Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1998-09-30
2002-05-14
Schwartzman, Robert A. (Department: 1632)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S320100, C435S325000, C536S023100, C536S023500
Reexamination Certificate
active
06387888
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
Not Applicable.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not Applicable.
BACKGROUND OF THE INVENTION
(1) Field of the Invention
The present invention relates to compositions and methods for immunotherapy of human cancer patients.
(2) Description of Related Art Including Information Disclosed Under 37 CFR 1.97 and 37 CFR 1.98.
All normal human nucleated cells express on their membrane small protein fragments derived from de novo protein synthesis. These so-called peptides are associated with the major histocompatibility complex (MHC) class I molecules and form the antigens which are recognized by CD8 cytotoxic T-lymphocytes (CTLs). Such recognition is important for the elimination of virally infected cells, of tumor cells, or of cells that contain intracellular parasites. For this to occur potentially antigen-reactive T cells need to be “pre-educated” by recognizing the antigen in question on the membrane of professional antigen-presenting cells (dendritic cells) (APCs, DCs) which, in addition to the antigen, provide co-stimulatory “maturation” signals to the T cells. In the absence of such signals the T cells become paralyzed and tolerant to the antigens in question.
Tumor cells, which are not professional APCs, do not stimulate CTL generation and are not rejected by the immune system. For the generation of an immune response against a tumor the tumor antigen(s) need(s) to be expressed by professional APCs. This presentation has been accomplished by in vitro exposure of dendritic cells to tumor lysates that presumably contain tumor antigens, to purified tumor antigens or, to peptides derived from such antigens.
Another possibility to achieve expression of antigen-derived peptides is by introducing into dendritic cell desoxy—(DNA) or ribonucleic acid (RNA) that encodes the antigen of interest. Cells transfected with the plasmid DNA transiently synthesize the protein and the peptides that are obtained during the synthesis are then expressed in association with MHC. For example, patient's cells grown in vitro are transfected with plasmids, containing the DNA, or with the RNA of interest or infected with a recombinant viral vector that contains the DNA or RNA, and then returned to the patient. Another possibility is to directly immunize the patient with the plasmid (“nude” DNA immunization) or with the recombinant viral vector.
A major problem with this technique comes from the possible adverse effects of the expressed products on the patient's health or on cell viability. Since the function of these tumor-associated or tissue-specific antigens is largely unknown, their synthesis and release by patient's cells in vivo may lead to serious side effects. Furthermore, in cases where dendritic cells are transfected in vitro, expression of a functional protein may alter dendritic cell viability, change their migration pattern or their ability to provide co-stimulation to T cells.
The present invention discloses the idea for the introduction of specific changes in the DNA or RNA encoding the antigen in question as a way of solving this problem. Such changes result in the expression of functionally inactive products without affecting the efficiency of transcription and translation of the DNA, the translation of the RNA, or the generation of antigenic peptides. Specifically the present invention discloses the development of a DNA, which leads to expression of a truncated form of the human prostate specific membrane antigen (PSMA). In particular, we have developed a DNA construct with deletions of the membrane and the intracellular portions of the human PSMA. The resulting DNA encoding the extracellular portion of the PSMA (XC-PSMA) has been incorporated in mammalian expression vectors. PSMA is a type II protein, it lacks a hydrophobic signal sequence and therefore is not secreted by the cell that produces it. Since our construct lacks membrane and cytoplasmic sequences, the resulting protein is not expressed on the membrane, therefore does not transduce signals and is not released from the membrane. Cells transfected with the XC-PSMA plasmid retain viability and express PSMA-derived peptides.
Furthermore, since the synthesized protein is not released but remains confined to the intracellular milieu, there is no production of antibodies directed against the protein and the immune response remains strictly cell-mediated. The exquisite engagement of cell-mediated immunity against a particular antigen is very important especially in cases where the target antigen of interest is expressed on normal tissues that are anatomically sequestered in immuno-privileged sites such as the eye, brain, testis etc. Those tissues are inaccessible to cell mediated injury, but readily damaged by antibodies. Immunotherapy based on eliciting cellular responses to differentiation (tyrosinase; gp100; TRP1; TRP2; MART-1/Melan-A; membrane-associated mucin, MUC-1 mucin) or normal tissue-specific (PSMA, PSA) antigens constitute an example where the production of antibodies against the target must not occur.
In the first method of treating of prostate cancer patients, the plasmid is injected intradermally. In a second method of treatment, the plasmid is incorporated into the genome of a replication-deficient adenovirus, which is injected intradermally into a patient. In a third method of treatment, CD14+ monocyte cells of a prostate cancer patient are isolated and matured into dendritic cells (DC) and transfected with either the plasmid or the adenovirus of the first two methods. The DC are then stimulated to express MHCs and are infused back into the prostate cancer patient where they stimulate autologous T-cells. These stimulated T-cells then destroy both normal and malignant prostate cells.
The effect of all of these treatments is to either by-pass the normal tolerance for self-antigens or the tolerance to tumor antigens. This will enable the cytolysis of target normal and malignant prostate cells normally shielded from immune recognition. The destruction of normal prostate cells by this procedure is not detrimental to the patient. A malignant prostate (with its mixture or normal and malignant cells) customarily is destroyed through surgery or radiation in the conventional primary treatment for this disease.
U.S. Pat. No. 5,227,471 discloses the structure of the prostate-specific membrane antigen. A method for treating prostate cancer was disclosed which involves an antibody directed against the prostate-specific membrane antigen and a cytotoxic agent conjugated thereto. However, since the PSMA is expressed on normal brain cells, use of antibodies which can transverse through the blood-brain barrier and damage normal brain cells is not acceptable. Methods for imaging prostate cancer and an immunoassay for measuring the amount of prostate-specific membrane antigen also were disclosed.
U.S. Pat. No. 5,788,963 discloses the use of human dendritic cells to activate T cells for immuno-therapeutic response against primary and metastatic prostate cancer. Human dendritic cells are isolated and exposed to PSMA or peptides derived thereof in vitro. The PSMA or peptides are believed to exchange with peptides already bound to MHC molecules on the dendritic cells and thereby to be expressed in an imunogenic manner, enabling the DC to stimulate killer cells which then lyse prostate cells.
U.S. Pat. Nos. 5,227,471 and 5,788,963 are incorporated by reference herein.
The present invention differs from the prior art in that it causes the DC to present an antigen derived from prostate cancer cells on their surface through transfection with a plasmid or adenovirus. The transfection may occur in vivo using injected plasmid or adenovirus. Alternatively, the transfection may occur in vitro using purified DC precursor cells isolated from the prostate cancer patient's blood. If transfection is done in vitro, the transformed cells are injected into the patient. Transfected DC cells are superior to DC cells, which have been exposed to antigen in vitro beca
Loukinov Dmitri I.
Mincheff Milcho S.
Zoubak Serguei
American Foundation for Biological Research, Inc.
Clauss Isabelle M.
Foley Hoag & Eliot
Li Janice
Schwartzman Robert A.
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