Patent 6,759,036

There is disclosed a fibrocyte-based vaccine formulations made from isolated fibrocytes. There is further disclosed a method for establishing an immune response against a specific antigen by administering a fibrocyte-based vaccine formulation, such as one made by pulsing fibrocytes in culture with the antigen peptide or protein, or by fusing tumor cells (whole cells or membrane fragments thereof) with fibrocytes.

SUMMARY OF THE INVENTION

The present invention provides fibrocyte-based vaccine formulations made from isolated fibrocytes and an antigenic component, wherein the antigenic component is selected from the group consisting of pulsed antigen (protein, peptide, lipid, carbohydrate or a synthetic compound), a gene encoding specific antigenic determinants of proteins or peptides, tumor cells, and membrane fragments from tumor cells. Preferably, the antigenic component is a tumor cell or a tumor cell membrane that is fused with the isolated fibrocytes to form a fused cell that is the fibrocyte-based vaccine formulation. Preferably, the fusion process occurs ex vivo and the fibrocyte-based vaccine formulation is administered in vivo. Preferably the fibrocyte-based vaccine formulation is directed against an infectious disease and is formed by transfecting fibrocytes with a gene encoding a viral or a bacterial antigenic determinant that is displayed as an MHC class II antigenic determinant on the surface of the transfected fibrocytes.

The present invention further provides a method for establishing an immune response against a specific antigen by administering a fibrocyte-based vaccine formulation, such as one made by pulsing fibrocytes in culture with the antigen peptide or protein, or transfecting fibrocytes with genes encoding specific antigenic determinants of peptides or proteins, or by fusing tumor cells (whole cells or membrane fragments thereof) with fibrocytes. Preferably, the method provides an immune response is directed against a tumor antigen, a viral antigen, or a bacterial antigen. Preferably, the fibrocyte-based vaccine is produced by a process comprising pulsing fibrocytes in culture with an antigen peptide or protein, or transfecting fibrocytes with genes encoding specific antigenic determinants of peptides or proteins, or by fusing tumor cells (whole cells or membrane fragments thereof) with fibrocytes.

The present invention further provides a process for producing a fibrocyte-based vaccine formulation, comprising: (a) obtaining isolated fibrocytes, and (b) either (i) pulsing fibrocytes in culture with an antigen peptide or protein; or (ii) transfecting fibrocytes with genes encoding specific antigenic determinants of peptides or proteins; or (iii) fusing tumor cells (whole cells or membrane fragments thereof) with fibrocytes. Preferably, the fusion process (iii) is performed by mixing a population of isolated fibrocyte cells taken from a patient having cancer with tumor cells taken from the patient in a fusion catalyst, and isolating fused cells from non-fused fibrocytes or tumor cells by density gradient means to form a fibrocyte-based vaccine formulation. Most preferably, the process further comprises irradiating the isolated fibrocyte-based vaccine formulation to insure that it is incapable of growth after in vivo administration.

DETAILED DESCRIPTION OF THE INVENTION

Fibrocyte-Based Vaccine Formulation

The present invention provides a fibrocyte-based vaccine formulation made from isolated fibrocytes and an antigenic component, wherein the antigenic component is selected from the group consisting of pulsed antigen (protein, peptide, lipid or carbohydrate), a gene encoding specific antigenic determinants of proteins or peptides, tumor cells, and membrane fragments from tumor cells. Preferably, the antigenic component is a tumor cell or a tumor cell membrane that is fused with the isolated fibrocytes to form a fused cell that is the fibrocyte-based vaccine formulation. Preferably, the fusion process occurs ex vivo and the fibrocyte-based vaccine formulation is administered in vivo. Preferably the fibrocyte-based vaccine formulation is directed against an infectious disease and is formed by transfecting fibrocytes with a gene encoding a viral or a bacterial antigenic determinant that is displayed as an MHC class II antigenic determinant on the surface of the transfected fibrocytes.

Fibrocytes are potent antigen presenting cells that prime native cytotoxic T cells (CTLs). Fibrocytes are produced by a process for isolating fibrocyte cells from peripheral blood. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from human blood by centrifugation over a commercial density gradient (Ficoll-Paque.RTM. Pharmacia, Uppsala, Sweden) following the manufacturer's procedures. The isolated PBMCs were cultured overnight on either non-coated ("Control") or fibronectin-coated plastic (6-well, 5x10⁶ PBMCs/well) plates (both from Becton Dickenson Labware, Bedford, Mass.) in Dulbecco's Minimal Eagle Medium (Life Technologies, Gaithersberg, Md.) supplemented with 20% fetal bovine serum (Hyclone Labs, Logan, Utah). The non-adherent cells were removed by a single, gentle aspiration.

The cultures were incubated for ten days in continuous culture. The adherent cells were lifted by incubation in cold 0.05% EDTA (Sigma, St. Louis, Mo.) in phosphate buffered saline (PBS, Life Technologies) and analyzed for fibrocytes by flow cytometry. Cell samples (2x10⁵ cells/sample) were washed twice in PBS containing 0.1% by weight sodium azide (Sigma) and 1% by weight bovine serum albumin (Sigma) (FACS medium). The cells were incubated for 30 minutes on ice in 25 .mu.l of FACS medium containing the following antibodies: phycoerythrin-conjugated anti-CD34 monoclonal antibody (Becton Dickenson) and fluorescein-conjugated anti-collagen I mAb (Chemicon, Temecula, Calif.). The cells were then washed twice in PBS and then resuspended in 200 .mu.l of FACS medium. At least 100,000 cells were analyzed on a FACScan instrument (Becton Dickenson). Cell viability was determined by trypan blue exclusion.

The isolated fibrocytes are fused with tumor cells or tumor cell membranes displaying appropriate tumor antigens. Standard fusion methods, such as those used for hybridoma fused cells are used. Briefly, one method mixes the cells in flasks in 50% PEG (polyethylene glycol) in Dulbecco's phosphate buffered saline without Ca2+ or Mg2+ at pH 7.4. The fused cells were then plated in 24-well culture plates in the presence of HAT selection medium (Sigma) for 10-14 days.

Another method for forming fusion cells is to fuse the isolated fibrocytes with tumor cells or tumor cell membrane fragments is to mix the cell again in PEG for several days in culture, waiting for unfused fibrocytes cells to die off to better isolate a population of fused fibrocytes. The fused fibrocyte cells are separated from the unfused tumor cells by switching to HAT medium with aminopterin to prevent cells from making their own thymidine. The fused cells will grow well in this medium and double in every 1-4 days, whereas the tumor cells should die off. Thus, the surviving cells should be a fairly homogenous population of fused fibrocyte cells.

When fibrocytes are fused with tumor cells or tumor cell membranes displaying tumor antigens, the fused cells are positive for MHC class I and class II antigens. In an experiment showing the utility of the claimed invention, fibrocytes were fused with MC38 carcinoma cells which express the DF3/MUC1 tumor-associated antigen. The results show that the fusion cells stimulated native T cells in a primary mixed lymphocyte reaction (MLR) and induced MC38/MUC1 tumor specific CTLs in vivo. Antibody-mediated depletion experiments demonstrated that induction of CD4+ and CD8+ CTLs protected against challenge with tumor cells, and immunization with the inventive fusion cells induced rejection of established metastases, indication the therapeutic utility of he inventive fusion cells for treating established cancers. Therefore, the inventive fibrocytes fused with tumor cells or tumor cell membranes are effective as tumor vaccines for treating or preventing cancer.

Fibrocytes are fused with tumor cells to form a fused fibrocyte/tumor cell and the fused fibrocyte/tumor cells isolated, cultured and utilized as a tumor immunogen. For example, murine MC38 adenocarcinoma cells that stably express the marker DF3/MUC1 antigen were fused to peripheral blood derived fibrocytes. Cells were maintained in DMEM supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM glutamine, 100 U/ml penicillin and 100 .mu.g/ml streptomycin. Fusion efficiencies may be enhanced by pre-culturing tumor cells and fibrocytes prior to fusion as well as fusing at relatively low cell densities (<10⁶ /ml). However, fusion at low cell densities is not a requirement for fusion. Agents that act to enhance fiborycte:tumor cell adherence also enhance fusion efficiencies. These agents include, for example, IL-1.alpha. and IL-1.beta.. In addition, a fibrocyte may be modified prior to fusion with a tumor cell to increase the immuno-potency of the resultant hybrid. This modification can take several different means, including gene or protein transfer. Examples of proteins that can be expressed or inhibited in a fibrocyte include cytokines, chemokines, adhesion receptors, or co-stimulatory molecules.

Fibrocytes were isolated as described above. The cells were plated in six-well culture plates in RPMI medium supplemented with 20% FCS. Fusion was carried out with 50% PEG in Dulbecco's phosphate buffered saline without Ca²⁺ or Mg²⁺ at pH 7.4. The fused fibrocyte/tumor cells were plated in 24 well culture plates in the presence of HAT selection medium (Sigma) (100 .mu.M hypoxanthine, 0.8 .mu.M aminopterine, and 16 .mu.M thymidine) for 10-14 days. Other methods of fusion are also applicable, including other chemical means, varying the electric strength such is in electroporation and by alterations in external atmospheric pressure.

Method for Establishing an Immune Response

The exemplary fused fibrocyte/tumor cells, made according to the method described above, express DF3/MUC1 as well as class II and other co-stimulatory molecules. Injection of MC38/MUC1 cells in mice resulted in the formation of subcutaneous tumors. Similar findings were obtained with MC38/MUC1 cells mixed with fibrocytes. However, no tumors formed in mice injected with fused MC38/MUC1/fibrocyte cells and fibrocytes. These data show the therapeutic utility of a fibrocyte fusion cell over an unfused fibrocyte cell when used for autologous treatment of tumors.

Mice were immunized twice with fused MC38/MUC1/fibrocytes cells and fibrocytes to assess function in vivo. All animals remained tumor free after challenge with 2.5x10⁵ to 2x10⁶ MC38/MUC1 cells. Control animals immunized with fibrocytes alone or PBS and then challenged subcutaneously with 2.5x10⁵ MC38/MUC1 cells exhibited tumor growth within 10-20 days. CTL's isolated from mice immunized with fibrocytes or PBS exhibited no detectable lysis of the MC38/MUC1 targets. However CTL's isolated from mice immunized with the fibrocyte/tumor fusion cells induced lysis of the MC38/MUC1 cells.

A model of MC38/MUC1 pulmonary metastases was used to determine whether immunization with fibrocyte/tumor fused cells is effective for the prevention of disseminated disease. Immunization with the fibrocyte/tumor fused cells intravenously or subcutaneously completely protected against intravenous challenge with MC38/MUC1 cells. By contrast, all non-immunized mice similarly challenged with MC38/MUC1 cells developed over 250 pulmonary metastases. In a treatment model, MC38/MUC1 pulmonary metastasis were established 4 days prior to immunization with fused fibrocyte/tumor cells. Control mice, treated with vehicle, developed over 250 metastases. Treated mice on the other hand had none.

The present invention discloses the capacity of a fibrocyte/tumor fusion cell to stimulate anti-tumor responses and T-cells. This capacity can be used for both in vivo and ex vivo stimulation of T-cells. Ex vivo stimulation of a T cell using a fibrocytes/fusion product can be used as a means of amplifying T-cells with a tumor specificity prior to infusion of such T-cells into patients. Methods are well known in the art for delivering T-cells into patients.

The fused fibrocyte/tumor fusion cells may be used either in a prophylactic or treatment procedure generally with autologous but also allogeneic procedures. The use of fusion with fibrocytes provides substantial advantages for practicing tumor cell engineering and enhancing tumor cell immunogenicity. There is no need for decoding the precise tumor antigens that may be expressed in the particular tumor. Cell fusion is applicable to diverse tumor types, including solid and blood tumors. It is a simple process and can utilize fusion methods involving chemical fusion, or methods of generating cell fusion that involve electric fields or high pressure. The tumor cell contributes relevant tumor-specific antigens to the hybrid cell or membrane preparation and the fibrocyte-like cell contributes cell surface co-stimulators, soluble cytokines, MHC molecules, adhesion molecules, chemokines and other undefined molecular factors to the hybrid cell. This results in a highly antigenic and immunogenic phenotype for the hybrid cell or hybrid cell membrane preparations. In turn, the hybrid cell can be used as an effective cellular vaccine.

The present invention is, in part, based upon the finding that enhanced immunogenicity can be induced by fusing a cell membrane from a tumor cell with a cell membrane from a fibrocyte or an isolated population of fibrocyte cells. The resulting a fibrocyte-tumor cell fusion is shown to stimulate anti-tumor T-cells and to have significant anti-tumor activity in relevant animal tumor models. Through such fusion the full complement of molecular factors that are normally produced by professional APC's and that are required for effective T-cell activation are combined with a full set of potential tumor antigens associated with a particular tumor.

Fibrocyte-Based Vaccines Made by A Transfection Process

This invention also provides a fibrocyte-vaccine formulation wherein the antigenic component is a pulsed antigen protein or peptide, or a gene encoding specific antigenic determinants of proteins or peptides for uses in tumor immunotherapy. Fibrocytes can be induced in vitro to secrete, for example, tumor antigens, viral markers, or T-cell attractants in vivo for enhanced immunogenicity. These T-cell attractants include, for example, various chemokines, cell surface proteins and secreted cytokines. For instance, IL-1.beta. can induce fibrocyte expression of MIP-1.alpha., a powerful T-cell chemoattractant. IL-1.beta. also induces fibrocyte migration which is important for T-cell interaction with the fibrocyte.

Fibrocytes may be used for generating immune response against specific antigens not only involved in viral, but also bacterial, fungal and parasitic infections. In another approach, fibrocytes may be loaded with a source of tumor antigen. Where aberrant expression of specific antigen is known, this approach is useful for generating immune responses against specific tumors. Tumor specific antigens which may be used in peptide pulsing of fibrocytes for tumor immune therapy may include, but are not limited to, MAGE, CEA, PSA, Her2/neu, and other tumor derived peptides (Wang and Rosenberg, J. Leuk. Biol. 60:296-309, 1996). Alternatively, fibrocytes may be transfected with the genes encoding such tumor derived peptides.

This method is useful for more standard ex vivo immunotherapy protocols, such as tumor peptide or protein "pulsing" of fibrocytes. However, with the tumor-fibrocyte fusion there is no requirement for identification of tumor specific antigens, nor the production and purification of the corresponding recombinant proteins. In addition, tumor antigens are displayed in a more physiological state, within a cellular membrane.

Process for Producing a Fibrocyte-Based Vaccine Formulation

Advantage of Fibrocytes Versus Dendritic Cells

Use of the fibrocyte for tumor, viral, fungal, bacterial or parasitic immunization has distinct advantages over the use of other APCs (antigen presenting cells). First, unlike dendritic cells, fibrocytes can be conveniently separated from blood and the separation involves straightforward means, without the need for complicated selection, such as a selection based on the CD34 antigen. Second, fibrocytes constitute upwards to 0.3% of the circulating PBL's, a frequency much higher than dendritic cells. Third, fibrocytes are potent APCs, at least equivalent to dendritic cells and superior to monocytes. Fourth, fibrocytes do not require sophisticated and complex cocktails of cytokines and growth factors (such as GM-CSF, IL-3, TNF, IL-1 Flt-3, etc.) for ex vivo expansion, as required for dendritic cells and other APCs. Fifth, fibrocytes (unlike dendritic cells) divide with rapid doubling times ex vivo, which allows for harvesting large numbers of cells useful for immunotherapy and allowing cell banking of fibrocytes for later use.

Claim 1 of 4 Claims

We claim:

1. A fibrocyte-based vaccine formulation comprising isolated fibrocytes having an antigenic component associated therewith, wherein the antigenic component is a tumor antigen selected from the group consisting of pulsed antigen protein, peptide, lipid, carbohydrate or a synthetic antigen thereof, a gene expressing specific tumor antigenic determinants wherein said determinants are proteins or peptides, tumor cells and membrane fragments from tumor cells, wherein the fibrocyte-based vaccine formulation displays at least one tumor antigen of the antipenic component, with the proviso that when the antigenic component is tumor cell or membrane fragments from tumor cells the antigenic component is fused with the isolated fibrocytes to form a fused cell that is the fibrocyte-based vaccine formulation.

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

[ Outsourcing Guide ] [ Cont. Education ] [ Software/Reports ] [ Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ]

[ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ]
[ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ]
[ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ]
[ Site Map ]