski is on the vertical axis. Mice in Figures C to F were genetically immunized with OVA (filled squares) or with lacZ (open circles). Booster immunization was performed after 7 days. 7 days after the last immunization (which took place on day 0), mice were challenged with a dose of either melanoma B16 (D) or its OVA expressing subclone MO4 (C). Optionally, mice were divided into two groups, one of which was depleted of CD8 ⁺ lymphocytes by intraperitoneal injection of anti-CD8 monoclonal antibody 7 and 9 days after the last immunization. Intact mice (E) and mice depleted of CD8 ⁺ lymphocytes -were exposed to a dose of MO4 on day 1.0 after the last immunization.

PrežTEi j~e~^vyj~ádreiro—j-as—preeenťe—survival-i-ovi-íclch animals (C to F) . No signs of tumor growth were observed in animals that survived day 60. In all experiments, experimental groups consisted of five animals and were repeated three times. Terminally ill mice were killed in accordance with laboratory animal care regulations. Horizontal timelines are calibrated in days. The vertical axes are the percentage of surviving animals.

Figure 4 shows that antigen presenting cells internalize and then express particle-bound polynucleotides. The expressed antigen is processed by these cells and presented by the MHC class I pathway. Dendritic cells were prepared by separating lymphocytes from bone marrow and culturing it overnight in RPMI 1640 medium supplemented with 10% FCS (fetal calf serum), L-glutamine, antibiotics and 2-mercaptoethanol in 24-well plates at a concentration of 10θ cells per well. After one day, cells were supplemented with GM-CSF (10 ³ U/ml, Sigma St. Luis, MO) and mouse rIL-4 (10 3 u/ml, Genzyme, Cambridge, ΜΆ) at a concentration of 2.5x10^ cells per well ). Loosely adherent cells were collected on day 8 of culture.

-17 Flow cytometry determined that these dendritic cells express CD45, CD44, CD11b (Mac-1), CD18, CD80, CD86 and MHC I and II antigens. classes. Dendritic cells were then pulsed for 2 h at 37°C in the presence and absence of OVA peptides (20 ng/ml) + β2-microglobin (human β2-Μ, 10 μΐ/ml, Sigma) in serum-reduced medium (Optimen, Gibco, Grand Island , NY). Cells were then thoroughly washed in PBS and irradiated (2000 rads) and injected into naïve mice. The indicated number of bone marrow-derived dendritic APC cells were co-cultured with OVA encoding polynucleotides attached to particles (for their preparation, see example 2) (50 µl/ml/10 ⁶ cells of a preparation containing 7 mg of particles per ml). Either iron beads (filled squares) or gold beads' (filled—circle-yj—or soluble OVA protein (2mg/ml, open squares) were used as nucleotide-binding substrate. Incubation was for 24 h, after which APC cells were washed and the indicated amount of APC was co-cultured with T-lymphocytes from hybridoma RF33.70 (anti OVA +K ^b ). After 18 hours of incubation, the supernatants were collected and IL-2 was determined in them using the indicator cell line HT2 (see Rock and et al. 1990, J. Immunol. 45, pp. 804-811).

Figure 5 shows a comparison of immunization with particle-bound polynucleotides by bioballistic delivery and by subcutaneous injection. C57B1/6 were immunized and po

Groups of 5 mice line days the dose was repeated. Then, they were exposed by intradermal injection

5x1O ⁵ MO5 tumor cells into each groin another 7 days later. The immunization doses contained: (A) a polynucleotide encoding β-gal bound to particles, prepared and bioballistically administered according to the method described in Example

2; (B) polynucleotide encoding prepared and bioballistically

OVA bound to particles, applied according to the method described in Example 2; (C) increased amount of polynucleotide

• · • 99 • • · 99 • • • 9 9 9 • · 9 9 9 in • • · • · 9 ♦ • · • ···· • · · ···· 9 9 9 9 9 • • 9· • 9 9 • ··· • ♦ ·· 99 • ·

encoding OVA without carrier particles administered by subcutaneous injection; (D) an equivalent amount of polynucleotide encoding OVA, which was prepared according to the method of Example 2, but was administered by subcutaneous injection. Data shown indicate the % of animals in each group that were tumor free at 50 days post-challenge.

Figure 6 shows immunization with APCs co-incubated with particle-bound polynucleotide encoding OVA. Such immunization protects animals against melanoma of the MO5 line, which expresses OVA peptides on its surface. Groups of C57B1/6 mice were immunized once subcutaneously as in Figure 5 and then challenged with a dose of 10 ⁵ MO5 tumor cells into each groin 10 days after immunization. Immunization doses contained: control -po±ynu-kle©-ti-d-encoding β-gal attached to particles (open squares), 100 µl of a particle solution with a concentration of 7 mg/ml was applied to each hind limb; soluble pAc-Neo-OVA (100 μΐ into each hindlimb, approximately the same DNA/animal ratio was maintained, open circles); or 5x104 bone marrow-derived dendritic cells (filled squares), again 100 μΐ was applied to one hind limb of the animal. Survival is expressed as a percentage of surviving animals. In all experiments, experimental groups consisted of five animals. Terminally ill mice were killed in accordance with laboratory animal care regulations. The horizontal time axis is calibrated in days and the vertical axis is calibrated in percentage of surviving animals.

Detailed description of the invention

The term mammalian host, as used herein, includes representatives of the animal kingdom including, but not limited to, humans.

-19ΦΦΦ · · φ « φ φφ ··· · · r φ ·· ·· • φφφφ φ φ φ φφφφ · ··· ®φ • · φφφ · ·· φφφφ φ φφ · φφ φφ

The term DNA fragment, as used in the present invention/ means any DNA or RNA nucleotide sequence that contains a suitable coding region and regulatory sequences that induce in the target cell the expression of an antigenic protein or a fragment of an antigenic protein presented on the cell membrane via the endogenous MHC antigen presentation pathway I.class.

The term particle-bound polynucleotide as used herein refers to particles made of various materials, including, for example, gold, iron, and synthetic plastic, where each individual particle contains a population of DNA fragments, as defined in the preceding paragraph.

----------Invention ^- pop±sug-e—l-therapeutic—or-preventive-vnl— genetic------- immunization of a mammalian host that involves transfer of a DNA fragment to a target cell within mammalian host, the expression of this DNA fragment inside the target cell and the subsequent presentation of the recombinant antigenic peptide(s) inside the target cell, which leads to the stimulation of cellular or humoral immunity, or both.

The invention further relates to the therapeutic or preventive gene immunization of a mammalian host, which involves the transfer of a DNA fragment encoding a protein or its biologically active fragment to a specific target cell within the mammalian host.-Antigenic peptides resulting from expression from the given DNA.

the sequences are specific for altered cells, such as tumor cells or virus-infected cells. There is activation of cytotoxic T-lymphocytes (CTL) specific for the given antigen and subsequently cytotoxic destruction of target cells such as tumor cells or cells infected with a virus.

-20 The invention describes a method of gene immunization, which serves to treat or prevent cancer or viral infections. Cytotoxic T-lymphocytes are an important component of the immune response directed against tumors and viral infections. Cytotoxic T-lymphocytes kill tumor cells and virus-infected cells due to the fact that they can recognize antigenic peptides presented by MHC class I molecules on the surface of tumor cells. These peptides are derived from tumor antigens that are synthesized by altered cells and degraded in the cytosol. Attempts to induce tumor-specific responses of cytotoxic T-lymphocytes in vivo by immunization with dead tumor cells or their structural proteins are usually unsuccessful, probably because the proteins from the extraction fluid ^cannot enter the cytosol and therefore, they do not have the possibility to be presented through MHC class I molecules.

A specific embodiment of the present invention is associated with genetic immunization using particles containing a DNA fragment, the expression of which produces the desired protein. Such particles containing a DNA fragment are further referred to as particle-bound polynucleotides. In an in vivo embodiment of the invention, delivery of the DNA fragment or desired protein particle to the target host cell in a complex form, e.g. as coated beads or gold particles, appears to be the best. Following administration into the cytosol of the target host cell, the protein or biologically active fragment is expressed by this cell. The expressed protein or protein fragment specific for the altered cell provides a substrate for the creation of antigenic peptides (antigenic peptides), presented to T-lymphocytes via endogenous MHC class I molecules. Appropriate presentation of the desired antigenic peptide or antigenic peptides through MHC class I molecules stimulates ·· ·♦ · ·«··

• · · • ···· · • « · · • · 4 · · • · · ·· ·· production of cytotoxic substances by T-lymphocytes and thus increases the destruction of altered cells.

The invention is based on the premise that a DNA fragment expressing a TRA antigen or a viral antigen or their active fragments can be specifically directed to the target cell and there trigger a cascade of processes leading to anti-tumor immunity mediated by cytotoxic T-lymphocytes. The present invention elucidates the induction of cytotoxic T-lymphocyte-mediated immunity by transfection of a target host cell with a particle containing a DNA fragment that encodes an antigenic protein. The immunizing protein is produced intracellularly and thus has access to class I MHC molecules. As a result, naturally processed epitopes and transfected ^cells can produce the immune ^- stimulating protein for several days, potentially allowing for more intense immunogenic stimulation.

In a specific embodiment of the invention, the mammalian host is immunized with a particle-bound polynucleotide by means of “microprojectile bombardment. This non-specific bombardment is performed so as to result in the entry of the particle-bound polynucleotide into the cytoplasm of a sufficient number of host cells. In a preferred embodiment of the invention, these host cells include antigen presenting cells (APC), but the invention is not limited to them. In the case of skin bombardment, APCs that may be affected by bombardment include epidermal Langerhans cells, keratinocytes, or skin dendritic cells. In the case of lymphoid tissue bombardment, antigen presenting cells that may be affected by bombardment include dendritic cells, macrophages, stromal cells, T-lymphocytes or B-lymphocytes. The transgenic polynucleotide is expressed in a biologically effective amount, so that the processing of antigenic peptide fragments and

-22 their presentation through endogenous MHC molecules

I.class on the membranes of APC antigen on the membrane of APC specific CTL either directly of cells. Endogenous presentation promotes antigen induction at the site of bombardment or in the lymphoid tissue where the bombarded cells are transferred.

Induced antigen-specific CTLs circulate throughout the mammalian, preferably human, host with the aim of destroying tumor cells or virus-infected cells.

Immunization of a mammalian host with a linked polynucleotide_E on particles can be performed by biolistic (bioballistic) delivery such that the polynucleotide bound to the particles enters host cells, which include APCs, via phagosomes. Polynucleotides attached to the -----------part of the CTch—enter^-!—through—phagosomes—into—cells—either after they are transferred to the lymphoid tissue, or directly at the site of bombardment (in the skin or in the lymphoid tissue). Thus, polynucleotides attached to particles can enter the lymphoid tissue in two ways. Either the polynucleotides attached to the particles are transported to the lymphoid tissue, where they are subsequently accepted by the cells of this tissue, or the cells that have accepted the polynucleotides attached to the particles are transferred to the lymphoid tissue. The polynucleotide attached to the particles is expressed in a biologically effective amount after being accepted by the host cell, which ensures the processing of antigenic peptide fragments and their presentation through .. ........ endogenous MHC class I molecules on the membranes of antigen-presenting cells. Endogenous presentation of antigen on the membrane of antigen-presenting cells promotes the induction of antigen-specific cytotoxic T-lymphocytes either directly at the site of bombardment or in lymphoid tissue. Induced antigen-specific cytotoxic T-lymphocytes circulate throughout the mammalian, in a preferred embodiment of the invention human,

-23·· * • · · ♦ · ···· ···· »· • · • · · • ···· • · ·· ·♦ 9 · ·· •· ··

999 ·· • ·· ·· ·· hosts, with a target infected by the virus.

to destroy tumor cells or cells, for example, dermal, lymphatic bound to particles of cells expressed in a mammal for injection or a host of particles using subcutaneous, intravenous.

after entering the biologically effective processing of antigenic fragments and their presentation through MHC molecules of class I on the membranes of APC cells.

In another embodiment of the invention, immunized with an injection linked polynucleotide. Can epidermal, Polynucleotide host amounts, peptide endogenous

Endogenous presentation of antigens on the APC membrane promotes the induction of antigen-specific cytotoxic T-lymphocytes, “which circulate in the mammalian forehead; in an advantageous design of the invention to a human, a host, with a target or cells infected with a virus.

destroy tumor cells

Mammalian immunized by specifically entering through phagosomes. particles enter the host can be bound to cell particles, including APC,

Particle-bound polynucleotides enter host cells via phagosomes either after being transferred to lymphoid tissue or directly at the site of injection. Thus, polynucleotides attached to particles can enter the lymphoid tissue in two ways. Either the polynucleotides attached to the particles are transported to the lymphoid tissue, where they are subsequently taken up by the cells of this tissue, or the polynucleotides are transferred to the lymphoid tissue.

acceptance effective

After biological processing of the antigen bound by the cell, the amount is peptide fragments and their presentation through endogenous MHC molecules of class I on the membranes of APC cells.

Endogenous antigen presentation on the APC membrane promotes the induction of antigen-specific CTL either directly at the injection site or in lymphoid tissue. Induced antigen-specific CTLs circulate throughout the mammalian, preferably human, host with the aim of destroying tumor cells or virus-infected cells.

In another embodiment of the present invention, the mammalian host is immunized with the particle-bound polynucleotide by injection. It can be, for example, a subcutaneous, epidermal, dermal, lymphatic or intravenous injection. The composition of the polynucleotide attached to the particles is designed so that it contains specific molecules that ensure easy phagocytosis (i.e., for example, through receptors for ^- mannose—or—through medium±ct vim—Fc—receptors) or—so-k-> —to facilitate the accessibility of the particle in the cytosol (i.e., for example, using membrane fusion proteins, such as the viral HA protein or the listerolysin protein). Particle-bound polynucleotides enter host cells via phagosomes either lymphoid bound enter thus

Either tissues or directly on the particles in two ways.

transported on particles subsequently accepted by cells of lymphoid tissue transferred polynucleotides attached to Polynucleotide attached. r.a host cell amount, so it is until after they are transferred to the injection site. Polynucleotides can enter the lymphoid tissue, the polynucleotides are bound to the lymphoid tissue, this tissue, or the cells, which particles are already expressed.

to adopt effective po.

in biological processing of antigenic peptide fragments and their presentation through endogenous MHC class I molecules on the membranes of APC cells. Endogenous antigen presentation on the APC membrane promotes the induction of antigen-specific CTL either directly at the injection site or in lymphoid tissue. Induced antigen-specific CTL • ·

-25 circulate in the entire mammalian, in a preferred embodiment of the invention human, host, with the aim of destroying tumor cells or virus-infected cells.

The invention is illustrated by several examples using the murine melanoma model described in detail in example 2. The main limitation in the study of antigen-specific tumor immunity in the murine model is the absence of a uniform tumor antigen that would be recognized by CTL cooperating with MHC class I. Since TRAs are not fundamentally different from other cellular proteins except for the fact that the host is not tolerant to them, a foreign antigen synthesized by the tumor can be used for the same purpose. The methods of tumor immunization according to the invention are very useful of the la-nome--------model------------ with a defined, endogenously synthesized. TRA which is the OVA gene transfected into B16 melanoma derived from the C57B1/6 mouse line. This system is advantageous for several reasons: *(1) melanoma B16 is a very well studied murine tumor, (2) the in vivo growth characteristics and ability to metastasize are very well known for this tumor, (3) ovalbumin also has a well-defined structure. The manner of processing and presentation of OVA in C57BL/6 mouse line cells is also well known. The structure of the processed peptide presented by MHC class I K* ⁵ is even known. Methods for determining the functional expression of the ovalbumin peptide [SIINFEKL] associated with H2-K ^b using TT hybridoma 33.70 are also known. AI an ti -OVA-K* ³ , see Koyacoyics-Banjcqwski____and others,

1993, Proc. Nati. Acad. Sci. USA. 90: page 4942 to 4946).

Methods for determining the in vivo induction of cytotoxic T-lymphocytes with specificity against OVA are also well described in this system (Moore et al., 1988, Cell 54, pages 777-785).

• · • β

One embodiment of the invention is immunization with a selected polynucleotide bound to particles using a biolistic (bioballistic) device. A mammalian host is bioballistically immunized with a particle-bound polynucleotide such that uptake of this polynucleotide by transfected cells triggers a series of biological events that lead to protective immunity against a lethal cancer. The invention is described several times on the example of skin administration of antigen using a bioballistic device. First, penetration of a particle-bound polynucleotide encoding a foreign β-gal protein leads to the expression of the β-gal protein both in the epidermis and in the lymph nodes. Also, immunization with OVA leads to the induction of cytotoxic T-lymphocytes with _______specificity for ovalbumin. Third, when using the OVA model --------B—;1-6—it has been shown that the immune systems induced by OVA are ___________ protected against tumor formation expressing OVA protein and this protection is antigen-specific and CTL-dependent. The mechanism of this protection is more complex than general skin delivery on particles of bound polynucleotides using a bioballistic device. Specific targeting of the polynucleotide to phagocytic APCs and/or antigen expression in lymphoid tissue is necessary for the induction of antigen-specific CTLs from their precursors. Therefore, in a preferred embodiment of the invention, the target cell is a phagocytic APC and preferably an APC located in the lymphoid tissue or an APC that can migrate into it.

In another embodiment of the invention, the mammalian host is immunized with a directly injected polynucleotide bound to _ ₌ particles, the injection administration including, for example, subcutaneous, epidermal, dermal, lymphatic and intravenous injection. The transgenic polynucleotide is expressed in a biologically effective concentration so that fragments of antigenic peptides are produced and presented by the endogenous MHC class I pathway and exposed on the surface of APC cells.

• · • ·

-27 Presentation on the cell membrane of the APC accelerates the induction of antigen-specific cytotoxic T-lymphocytes that circulate in the body of the mammalian host, in a preferred embodiment of the invention the human host, and destroy tumor or virally infected cells.

In a preferred embodiment of the invention, target mammalian cells are immunized by means of direct injection of polynucleotides bound to APC phagocytizing particles. The transgenic polynucleotide is expressed at a biologically effective concentration so that antigenic peptide fragments are presented by the endogenous MHC class I pathway and exposed on the surface of the membrane of antigen presenting cells (APC). Presentation on the cell membrane of APC triggers and enhances the induction of antigen-specific cytotoxic T-lymphocytes (CTL), which are known as mammalian host cells. by a preferred___________ embodiment of the invention the human host and destroys neoplastic or virally infected cells.

In a preferred embodiment of the invention, the particle-bound polynucleotide is administered to the human host by subcutaneous injection. The invention describes the fact that direct injection of a particle-bound polynucleotide complex leads to targeted delivery of phagocytic APC and gene expression in lymphoid tissue. This targeted delivery to phagocytic antigen-presenting cells and/or this gene expression in lymphoid tissue after subcutaneous administration results in significantly higher induction of antigen-specific cytotoxic T-lymphocytes (CTL) from naïve precursors. _ .....___ _

The invention therefore mainly describes DNA fragments encoding a protein or its biologically active fragment, which is able to enter antigen-presenting cells and be expressed in these cells. The desired antigen is expressed in the APC cytoplasm and then further processed by the MHC class I pathway, allowing these antigen-presenting cells to participate in

-28 induction of antigen-specific CTLs. The data presented herein support the novel premise that the most efficient way to induce such antigen-specific cytotoxic T-lymphocytes is direct transfer of genetic material to antigen-presenting cells in lymphoid tissue or to antigen-presenting APC cells capable of migrating to lymphoid tissue.

In another embodiment of the invention, the mammalian host is immunized by injection, including, for example, subcutaneous, epidermal, dermal, lymphatic and intravenous injection, containing syngeneic antigen-presenting APCs obtained after in vitro administration of a particle-bound polynucleotide. In a particular embodiment of the invention, the mammalian host is immunized

-----pomeeí--A-PC-----which-have__been__transfied__with a particle-bound polynucleotide so that this polynucleotide is taken up by APC cells in vitro and these antigen-presenting cells are then injected into the host. After the entry of the transgenic polynucleotide into the APC, it is expressed to reach a biologically effective concentration so that the antigenic peptide fragments are processed and presented by the endogenous MHC pathway. Class I and exposed on the surface membrane of antigen-presenting cells. After injection of these APCs, antigen presentation on their cell membranes induces the formation of antigen-specific cytotoxic T-lymphocytes either at the injection site or in the lymphoid tissue. The induced CTLs are circulated throughout the body of the mammalian host, preferably the human host, and destroy neoplastic or virally infected cells.

In another specific embodiment of the invention, the mammalian host is immunized by injection, including, for example, subcutaneous, epidermal, dermal, lymphatic and intravenous injection, containing syngeneic antigen-presenting APCs obtained in vitro by co-incubation with a polynucleotide bound to

particles. In a particular embodiment of the invention, a mammalian host is immunized with APCs that have been transfected with a particle-bound polynucleotide such that the polynucleotide is taken up into the cytosol of the APC cells via phagosomes, and these antigen-presenting cells are then injected into the host. After the transgenic polynucleotide enters the APC, it is expressed to reach a biologically effective concentration so that antigenic peptide fragments are processed and presented by the endogenous MHC class I pathway and exposed on the surface membrane of antigen-presenting cells. After the injection of these APCs, the presentation of antigens on their cell membranes induces the formation of antigen-specific cytotoxic T-lymphocytes either at the injection site or in the lymphoid tissue. Induced CTLs are distributed in the circulation. throughout the body of the mammalian host, in a preferred embodiment of the invention, the human host and destroys neoplastic or virally infected cells.

In another specific embodiment of the invention, the mammalian host is immunized by injection, including, for example, subcutaneous, epidermal, dermal, lymphatic and intravenous injection, containing a syngeneic APC presenting an antigen obtained in vitro using a particle-bound polynucleotide. In this particular embodiment of the invention, the mammalian host is immunized with APCs that have been transfected in vitro by bombardment with microprojectiles containing particle-bound polynucleotide. After this exposure to microprojectiles with transgenic polynucleotide, these antigen-presenting cells express the desired antigen in a biologically effective concentration so that the antigenic peptide fragments are processed and presented by the endogenous MHC class I pathway and exposed on the surface membrane of the antigen-presenting cells. After the injection of these APCs, the presentation of antigens on their cell membranes induces the formation of antigen-specific cytotoxic T-lymphocytes either at the injection site or in the

-30 lymphoid tissues. Induced CTLs are circulated throughout the body of a mammalian host, preferably a human host, and destroy neoplastic or virally infected cells.

In another specific embodiment of the invention, the mammalian host is immunized by injection, including, for example, subcutaneous, epidermal, dermal, lymphatic and intravenous injection, containing syngeneic APC transfected in vitro with a particle-bound polynucleotide. In this particular embodiment of the invention, a mammalian host is immunized with APCs that have been transfected in vitro with a particle-bound polynucleotide and then these APCs are injected into the host. These bound on particles ---po-l-y-n-u-k-l-eo-fe-i-d-y---enter---de---cells---either---by bombardment, microprojectiles or via phagosomesT APC cells are transfected in vitro with polynucleotides that they encode an antigen and/or a molecule or molecules that increase the efficiency of antigen presentation on the surface of APCs. Such molecules are, among others, for example cytokines such as IL-12, IL-2 and/or IL-4 and/or costimulatory molecules such as CD80 and/or CD86. After entering the antigen-presenting cell, these polynucleotides encoding transgenic antigens are expressed so that a biologically effective concentration is reached and the peptide fragments of these antigens can be. processed and presented via the endogenous MHC class I pathway on the APC surface membrane. After injection of such an antigen. presenting cells and antigen presentation on _ their cell membranes, the induction of antigen-specific cytotoxic T-lymphocytes (CTL) will be enhanced either at the injection site or in the lymphoid tissue. Induced antigen-specific CTLs are distributed throughout the body of a mammalian host, preferably a human host, and destroy neoplastic or virally infected cells. Polynucleotides encoding transgenic cytokines and/or costimulatory factors ·· • ·

-31 are also expressed after entering the APC to such an extent that their concentration is biologically effective and thus that the presentation of the antigen on the surface of the APC leads to the induction of an antigen-specific immune response either at the injection site or in the lymphoid tissue.

One skilled in the art can insert a tumor rejection antigen (TRA) or any viral antigen into the system of the invention. Examples of currently available TRAs include, but are not limited to, MAGE-1, MA.GE-3, BAGE, GAGE-1, GAGE-2, tyrosinase, Melan-A(MART-1), gp100(pmell7), gp75(TRP1), CEA (carcinoembryonic antigen) as well as antigens from tumors caused by HPV, HBV, and EBV viruses as well as tumor-associated oncogenes/antigens encoded by mutated genes for up-resors of tumors such as p53, p!6, RAS, HER2/neu, C-___________

ABL, ar polymorphic antigens θη^ΡίύΓθΐΤΑΙηί^ο—mucin—(vi-z---------summary article Maeurer et. al., 1996, Cancer Vaccines in Clinical Immunology Principles and Practice, editors R.

Rich, Mosby Publishers, Chapter 123, pp. 1904 to 1918 and Van den Eydne et al., 1995, J. Exp. Copper. 182: pages 689 to 698. Examples of available viral antigens include, but are not limited to: influenza virus nucleoprotein, (Donnelly et al., 1995, Nature Med. 1, pages 583 to 587), HIV gp120, HIV gp160, and hepatitis B virus surface antigen (see summary article Pardoll and Beckerleg, Immunity, 1995, 3: page 165 to 169).

Those skilled in the art will recognize that any available eukaryotic promoter and/or enhancer capable of enhancing the expression of a transgenic DNA sequence along with any particle to form a particle-bound polynucleotide of the invention may be used to construct a recombinant vector. Examples of such fragments of promoter sequences include: cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, murine virus promoter

leukemia (MLV), the β-actin promoter as well as any cell-specific promoter or enhancer known to be active in the target cell.

A preferred embodiment of the invention is the use of particle-bound DNA to deliver nucleic acid encoding a tumor or viral antigen specifically to APCs in lymphoid tissue, or to antigen-presenting cells capable of entering lymphoid tissue so as to stimulate antigen-specific CTLs. These cytotoxic T-lymphocytes are then distributed into the host's body and destroy neoplastic or virally infected cells.

Antitumor immunity based on a preferred method of using subcutaneous injection inducing a specific response in antigen-presenting cells of lymph nodes is described in Example 1, 2 and 3. The following examples are given to illustrate the implementation of the invention and are in no way limiting.

Example 1: 0VA/B16 mouse tumor-based model

The 0VA/B16 mouse model was used to obtain the data presented in Examples 2 and 3. The attractiveness of the 0VA/B16 mouse system lies in several facts: (1) B16 melanoma is a well-studied murine tumor, (2) the growth characteristics and ... the ability to metabolise and act in vivo are " u ." the cancerous one. lines well characterized and (3) the structure of ovalbumin is well defined. The manner of processing and presentation of OVA in C57BL/6 mouse line cells is also well known. The structure of the processed peptide presented by MHC class I is even known. Methods of determining the functional expression of the ovalbumin peptide [SIINFEKL] associated with Η2-Κ^ using T-T hybridoma 33.70.AI anti-OVA-Kb are also known, see

Kovacovics-Bankowski et al., 1993, Proc. Nati. Acad. Sci. USA. 90: page 4942 to 4946). Methods for determining the in vivo induction of cytotoxic T-lymphocytes with specificity against OVA are also well described in this system (Moore et al., 1988, Cell 54, pages 777-785).

Mice and cell lines. Female 5- to 8-week-old C57BL/6 mice were purchased from Jackson Laboratories, Bar Harbor, ME. The EL4 line is a C57BL/6 T-lymphoma and the EG7 line is a subclone of EL4 transfected with chicken egg ovalbumin (OVA), see Moore et al., 1988, Cell 54, pages 777 to 785. The B16 mouse melanoma derived from the C57BL/6 mouse line was obtained from American Tissue Collection (ATCC), see Fidler et al., 1976, .Cancer Res. 36, page 3160 to 3165). MO4 was constructed by —trans-fec-GÍ—line—Bi6—plasmid—pAc^Nco—OVA_viz__Ealo__and____ others, 1995, Nátúre Med. Γ, page 649 to 65'3") (Moore et al., 1988, Cell 54, 777 to 785). Monoclonal antibodies were prepared from hybridomas GK1.5 (anti-CD4, ATCC TIB-207), 2.43 (anti-CD8 prepared after immunization of Balb/c nu/nu mice by ip injection of GKfl.5 cells (3x10 ⁶ ) and IFA ( 0.5 ml/mouse).

Subclone MO4 was isolated after transfection of melanoma B16 with OVA and subsequent selection. Both the parental B16 melanoma line and the OVA-transfected line express a similar amount of functional determinants on the cell surface, as determined from the presentation of OVA-derived peptides (SIINFEKL) to RF33.70 T-lymphocytes (see Figure 1). The MO4/5 line is able to stimulate hybridoma cells even in the absence of exogenously added peptides (see Figure 1), whereas the parental B16 line is not. This demonstrates the endogenous production, processing, and presentation of the transfected antigen. It is important to note that endogenous expression of OVA in cells of the MO4 line did not significantly alter in vivo tumor immunogenicity (see Figure 2). Tumor growth (see Figure 2A) B16 a

M04 is comparable in naïve mice, as is host animal survival (see Figure

Example 2: Gene immunization using biolistic (bioballistic) administration

Localization of foreign gene expression: Tissue samples (skin or fluid aspirated from lymph nodes) were collected 24 to 48 hours after immunization, washed and fixed in 2% formaldehyde - 0.2% glutaraldehyde in PBS (phosphate buffered saline). for 3 minutes and at a temperature of 4 ^° C. Fixed samples ^- were then thoroughly washed in PBS and incubated for 18 hours in a staining bath with X-gal (1 mg/ml X-gal, 5 mM potassium ferricyanide, 5 mM ferrocyanide, 2 mM MgClg in PBS) at 37°C. The stained tissue was embedded in paraffin and cut into thin sections, which were further stained with 0.1% nuclear fast red.

Gene immunization: DNA-coated gold particles were prepared by mixing 50 mg of 0.95 µm diameter gold beads and 100 µl of 0.1M spermidine. This mixture was sonicated for 5 seconds and then 132 µg of plasmid DNA and 200 µl of CaCl 2 · were added in portions while vortexing. The mixture was allowed to precipitate for 5-10 minutes at room temperature. This material was centrifuged for 30 seconds at 10,000 rpm and then washed 3 times in cold ethanol and resuspended in 7 ml of ethanol to produce a preparation with a concentration of 7 mg of gold per 1 ml. This solution was then placed in a Tefzel® (Agracetus) tube and allowed to settle for 5 minutes. The ethanol was then removed and the gold particles were attached to the walls of the tube by spinning for 30 seconds at 20 rpm and dried with N2. A dried tube with deposited gold

the particles were then uncut into 0.5 inch (1.27 cm) long sections. which were kept in paraffin-sealed glass containers together with a desiccant. Animals were immunized with two shots (each shot containing 0.5 mg of gold particles, i.e., 1.27 cm of the original tube into the shaved abdominal (belly) area using the Accell Gene Delivery device at a working pressure of 300 psi (2.067 MPa). Animals were immunized with either plasmid pAcNeo-OVA (Moore et al., 1988, Cell 54, pages 777-785) or plasmid pIEglacZ (obtained from Nadia Jouroud), which contains the lacZ gene under the control of the CMV promoter, for experiments with subcutaneous injection of particle-bound polynucleotides (see Figure 5) the beads were coated with DNA in the same manner as above and the same amount of DNA was also injected (see Figure 5~) with 5 µl of additional free plasmid administered in 100 μΐ PBS to For experiments with in vitro transfection of antigen-presenting cells (Figure 4), DNA-coated particles were prepared in a similar manner, and Biomag iron oxide beads of the same weight were used together with the gold ones.

In experiments with subcutaneous injection of polynucleotides according to the invention (see Figure 5 and 6), DNA-coated particles were prepared in the same manner as above and the same amount of DNA and/or amounts indicated in the figure captions were also injected. Some animals (see Figure 5 and 6) were injected subcutaneously with additional free plasmid. Doses were administered in 100 μΐ PBS to both hindlimbs.

For experiments with in vitro transfection of antigen-presenting cells (Figures 4 and 6), DNA-coated particles were prepared in a similar manner, using Biomag iron oxide beads (Fe

-36 balls) of the same weight. Dendritic APCs were obtained from bone marrow as indicated in the figure captions except that IL-4 was not used in tissue culture medium containing 25 μΐ of a 7mg/ml particle-bound polynucleotide solution at 37°C for 18 hours. Dendritic cells with antigen uptake were prepared as indicated in the brief description of the figures. Briefly, dendritic cells were prepared by separating bone marrow from lymphocytes and culturing it overnight in RPMI 1640 medium supplemented with 10% FCS (fetal calf serum) Á L-glutamine, antibiotics and 2 mercaptoethanol in 24-well plates at a concentration of 10® cells per hole. After one day, cells were supplemented with GM-CSF (10®U/ml, Sigma St. Luis, MO) and mouse rIL-4 (10®U/ml, Genzymeý Cambridge, MA) at a concentration of 2.5x10® cells per well. ^— . The free-playing cells were collected on the 8th day of cultivation. It was determined by flow cytometry that... these dendritic cells express antigens CD45, CD44, CD11b. (Mac-1), CD18, CD80, CD86 and MHC I and II antigens. classes. Dendritic cells were then pulsed for 2 h at 37°C in the presence and absence of OVA peptides (20 ng/ml) + β2-microglobin (human β2-Μ, 10 μΐ/ml, Sigma) in serum-reduced medium (Optimen, Gibco, Grand Island , NY). Cells were then thoroughly washed in PBS and irradiated (2000 rads) and injected into naïve mice.

Cytotoxicity Assays - Splenocytes from immunized mice were re-stimulated by methods similar to those already described with only minor modifications (Falo et al., 1995, Nature Med. 1, pages 649-653). One week after immunization, splenocytes (30x10®) were re-stimulated by co-culture with irradiated (20,000 rad) EG7 cells (10x10®). Effector cells were harvested 5 days later and cultured with 2x10^ target cells labeled with ®^Cr. This cultivation was carried out in microtiter plates with a round bottom (200 μΐ) in the indicated ratio of target and effector cells. In ·♦

in some cases, effector cells were depleted of T-lymphocyte subpopulations using a monoclonal antibody and complement prior to the test, see Rock et al., 1993, J. Immunol. 150, page 1244 to 1251). After culturing for 4 hours at 37°C, 100 μΐ amounts of supernatants from three identical wells were pooled and cells were counted. The percentage of specific releases was determined, see Falo et al., 1995, Nature Med. 1, pages 649 to 653. Results are reported as the average of three cultures. The standard error of the mean was never higher than 15%.

Testing of the protective function - Mice of the C57BL/6 line were immunized with the indicated antigenic gene construct in the described manner. Animals were given tumors and their survival was determined as described. 7 days after the last __________ immunization (day 0j) animals immunized with OVA or ia<aZ were intradermally injected with melanoma cells (2xio4) in the center of both groins, which is twice the LD5Q or were injected with a dose of tumor cells according to Section 4. Survival is shown in % surviving animals. Melanoma cells were washed three times in PBS and determined to be more than 95% viable by trypan blue staining in five mice and were repeated at least three times. Mortally ill animals were killed in accordance with the University of Pittsburgh Center for Animal Care guidelines. In some cases, mice were depleted of the CD8+ cell population by intraperitoneal injection of anti-CD8 mAb (2.43). on the seventh and ninth days after immunization, the injection of the tumor occurred on the tenth day, see Falo et al., 1995, Nature Med. 1, page 649 to 653.

The ability of bioballistic (bioballistic) immunization to induce an antigen-specific cytotoxic response was also determined. Naïve mice of the C57BL/6 line were

-38 were immunized with two overlapping pulses into the abdomen, with a total dose of 2.64 pg of OVA-encoding DNA. Seven days later, the immunization dose was repeated. Splenic cells from these mice restimulated in vitro were able to lyse syngeneic OVA-expressing EG7 murine thymoma cells, but were no longer able to cause lysis of untransfected parental cells of the EL4 line (see Figure 3A). This suggests that the cell lysis was antigen-specific and directed at the OVA expressed by the transfected tumor cells. Removal of T-lymphocytes from effector cell populations using monoclonal antibodies further demonstrated that cell lysis was dependent on Thy 1 ⁺ , CD8 ⁺ subpopulations. This is characteristic of MHC class I-restricted effector CTLs.

Groups of mice immunized in the manner described above were injected intradermally with M04 melanoma cells after 7 days at a site distant from the site of immunization so that the ability of bioballistic immunization to provide protective immunity against tumors could be evaluated. Mice immunized with OVA were protected from lethal tumor development, whereas in control mice immunized similarly, only with the lacZ reporter gene used instead of OVA, tumors grew very rapidly and were lethal in about 60% of cases after 40 days of experimental duration (Figure 3C ). However, mice immunized with DNA for OVA showed no protection against untransfected parental B16 melanoma (Figure 3D), suggesting that this protective immunity was antigen-specific and directed against OVA on the tumor cell surface. The contribution of CD8 ⁺ effector cells was also determined by repeated ip injections containing an anti-CD8 monoclonal antibody before tumor administration. This selectively eliminated the subpopulation of CD8 ⁺ lymphocytes in groups of immunized and control (immunized with lacZ) animals, see

-39 Falo and others, 1995, Nature Med. 1, pages 649 to 653. While animals immunized with OVA were protected from M04 tumor development, the survival of animals immunized with OVA but depleted of the CD8 ⁺ T-lymphocyte subpopulation was similar to that of control animals with or without these T-lymphocytes. It follows that CD8 ⁺ T-lymphocytes are absolutely necessary for the protective antitumor response induced by the gene immunization according to the invention.

_ . . ___________.__Another___evidence___supporting___this___mechanism___________ / of antitumor immunity is the demonstration of β-galactosidase expression in the epidermis of immunized animals after bioballistic insertion of the lacZ construct and interesting discrete areas of specific expression in lymph nodes.

___________Since these nodes were remote from the site of ______ immunization, it seems highly unlikely that~this expression ^- in the lymph nodes would be a direct result of physical bombardment. The predominance of gold particles in the epidermis was demonstrated. and dermis of immunized skin (unstained) 24 h after immunization with DNA and lacZ expression in epidermal keratinocytes at 48 h. Discrete specifically stained areas in the lymph nodes were also observed. Similarly treated animals that were immunized with inappropriate DNA showed no expression of lacZ. This observation led to another experiment, which is described in Example 3.

Example 3: Gene immunization after subcutaneous administration

Material and methods used for the induction of antitumor immunity by subcutaneous administration of a polynucleotide ♦ · · · · · * • · · · · · ·· • · ···· · · · · · *

-40 bound to the particles are described in detail in Example 1 and 2.

These data demonstrate the ability to specifically target phagocytic APCs in lymphoid tissue or APCs capable of migrating to lymphoid tissue by subcutaneous injection of particle-linked polynucleotides in vivo.

Protection against tumors was comparable in groups of mice immunized with particle-bound pAc-Neo-OVA polynucleotide both by direct subcutaneous administration and by bioballistic administration (see Figure 5). Consistent with the results of Example 2, immunization with a polynucleotide encoding an inappropriate antigen (eg, pIEglacZ) did not elicit protective immunity. Suhku/tanny irTj“ection of pAc^Něo^OVAr ^- be ^- z ^— carrier ^- particles also did not lead to protective immunity against the corresponding tumor (see Figure 5). These data suggest that subcutaneous injection of particle-bound polynucleotides is at least as effective as bioballistic administration. Considering that during subcutaneous injection there is no direct physical forcible penetration of the particle with bound polynucleotide into the cytoplasm of the target host cell, it is clear that some way of active transport of polynucleotides by cells capable of phagocytosis/endocytosis is necessary for expression. The observation of the absence of immunity after injection of pAc-Neo-OVA polynucleotides, which was not bound to particles, also leads to the conclusion that transport of the particle by phagocytosis is the main mechanism of transduction. It also follows from this transduction mechanism that cells capable of phagocytosis, including APC, are more suitable for transgene expression.

Further evidence for this putative mechanism of action of particle-bound polynucleotides is the observation of APCs from bone marrow-derived dendritic cells that, after co-culture in vitro with a polynucleotide encoding

-41ovalbumin (pAc-Neo-OVA, prepared according to Example 2), which was attached to the particles, was able to stimulate OVA-specific T-lymphocytes (SIINFEKL+ Kb) from the RF33.70 hybridoma to produce IL-2 (see Figure 4).

It follows that these APCs functionally express the gene for ovalbumin and a complex of ovalbumin peptides and K^ is formed in them. Quantitatively, this process is comparable to a similar process observed during the pulsed transfer of 2 mg/ml soluble —El-------protein___OVA___into____antigen-presenting cells._________

Polynucleotides attached to gold or iron particles are comparably effective in this case. It follows that incubation of polynucleotide bound to particles together with phagocytic APCs in vitro conditions leads to endogenous ___________production,_processing and presentation of transfected antigens. -------— and expressed by the antigen presenting cell and the corresponding peptides can be functionally,; presented on the surface of these cells to induce an antigen-specific immune response.

Figure 6 shows that APCs, in this case subcutaneously injected dendritic cells derived from bone marrow, which have been incubated in vitro with the polynucleotide encoding the OVA antigen attached to the particles as described above, are able to induce antitumor immunity against tumor cells expressing the given antigen. In this case, one-time immunization with irradiated , . .transfected dendritic cells (10°) per animal _ (5x10^ cells to each hind limb) induced complete antitumor immunity. In vivo administered dendritic APCs, which are good stimulators of antigen-specific CTLs and antitumor immunity, are at least as immunogenic after peptide saturation in vitro as when saturated ·· ♦

-42 antigen when co-incubated with polynucleotide bound to particles.

Subcutaneous application of a polynucleotide attached to particles is thus advantageous mainly due to the fact that targeted transport of the antigen to the phagocytic antigen-presenting cells (APC) of the host is possible, which can increase the efficiency of gene immunization and at the same time reduce adverse effects such as, among others, the induction of tolerance or mutagenesis after transfection of normal^ antigens non-presenting cells.

Claims (22)

A vaccine comprising a polynucleotide bound to particles with a surface on which DNA fragments are expressed which express an antigenic protein or antigenic protein fragment, said vaccine being characterized in that it is able to penetrate into the cytoplasm of the target cell in a mammalian host such that wherein said antigenic protein or antigenic protein fragment is expressed and presented on the membrane surface of said target cell by an MHC class I mechanism. 2. A vaccine substance according to claim 1, wherein expression of the DNA fragment produces a tumor rejection antigen (TRA), a viral antigen, or antigenic protein fragments thereof. 3. The vaccine of claim 2, which is capable of penetrating the cytoplasm of target antigen-presenting cells. Vaccine according to claim 3, characterized in that it is capable of penetrating the cytoplasm of antigen-presenting cells located or --- migrating into lymphoid tissue ... of a mammalian host. -t The vaccine of claim 2, wherein said TRA antigen is selected from the group consisting of MAGE 1 and MAGE 3. The vaccine of claim 2, wherein said TRA antigen is Melan-A. 7. The vaccine of claim 2, wherein said TRA antigen is gp100. The vaccine of claim 2, wherein said TRA antigen is p53. 9. The vaccine of claim 2, wherein said TRA antigen is CEA. 10. The vaccine of claim 2, wherein said TRA antigen is HER2 / neu ---- The vaccine of claim 2, wherein said viral antigen is gp120 or HIV gp160. The vaccine of claim 2, wherein said viral antigen is an influenza virus nucleoprotein. The vaccine of claim 2, wherein said viral antigen is hepatitis B surface antigen. .......... ´ - - - - = - Vaccine according to any one of claims 1 to 13, characterized in that it is useful for vaccinating a mammalian host with a biobalistic device or is suitable for vaccinating a mammalian host by injection. Vaccine according to any one of claims 1 to 14, characterized in that it is capable of infiltrating target cells in vitro or in vivo. 16. A vaccine comprising a polynucleotide bound to particles with a surface on which DNA fragments are broken down, expressing a protein that enhances the antigen-presenting function of the antigen-presenting antigen by a cell-like cell. the vaccine is characterized in that it is capable of penetrating the cytoplasm of the target cell in a mammalian host so as to express said antigen-enhancing protein in a biologically active form, and ------- b-i-oi-og-i-c-k-y — effective-concentration — ------ 17. The vaccine of claim 16, which is capable of penetrating the cytoplasm of target antigen-presenting cells. 18. The vaccine of claim 17, wherein said vaccine is capable of penetrating into the cytoplasm antigen presenting cells located or migrating into the lymphoid tissue of said human host. 19. The vaccine of claim 16, wherein the expression of said DNA fragment results in a costimulatory molecule. 20. The vaccine of claim 19, wherein said costimulatory molecule is selected from the group consisting of CD80 and CD86 molecules. 99 • · 9 9 • · • 9 e · • · • 9 • 9 99 • 9 9 9 ' 9 99 • 99 • * ··· • • 9 9 9 9 9999 9 9 999 9 9 • • 9 9M9 • • 9 • 9 · 9 9 21. The vaccine of claim 16, wherein said DNA fragment produces a cytokine molecule. 22nd vaccine according to claim 21 characterized in that said molecule is a cytokine selected from the group consisting of ^- Hr ^ l "= 27 Τΐτ 4 ^- 2- EII -