Combined Tumor Suppressor Gene Therapy and Chemotherapy in the Treatment of Neoplasms

专利摘要:
The present invention provides a method of treating a cell comprising contacting a mammalian cancer cell or hyperproliferative cell with a tumor suppressor protein or a tumor suppressor nucleic acid and further contacting the cell with one or more accessory anticancer agents. The present invention also provides a pharmaceutical composition comprising a tumor suppressor protein or tumor suppressor nucleic acid and at least one adjuvant anticancer agent and a kit for treating mammalian cancer cells or hyperproliferative cells.
公开号:KR20000071185A
申请号:KR1019997007475
申请日:1998-02-17
公开日:2000-11-25
发明作者:로레타 닐센；조 앤 호로위쯔；다니엘 씨. 마네발；지.윌리암 데머스；메리 엘렌 리박；진 레스니크
申请人:이. 엘. 나가부샨, 리차드 비. 머피；캔지, 인크.；
IPC主号:

专利说明:

Combined Tumor Suppressor Gene Therapy and Chemotherapy in the Treatment of Neoplasms}
Chromosomal abnormalities are often associated with genetic diseases, degenerative diseases and cancer. In particular, deletions or increases in the number of copy chromosomes or chromosomal segments of large numbers and high levels of amplification of certain regions of the genome are common in cancer (see, for example, Smith (1991) Breast Cancer Res. Treat., 18: Suppl. 1: 5-14; van de Viler (1991) Became. Beefiest. Acta. 1072: 33-50, Sato (1990) Cancer. Res., 50: 7184-7189). Indeed, amplification of DNA sequences containing the proto-oncogenes and deletion of DNA containing tumor suppressor genes is a common feature of tumor development (Dutrillaux (1990) Cancer Genet. Cytogenet. 49: 203-217).
Mutations in the p53 gene are the most common genetic variations in human cancer (Bartek (1991) Oncogene 6: 1699-1703, Hollstein (1991) Science, 253: 49-53). In addition, introduction of wild type p53 into mammalian cancer cells lacking the endogenous wild type p53 protein inhibits the neoplastic phenotype of such cells (see, eg, US Pat. No. 5,532,220).
Among many useful chemotherapeutic drugs, paclitaxel, marketed as Taxol (registered trademark; NSC No. 125973), has been of interest because of its efficacy in clinical trials for drug resistant tumors, including ovarian and mammary tumors (Hawkins). 1992) Oncology, 6: 17-23, Horwitz (1992) Trends Pharmacol.Sci. 13: 134-146, Rowinsky (1990) J. Natl. Canc. Inst. 82: 1247-1259). Recent studies on the interaction of paclitaxel with tumor suppressor gene therapy have shown that a decrease in the concentration of tumor suppressor (ie, p53) is associated with G2 / M phase arrest, micronucleation and p53-independent paclitaxel-induced apoptosis. Showed that there is. In contrast, surviving cells with complete p53 progress through mitosis and temporarily accumulate in subsequent G1 phases with increasing p53 and p21 ^cipl.wafl protein concentrations (Wahl (1996) Nature Med. 2: 72-79 ). Similarly, Hawkins (1996) Canc. Res. 56: 892-898 showed that inactivation of p53 increases susceptibility to certain antimitotic agents, including paclitaxel. The literature suggests that p53 plays a specific role in DNA repair, making it easier to advance cells through the S phase even in the presence of drugs. The study suggests that tumor therapy gene therapy and drug therapy with antimitotic agents (particularly paclitaxel therapy) work contradictory.
Summary of the Invention
The present invention provides a method of treating a hyperproliferative mammalian cell. The present invention surprisingly finds that when adjuvant anticancer agents are used in combination with tumor suppressor (eg p53) gene therapy, they provide an increased effect in inhibiting the proliferation of neoplastic cells or other cells lacking the activity of the tumor or tumor suppressor. Based in part.
Thus, in one embodiment the present invention provides a method of treating cancer cells or hyperproliferative cells by contacting cancer cells or hyperproliferative cells with a tumor suppressor protein or tumor suppressor nucleic acid, and one or more anticancer agents. In some embodiments, the method comprises simultaneous administration of a tumor suppressor protein or nucleic acid and an adjuvant anticancer agent with one or more chemotherapeutic agents. For example, tumor suppressor nucleic acids (eg, nucleic acids encoding p53) may be used in conjunction with adjuvant anticancer agents (eg paclitaxel) and DNA damaging agents such as cisplatin, carboplatin, navelbine (vinorelbine tartate). May be administered.
Cancer or hyperproliferative cells are often neoplastic cells. When the cells are present in the tumor, the method inhibits tumor growth, thereby providing a method of treating cancer. Examples of the cancer include ovarian cancer, pancreatic cancer, non-small cell lung cancer, small cell lung cancer, liver cancer, melanoma, retinoblastoma, breast tumor, colorectal cancer, leukemia, lymphoma, brain tumor, cervical cancer, sarcoma, prostate tumor, bladder tumor Tumors of reticuloendothelial tissue, Wilm's tumor, astrocytoma, glioblastoma, neuroblastoma, ovarian carcinoma, osteosarcoma, kidney cancer, and head and neck cancers.
Preferred adjuvant anticancer agents are paclitaxel or paclitaxel derivatives, and preferred tumor suppressor nucleic acids are nucleic acids encoding tumor suppressor proteins selected from the group consisting of p53 protein and its analogs and retinoblastoma (RB) proteins. Particularly preferred tumor suppressor nucleic acids encode wild type p53 protein, and particularly preferred retinoblastoma protein is p110 ^RB or p56 ^RB .
Tumor suppressor nucleic acids are preferably delivered to target cells by a vector. The vector is modified by recombinant DNA technology to enable expression of tumor suppressor nucleic acid in a target cell. These vectors can be derived from nonviral vectors (eg plasmids) or vectors of virus (eg adenoviruses, adenovirus viruses, retroviruses, herpes viruses, vaccinia viruses). In a preferred embodiment of the invention, the vector is a recombinantly modified adenovirus vector. Non-viral vectors preferably form complexes with substances that promote the passage of DNA through the cell membrane. Examples of such nonviral vector complexes include agents with polyvalent cationic materials and lipid based delivery systems that facilitate enrichment of DNA. Examples of lipid based delivery systems include liposome based delivery of nucleic acids.
Particularly suitable adenovirus vectors (eg for delivery of nucleic acids encoding wild type p53 protein) include some or all deletions of Protein IX DNA. In one embodiment, the deletion of the Protein IX gene sequence ranges from about 3,500 bp from the 5 'virus end to about 4,000 bp from the 5' virus end. The vector may also comprise deletions of non-essential DNA sequences in adenovirus initial region 3 and / or in adenovirus initial region 4, in one embodiment the deletions are DNA sequences Ela and / or Elb. Particularly preferred recombinant adenovirus vectors for delivery of human p53 cDNA include adenovirus type 2 late main promoters or human CMV promoters, and adenovirus type 2 tripartite leader cDNAs. One preferred adenovirus vector is A / C / N / 53.
Preferred paclitaxel or paclitaxel derivatives include paclitaxel and / or Taxotere®, with paclitaxel most preferred. Another preferred adjuvant anticancer agent is Epothilone. In one particularly preferred embodiment, the tumor suppressor is A / C / N / 53 and the adjuvant anticancer agent is paclitaxel.
Tumor suppressor proteins or tumor suppressor nucleic acids may be dispersed in pharmaceutically acceptable excipients. Similarly, adjuvant antiangers (eg paclitaxel or paclitaxel derivatives) may be dispersed in pharmaceutically acceptable excipients. Tumor suppressor proteins or tumor suppressor nucleic acids and the paclitaxel or paclitaxel derivatives can all be dispersed in one composition (including one or a plurality of excipient (s)).
Tumor inhibitors (proteins or nucleic acids) and / or adjuvant anticancer agents may be administered simultaneously or sequentially in intraarterial, intravenous (eg injection), intraperitoneal and / or tumors. Preferred sites of administration include intraarterial administration of the arteries when treating intrahepatic, intraperitoneal, or head cells (eg neurons).
Tumor suppressor proteins or nucleic acids may be administered in multiple doses of one or more or three times, each at a time interval of at least about 6 hours, preferably at least about 24 hours.
In another preferred embodiment, the tumor suppressor protein or tumor suppressor nucleic acid is from about 1 x 10 ⁹ to about 1 x 10 ¹⁴ , or from about 1 x 10 ⁹ to about 7.5 x 10 ¹⁵ , preferably about 1 x 10. ¹¹ to about 7.5 x 10 ¹³ of the total daily dose once daily, 5 days a daily administration, 15 days treatment regimen is selected from the method of administration every day for every day of administration, and 30 days (with or without the auxiliary anticancer agent with a secondary anticancer agent for ) Is administered. In addition, the dosage may be administered continuously for 1 to 30 days. Paclitaxel or paclitaxel derivatives are administered once in a total dose of 75 to 350 mg / m ² , daily dose for 2 days, daily dose for 3 days, daily dose for 15 days Dosing for 1 hour, 3 hours, 6 hours or 24 hours in a dosing regimen selected from 30 days of daily dose administration, daily continuous infusion for 15 days, daily continuous infusion for 30 days. Preferred dosages are 100 to 250 mg / m ² in 24 hours. Alternatively, paclitaxel or derivatives may be administered weekly at 60 mg / m ² . The method of administration may be repeated for two or more cycles (preferably three cycles) and the two or more cycles may be performed at three or four week intervals.
In some preferred embodiments, the daily dose of 7.5 × 10 ⁹ to about 7.5 × 10 ¹⁵ , preferably about 1 × 10 ¹² to about 7.5 × 10 ¹³ adenovirus particles may be administered daily for a period of up to 30 days. (Eg dosing regimen of 2 or 2 or 5 to 14 or 30 days of the same dose daily). Multiple dosing regimens may be repeated in cycles of 21 to 28 days. Preferred routes of administration include intraarterial (eg intrahepatic artery), intratumoral and intraperitoneal.
Adenovirus vectors when the tumor suppressor nucleic acid (eg p53) is administered with an adenovirus vector containing a secondary anticancer agent (eg paclitaxel) and a DNA damaging agent (eg cisplatin, carboplatin or nabelbin) Is administered from about 7.5 × 10 ¹² to about 7.5 × 10 ¹³ adenovirus particles per day for 5-14 days. When adenovirus vectors and paclitaxel are administered with carboplatin, the dosage is generally 7.5 × 10 ¹³ adenovirus particles per day. For example, a daily dose of about 7.5 × 10 ¹² adenovirus particles can be used to administer to the lungs.
The present invention also provides a kit for treating mammalian cancer cells or hyperproliferative cells. The kit may comprise a tumor suppressor protein or nucleic acid (preferably a wild type p53 protein or nucleic acid (eg in a viral or nonviral vector), or a retinoblastoma (RB) protein or nucleic acid); And the aforementioned adjuvant anticancer agents (eg paclitaxel or paclitaxel derivatives) and / or optionally the chemotherapeutic agents described above. The kit may optionally further comprise instructions describing administration to inhibit the growth or proliferation of cancer or hyperproliferative cells of tumor suppressor proteins or nucleic acids and adjuvant anticancer agents (and optionally other chemotherapeutic agents). One particularly preferred kit includes A / C / N / 53 and paclitaxel.
In another embodiment, the present invention provides a pharmaceutical composition comprising a tumor suppressor protein or tumor suppressor nucleic acid and an adjuvant anticancer agent. In different embodiments, the pharmaceutical composition optionally comprises a chemotherapeutic compound as described above. One particularly preferred composition comprises p53 nucleic acid (eg A / C / N / 53) and paclitaxel. Tumor inhibiting nucleic acids or proteins and chemotherapeutic agents (eg paclitaxel) can be present in different excipients or contained in one excipient as described above. If multiple excipients are present, the excipients may be intermixed or separated (eg in microcapsules).
In another embodiment, the present invention provides a composition comprising a cancer or hyperproliferative cell of a mammal containing an exogenous tumor suppressor nucleic acid or tumor suppressor protein. The cells optionally comprise adjuvant anticancer agents such as paclitaxel or paclitaxel derivatives. The exogenous tumor suppressor nucleic acid or tumor suppressor protein may be one or more tumor suppressor nucleic acids and / or proteins described above. Similarly, the cells may be one or more hyperproliferative cells and / or cancer cells as described above.
In another embodiment, the present invention provides a method of treating metastatic cells. This method involves contacting a cell with a tumor suppressor nucleic acid or a tumor suppressor polypeptide. Suitable tumor suppressor nucleic acids or polypeptides include tumor suppressor nucleic acids and / or polypeptides as described above. The method further includes the step of contacting the cell with a secondary anticancer agent as described above. In a particularly preferred embodiment, the method comprises topically administering a tumor suppressor nucleic acid and / or polypeptide to the trauma.
In another embodiment, the present invention provides particularly preferred dosage regimens. In one embodiment, the present invention provides a method of treating mammalian cells, comprising multiple administrations of the total dose of tumor suppressor protein or tumor suppressor nucleic acid to the mammalian cell as an incremental dose. Preferred multiple administrations are performed at intervals of about 6 hours or more. One preferred administration includes three or more treatments at intervals of about 24 hours.
In one embodiment, the present invention provides a method of treating mammalian cells. The method involves multiple doses of the total dose of tumor suppressor protein or tumor suppressor nucleic acid to the mammalian cells as incremental doses. Administration may be at intervals of about 6 hours or more. The method may comprise at least three incremental administrations, and the dosage may be administered daily. In one embodiment the method may comprise three or more treatments at intervals of about 24 hours. In another embodiment, the method comprises administering a tumor suppressor nucleic acid at a total dose of about 1 × 10 ⁹ to about 7.5 × 10 ¹⁵ or about 1 × 10 ¹¹ to about 7.5 × 10 ¹³ of adenovirus particles for 5 days. Administering intratumorally with a therapeutic regimen selected from daily administration, daily administration for 15 days, and daily administration method for 30 days. The method further comprises administering a total dose of paclitaxel or paclitaxel derivative of about 75 to about 350 mg / m ² once, daily for 2 days, daily for 3 days, daily for 15 days, for 30 days Dosing regimen selected from daily administration of, daily continuous infusion for 15 days, daily continuous infusion for 30 days, can include administration over 24 hours. The treatment regimen may be repeated for two or more cycles, and two or more cycles may be performed at three or four week intervals. The cells thus treated are ovarian cancer, pancreatic cancer, non-small cell lung cancer, small cell lung cancer, liver cancer, melanoma, retinoblastoma, breast tumor, colorectal cancer, leukemia, lymphoma, brain tumor, cervical cancer, sarcoma, prostate tumor, bladder Tumor cells including tumors, tumors of retinal endothelial tissue, Wilm's tumor, astrocytoma, glioblastoma, neuroblastoma, osteosarcoma, kidney cancer, and cancer selected from the group consisting of head and neck cancers. The treatment preferably inhibits the growth or proliferation of the tumor at the time of analysis by measuring the volume of the tumor.
The present invention also provides a pharmaceutical composition comprising a tumor suppressor protein or tumor suppressor nucleic acid and at least one adjuvant anticancer agent. Adjuvant anticancer agents may be paclitaxel or paclitaxel derivatives. The tumor suppressor protein or tumor suppressor nucleic acid is selected from the group consisting of nucleic acid encoding wild type p53 protein, nucleic acid encoding retinoblastoma protein (RB) protein, wild type p53 protein and retinoblastoma protein.
The retinoblastoma protein may be p110 ^RB or p56 ^RB . Nucleic acids can be contained in recombinant adenovirus vectors. The nucleic acid may be contained in a recombinant adenovirus vector comprising a partial or complete deletion of Protein IX DNA and comprising a nucleic acid encoding a P53 protein. In one embodiment, the deletion of the Protein IX gene sequence ranges from about 3,500 bp from the 5 'virus end to about 4,000 bp from the 5' virus end. DNA deletions may include the sequences designated E1a and E1b. The recombinant adenovirus vector may further comprise adenovirus type 2 late main promoter or human CMV promoter, adenovirus type 2 part leader cDNA and human p53 cNDA. In a preferred embodiment, the vector is A / C / N / 53. The composition may comprise paclitaxel, or paclitaxel derivatives or analogs.
The present invention further provides compositions comprising mammalian cancer or hyperproliferative cells containing exogenous tumor suppressor nucleic acids or tumor suppressor proteins and adjuvant anticancer agents. The tumor suppressor nucleic acid may be a nucleic acid encoding a tumor suppressor protein selected from wild type p53 protein and retinoblastoma (RB) protein. The retinoblastoma protein may be p110 ^RB or p56 ^RB . The cell may be present in a mammal. The cells may be neoplastic, and the neoplastic cells are ovarian cancer, pancreatic cancer, non-small cell lung cancer, small cell lung cancer, liver cancer, melanoma, retinoblastoma, breast tumor, colorectal cancer, leukemia, lymphoma, brain tumor, neck cancer, sarcoma , Prostate tumors, bladder tumors, tumors of retinal endothelial tissue, Wilm's tumor, astrocytoma, glioblastoma, neuroblastoma, osteosarcoma, kidney cancer, and cancer selected from the group consisting of head and neck cancers. .
The present invention provides a method of treating metastatic cells, comprising contacting the metastatic cells with a tumor suppressor nucleic acid or a tumor suppressor polypeptide and an adjuvant anticancer agent. Contacting may include topical administration to the trauma of the tumor suppressor nucleic acid. The method may further comprise simultaneous administration of the chemotherapeutic agent and the chemotherapeutic agent may be cisplatin, carboplatin or navelvin.
The term "adjuvant anticancer agent" refers to a drug having one or more of the following activities: ability to modulate microtubule formation or action, ability to inhibit polyprenyl-protein transferase activity, ability to inhibit angiogenesis, or inhibition of endocrine activity. Point. Adjuvant anticancer agents useful in the present invention are described in more detail below. As used herein, the adjuvant anticancer agent of the present invention does not include a compound having DNA damaging activity.
A "tumor suppressor gene" is a nucleic acid whose loss-of-function mutations are tumorigenic. Thus, lack of, normal mutation, or destruction of tumor suppressor genes in other healthy cells increases the likelihood that cells will maintain neoplastic phase. In contrast, the presence of a functional tumor suppressor gene or protein in a cell inhibits tumorigenicity, malignancy or hyperproliferative phenotype of the host cell. Examples of tumor suppressor nucleic acids within this definition include p110 ^RB , p56 ^RB , p53 and other tumor suppressor genes described in USSN 08 / 328,673 (filed Oct. 25, 1994), which are co-pending with the present application. It is not limited. Tumor suppressor nucleic acids include tumor suppressor genes or nucleic acids derived therefrom (eg, cDNAS, cRNAs, mRNA, and their subsequences encoding the active fragments of each tumor suppressor polypeptide), as well as vectors comprising these subsequences. have.
A "tumor suppressing polypeptide or protein", when present in a cell, refers to a polypeptide that reduces the tumor's tumorigenicity, malignancy or hyperproliferative phenotype.
The term "viral particle" refers to virion itself. Concentrations of infectious adenovirus particles are typically described, for example, in Huyghe (1995) Human Gene Ther. 6: 1403-1418] is measured by spectrophotometric detection of DNA.
The term “neoplastic” or “neoplastic” refers to the growth and / or division of cells at rates beyond the normal growth limits for the cell type.
The term "tumoral" or "tumorogenic" means the ability to form a tumor or the ability to cause tumor formation.
The phrase “treating cells” refers to inhibiting or ameliorating one or more disease characteristics of a diseased cell. When used for neoplastic cancer cells (eg, mammalian cancer cells lacking endogenous wild type tumor suppressor proteins), the phrase “treating cells” refers to alleviating or eliminating the neoplastic phenotype. Typically, such treatment is directed at inhibiting (reducing or stopping the growth and / or proliferation of cells) as compared to the same cells under the same conditions except for therapies (eg, anticancer agents and / or tumor suppressor nucleic acids or polypeptides). Cause Such inhibition includes cell death (eg, cell death). When used with respect to a tumor, this term refers to inhibiting the growth or proliferation of a tumor mass (such as, for example, by volume measurement). Such inhibition can be mediated through a reduction in the growth rate and / or the rate of diet and / or death of the cells that make up the tumor mass. Inhibition of growth or inhibition of proliferation can be achieved by modifying the cell phenotype (eg, restoring morphological properties of healthy cells, restoring contact inhibition, loss of invasive phenotype, inhibition of fixation-independent growth, etc.). . To this end, the diseased cell will have one or more mute traits. Such traits of diseased cells may in particular include incomplete expression of one or more tumor suppressor proteins. Incomplete expression may be characterized by complete loss of one or more functional inhibitory proteins or a decrease in the expression level of one or more functional tumor suppressor proteins. Such cells are often neoplasms and / or tumors.
The term "systemic administration" refers to the administration of a composition or drug, such as a recombinant adenovirus vector or adjuvant anticancer agent of the invention, or a chemotherapeutic compound as used herein, in such a way that they can be introduced into the circulation. "Regional administration" refers to the administration of a composition or drug to a particular anatomical space, such as intraperitoneal, intraperitoneal, subdural, or specific organs. For example, topical administration includes administering a composition or drug to the hepatic artery for topical administration to the liver. The term "local administration" refers to the administration of a composition or drug to a limited or limited anatomical space, such as intratumoral injection into a tumor mass, subcutaneous injection, intramuscular injection, and the like. Those skilled in the art will understand that the composition or drug may also be introduced into the circulation by local or topical administration.
As used herein, the term "reduced tumor formation" refers to the transition of a hyperproliferative (eg, neoplasia) cell to a state of self-proliferation. In the case of tumor cells, "reduced tumorigenicity" means tumorigenic cells that have reduced or eliminated their ability to convert to tumor cells, or tumor cells that become non-tumorigenic or non-tumorigenic cells. Cells with reduced tumorigenesis do not form tumors in vivo, or the retardation extends weeks to months before tumor growth occurs in vivo. Cells with reduced tumorigenesis are also compared with cells that are fully inactivated or have nonfunctional tumor suppressor genes grown in the same physiological environment (eg, tissue, organism age, organism sex, menstrual cycle, etc.). Three-dimensional tumor masses may grow slowly.
As used herein, an “active fragment” of a gene or polypeptide is a smaller portion (s) (partial sequence) of a gene or nucleic acid (eg cDNA) derived therefrom that has the ability to encode a protein with tumor suppressor activity. It includes. Similarly, an active fragment of a polypeptide refers to a partial sequence of a polypeptide containing a tumor suppressor protein. One example of an active fragment is, for example, p56 ^RB described in co-pending USSN 08 / 328,673 (filed Oct. 25, 1994).
The term "malignant tumor" refers to tumor cells with metastatic capacity.
As used herein, “nucleic acid” can be DNA or RNA. Nucleic acids can include modified nucleotides that can be read correctly by the polymerase and do not modify the expression of the polypeptide encoded by the nucleic acid.
The phrase “nucleotide sequence” includes each single chain or both the sense and antisense chains in a double chain.
The phrase “DNA sequence” refers to a single or double chain DNA molecule composed of the nucleotide bases adenosine, thymidine, cytosine and guanosine.
The phrase “coding nucleic acid sequence” refers to a nucleic acid that directs the expression of a particular protein or peptide. Nucleic acid sequences include zero DNA sequence sequences that are transcribed into RAN and then translated into proteins. Nucleic acid sequences include both full length nucleic acid sequences as well as fragment length sequences derived from full length nucleic acid sequences. It is also understood that sequences include synonymous codons of unique sequence (s) that can be introduced to provide all options in a particular host cell.
The expression “expression cassette” refers to a nucleotide sequence that can affect the expression of a structural gene of a host that can correspond to this sequence. Such a cassette includes at least a promoter and also a transcription termination signal. Additional factors necessary or helpful in performing expression may also be used as described herein.
As used herein, the term "operonally linked" refers to the linkage of a promoter upstream sequence from a DNA sequence such that the promoter mediates the transcription of the DNA sequence.
When referring to a nucleic acid sequence or fragment thereof encoding a tumor suppressor protein or polypeptide, "independent" or "virtually pure" refers to an independent nucleic acid sequence that does not encode a protein or peptide other than the tumor suppressor protein or polypeptide or fragment thereof. .
The term “recombinant” provides DNA that is isolated from natural or endogenous sources, or that is chemically or enzymatically modified to remove naturally occurring flanking nucleotides, or non-naturally occurring flanking nucleotides. Point to DNA. A flanking nucleotide is a nucleotide that is higher or lower than the described nucleotide sequence or partial sequence.
"Vector" includes nucleic acids that can infect, transfect, transiently or permanently transduce cells. It will be appreciated that the vector may be the nucleic acid itself or a nucleic acid complexed with a protein or lipid. Vectors may optionally include viral or bacterial nucleic acids and / or proteins, and / or membranes (eg, cell membranes, viral lipid coatings, etc.) Vectors often contain a nucleic acid of interest under the control of a promoter. Expression cassettes include, but are not limited to, replicons (eg, plasmids, bacteriophages) to which fragments of DNA can be attached or replicated. Including, but not limited to, replicative circular DNA (plasmids) and both expressed and non-expressed plasmids.If a recombinant microorganism or cell culture is described as containing a "expression vector," this is the original DNA of the extracellular chromosome and the host Includes all of the DNA introduced into the cell, when the vector is maintained by the host cell, the vector is an autonomic structure, It can be stably replicated or introduced into the genome of the host.
The term "effective amount" refers to the amount of a vector or drug that achieves a positive result in regulating cell growth and / or proliferation.
As used herein, the abbreviation “C.I.U.” refers to “cell infection unit”. C.I.U. is calculated by measuring viral hexon protein positive cells (eg 293 cells) after 48 hours of infection. Huyghe (1995) Human Gene Ther. 6: 1403-1416.
As used herein, the abbreviation “m.o.i.” refers to “multiplicity of infection” and is C.I.U. per cell.
The term "paclitaxel" as used herein refers to a commercially available drug known as Taxol®. Taxol® inhibits replication of eukaryotic cells by enhancing the polymerization of tubulin components into stabilized microtubules that cannot be reconstituted into structures suitable for mitosis.
When it comes to contacting a drug and / or nucleic acid, the term “contacting a cell” is used herein to refer to the internalization of the drug and / or nucleic acid into a cell. In this context, contacting a cell with a nucleic acid is equivalent to transfecting the cell with the nucleic acid. If the drug is lipophilic or the nucleic acid is a complex with a lipid (eg, cationic lipid), a simple contact will result in transport (eg, active transport, passive transport and / or diffusion) to the cell. . Alternatively, the composition will be actively transported to the cell if the drug and / or nucleic acid alone or in combination with a carrier. That is, for example, if the nucleic acid is present as an infection vector (eg, adenovirus), the vector can mediate the uptake of the nucleic acid into the cell. Nucleic acids can be complexed with drugs that specifically interact with extracellular receptors to facilitate delivery of nucleic acids to cells, examples of which are ligands / polys as described in US Pat. Nos. 5,166,320 and 5,635,383. Cation / DNA complex. In addition, delivery by the virus can be enhanced by recombinantly modifying the spherical or fibrous domains of the viral genome into the target component of the cell.
Components herein indicated as "A / C / N / 53", "A / M / N / 53", p110 ^RB , p56 ^RB are described in co-pending application USSN 08 / 328,673 (filed Oct. 25, 1994, International Designation indicated in Publication 95/11984).
When describing a protein, "conservative substitutions" refer to changes in the amino acid composition of the protein that do not substantially modify the activity of the protein. Thus, a "conservatively modified variant" of a particular amino acid sequence means that amino acid substitutions, or similar properties (e.g., amino acid substitutions in amino acids that are not important for the activity of the protein, do not substantially modify activity even if the important amino acid is substituted) , Acidic, basic, positive or negative charge, polar or nonpolar, etc.). Conservative substitutions that can provide functionally similar amino acids are well known in the art. For example, each of the six groups below includes amino acids that are conservative substituents on one another.
1) Alanine (A), Serine (S), Threonine (T)
2) aspartic acid (D), glutamic acid (E),
3) Asparagine (N), Glutamine (Q)
4) Arginine (R), Lysine (K)
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V) and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
(Creighton (1984) Proteins W.H. Freeman and Company). Also, each substitution, deletion or addition that modifies, adds or deletes one or a small percentage of amino acids in the encoded sequence is also a "conservatively modified variant".
<Brief Description of Drawings>
1 illustrates in vitro inhibition of SK-OV-3 ovarian tumor cells by various concentrations of p53 (A / C / N / 53) and / or Taxol®.
FIG. 2 provides isobologram analysis for the experiment illustrated in FIG. 1. Synergy between Taxol® and p53 (A / C / N / 53) was observed when cells were pretreated with Taxol® 24 hours prior to treatment with p53.
3A, 3B and 3C illustrate the efficiency of p53 Ad against human breast cancer xenografted into nude mice. Mice were dosed with a total of 2.2 × 10 ⁹ CIU adenoviruses (A / C / N / 53 or Ad) per mouse divided into 10 infusions on days 0-4 and 7-11. Mice were treated with p53 Ad, beta-gal Ad, or vehicle alone. 3A illustrates the results with MDA-MB-231 tumors. 3B illustrates the results by MDA-MB-468 (-468) tumors and FIG. 3C illustrates the results by MDA-MB-435 (-435) tumors.
4C and 4B provide p53 Ad (A / C / N / 53) dose response curves for MDA-MB-231 (-231) tumors (FIG. 4A) and MDA-MB-468 tumors. Mice were administered intraperitoneally in 10 doses of 1 × 10 ⁷ CIUp53 Ad (A / C / N / 53) on days 0-4 and 7-11. Average percent inhibition was tumor-treated with buffer volume at each p53 Ad dose at 14 / 15.18, 21, 24, 28, 30/32, and 35 days (MDA-MB-468 tumors only on day 35). -231 tumors averaged 22.5 ± 1.2 mm ³ on day 0, while -468 tumors averaged 33.1 ± 1.8 mm ³ on day 0.
5 provides a comparison of efficacy as a therapeutic when administered alone or in divided doses. Tumors (MDA-MB-231) were administered for 1 and 3 weeks followed by a total of 2.2 × 10 ⁸ CIUp53 Ad per week.
Figure 6 illustrates the efficiency of multiple cycles of low dose p53 Ad against large and well established tumors. Total 1.32 × 10 ⁹ CIUp53 Ad was administered to MDA-MB-468 xenografts over 6 weeks (P = stability level of control tumor growth rate; E = end of administration).
7A 7B and 7C show nude injection of 1 × 10 ⁹ CIUp53 Ad (A / C / N / 53) in a single cyclic infusion (FIG. 7A), or divided into three (FIG. 7B) or five (FIG. 7C) In vivo inhibition of MDA-MB-468 tumors in mice is illustrated.
Figure 8 illustrates the ability of low dose dexamethasone to inhibit the inhibition of tumor growth mediated by NK cells in seed mice. MDA-AB-231 tumors were administered at 14-18 days and 21-25 days divided by 10 injections of the total 2 × 10 ⁹ CIU beta-gal Ad (1.1 × 1011 virus particles). Subcutaneous dexamethasone (or placebo) pellets released 83.3 μg of steroid per day.
9 compares the combination p53 and cisplatin combination therapy to normal and tumor cells.
The present invention relates to a novel method of treatment of a subject with a hyperproliferative disease, such as a tumor or metastatic disease. In particular, the present invention provides methods for inhibiting hyperproliferation of cells, more specifically neoplastic cells, comprising the use of a combination of tumor suppressor genes or gene products and adjuvant anticancer agents.
The present invention relates to novel methods of inhibiting the growth and / or proliferation of cells, more specifically the growth and proliferation of cancer cells. In one embodiment, the invention comprises contacting a cell with a tumor suppressor nucleic acid or tumor suppressor protein and an adjuvant anticancer agent. Typically, the tumor suppressor protein or nucleic acid used will be of the same kind as the tumor suppressor protein lacking activity. That is, if the cell lacks endogenous p53 activity p53 protein or p53 nucleic acid will be used.
Surprisingly, unlike the results of previous studies (Wahl et al. (1996) Nature Med., 2 (1): 72-79 and Hawkins et al. (1996) Canc. Res. 56: 892-898) Researchers of the present invention lack or endogenous wild-type tumor suppressor proteins (ie, many neoplastic cells) with adjuvant anticancer agents (eg, paclitaxel (taxol, trademark) and tumor suppressor genes or polypeptides (eg, p53) or We have found that the treatment of defective mammalian cells results in inhibition of proliferation and / or growth of cells to a greater extent than treatment with chemotherapy or tumor suppressor constructs alone. Treatment has been found to dramatically increase the antiproliferative effect of tumor suppressor nucleic acids, although not wishing to be bound by a particular theory, the following effects may be achieved by possible means that an anticancer agent may contribute: Increase the efficiency of infection of various gene therapy vectors (eg, adenoviruses), increase the level of expression of tumor suppressor genes, stabilize microtubules to maintain intracellular viral transport, or various intercellular mechanisms. It is believed to increase the effect via the interaction of (e.g., signaling pathways, apoptosis pathways, cellular circulation pathways).
That is, in one embodiment, the invention inhibits mammalian cells lacking or binding to diseased endogenous wild type tumor suppressor proteins in contact with a secondary anticancer agent and a tumor suppressor nucleic acid and / or a tumor suppressor polypeptide. Provide a way to. If the cells are present in the tumor, the method inhibits tumor growth and thus provides a method for treating cancer. Particularly preferred tumor suppressor nucleic acids or polypeptides include p53, RB, h-NUC (see, eg, Chen (1995) Cell Growth Differ. 6: 199-210) or active fragments thereof (eg, p110 ^RB). , p56 ^RB ), and particularly preferred adjuvant anticancer agents (compounds) are paclitaxel and compounds with paclitaxel like activity, for example paclitaxel derivatives (eg analogs).
We also found that contacting cells with tumor suppressor nucleic acids and / or polypeptides can inhibit metastatic cells. Such inhibition may take the form of inhibiting the formation, growth, migration or regeneration of metastatic cells. In one embodiment, the inhibition can be characterized by inhibiting (eg, reducing and / or eliminating) the development of distant neoplasm from the primary tumor. The present invention thus provides a method for treating (mitigating or eliminating) the progression of metastatic disease. The present invention encompasses contacting metastatic cells with tumor suppressor nucleic acids and / or polypeptides. In a particularly preferred embodiment, the invention comprises contacting a cell at a surgical wound site (after removing (debulking) the tumor mass) with a tumor suppressor nucleic acid and / or a tumor suppressor polypeptide combined with a secondary anticancer agent. Can be. These cells can be further contacted with the adjuvant anticancer agents described herein.
In another embodiment, the present invention provides an advantageous therapeutic regimen using tumor suppressor genes and gene products. In part, these therapeutic regimens are based on the surprising finding that in inhibiting cells or tumors, it is more effective when tumor suppressor nucleic acids and / or polypeptides are delivered in multiple doses rather than in a single cycle.
The order in which tumor suppressors and adjuvant anticancer agents are administered is not critical to the invention. That is, the composition (s) may be administered simultaneously or sequentially. For example, in one embodiment, pretreatment of cells with one or more adjuvant anticancer agents (alone or combined with chemotherapeutic agents) can increase the efficiency of tumor suppressor nucleic acids and / or polypeptides that are administered sequentially. . In one embodiment, the chemotherapeutic agent is administered prior to the adjuvant anticancer agent and the tumor suppressor nucleic acid and / or polypeptide. In another embodiment, the adjuvant anticancer agent (either alone or in combination with chemotherapeutic agents) is administered simultaneously with the tumor suppressor nucleic acid and / or polypeptide. In further embodiments, the tumor suppressor nucleic acid and / or polypeptide is administered after the tumor suppressor nucleic acid and / or polypeptide.
The anticancer effects of the methods of the invention and the compositions to be administered include nonspecific effects called antitumor, bystander effects (see, eg, Zhang (1996) Cancer Metastasis Rev. 15: 385-401 and Okada (1996). Gene Ther. 3: 957-996). In addition, the immune system can also be modulated to selectively enhance (or lower) humoral or cellular cancer of the immune system, ie B cells and / or T cells (eg, cytotoxic lymphocytes (CTLs) or Tumor invasive lymphocyte (TIL) response can be modulated. For example, an increase in TIL is observed when p53-expressing adenovirus is administered to humans. In particular, an increase in TIL (typically T helper cells, CD3 ⁺ , and CD4 ⁺ ) was observed when hepatic arterial administration of p53-expressing adenovirus to treat metastatic hepatic cancer as described in detail below. Is observed.
The method of the present invention does not limit the use of a single anticancer agent or even a single chemotherapeutic agent. Accordingly, the present invention provides a method of inhibiting a diseased mammalian cell lacking an endogenous tumor suppressor protein, or a tumor comprising such cell, by contacting the cell or tumor with a tumor suppressor nucleic acid described herein and one or more accessory anticancer agents. To provide.
I. Adjuvant anticancer drugs
A) microtubule agents
As described in one embodiment, the present invention provides for endogenous tumor suppressor proteins by contacting cells with tumor suppressor proteins or adjuvant anticancer agents such as tumor suppressor nucleic acids, microtubule agents (eg, paclitaxel, paclitaxel derivatives or paclitaxel like compounds). Provided is a method for inhibiting a diseased cell lacking this disease. Microtubule agents used in the present cloud are compounds that act on microtubule formation and / or action to exhibit cell mitotic effects. Such drugs can be, for example, microtubule stabilizers, or microtubule forming disruptors.
Microtubule agents useful in the present invention are well known in the art and include allocolkicin (NSC 406042), haliconerin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), Dola Statin 10 (NSC 376128), maytansine (NSC 153858), lysine (NSC 332598), paclitaxel (taxol®, NSC 125973), taxol® derivatives (e.g., NSC 608832), thiocolkicin (NSC 361792), trityl cysteine (NSC 83265), vinblastine sulphate (NSC 49842), vincristine sulphate (NSC 67574), epothilone A, epothilones and discodimolides (see, eg, Service , (1996), Science, 274: 2009]), estramustine, nocodazole, MAP4, and the like. Examples of such drugs are described in the scientific and patent literature (eg, Bulinski (1997) J. Cell Sci. 110: 3055-3064; Panda (1997) Proc. Natl. Acad. Sci. USA 94: 10560-10564; Muhlradt ( 1997) Cancer Res. 57: 3344-3346; Nicolaou (1997) Nature 387: 268-272; Vasquez (1997) Mol. Biol. Cell. 8: 973-985; Panda (1996) J. Biol. Chem. 271: 29807-29812).
Particularly preferred drugs are compounds with paclitaxel like activity. Examples of these include, but are not limited to, paclitaxel and paclitaxel derivatives (paclitaxel like compounds) and analogs. Paclitaxel and its derivatives are commercially available. In addition, methods for preparing paclitaxel and paclitaxel derivatives and analogs are well known in the art (eg, US Pat. Nos. 5,569,729; 5,565,478; 5,530,020; 5,527,924, 5,508,447; 5,489,589; 5,488,116; 5,484,809; 5,478,854; 5,478,736; 5,475,120; 5,468,769; 5,461,169; 5,440,057; 5,422,364; 5,411,984; 5,405,972; 5,297,506)
Additional microtubule agents are semi-automated to evaluate tubulin polymerization activity of paclitaxel analogs with one of a number of assays known in the art, for example, cell assays to assess the ability of compounds to block cells in mitosis. The assay can be used to evaluate (see, eg, Lopes (1997) Cancer Chemother. Pharmacol. 41: 37-47).
In general, the activity of a test compound is determined by measuring whether the cell cycle is disrupted by contacting the compound with cells and, in particular, by inhibiting mitosis. Such inhibition can be mediated by disruption of mitotic organs, for example disruption of spindle formation. Cells with mitosis disruption are characterized by modified forms (eg, microtubule compaction, increased chromosome number).
In a preferred embodiment, compounds with potential tubulin polymerization activity were selected in vitro. In a preferred embodiment, the compounds were selected for cultured WR21 cells (cell line 69-2 web-las mice) for inhibition of proliferation and / or modified cell morphology, in particular microtubule compaction. In vivo selection of positive test compounds was then performed using nude mice containing WR21 tumor cells. Detailed protocols for this screening method are described in Porter (1995) Lab. Anim. Sci., 45 (2): 145-150.
Other methods for selecting compounds for the desired activity are well known to those skilled in the art. Typically such an assay involves analyzing the inhibition of microtubule association and / or degradation. Assays for microtubule associations are described, for example, in Gaskin et al. (1974) J. Molec. Biol., 89: 737-758. US Pat. No. 5569,720 also provides in vitro and in vivo assays for compounds with paclitaxel like activity.
B) Proprenyl-protein transferase inhibitors
In another embodiment, the present invention provides for the use of a tumor suppressor nucleic acid and / or polypeptide and a proprenyl-protein transferase inhibitor together. Particularly preferred polyprenyl-protein transferase inhibitors include, but are not limited to, panesyl-protein transferase (FPT) inhibitors, geranylgeranyl-protein transferase inhibitors, and other monoterpene protein transferases. Examples of compounds that are polyprenyl-protein transferase inhibitors are well known in the scientific and patent literature (see, eg, Zhang (1997) J. Biol. Chem. 272: 10232-10239; Njoroge (1997) J). Med. Chem. 40: 4290-4301; Mallamx (1997) Bioorg.Med. Chem. 5: 93-99).
Compounds which can be exemplified by farnesyl-protein transferase are shown below.
The FPT inhibitor designated "FPT39" described in International Publication No. 97/23478 (filed December 19, 1996) is a compound designated as Compound "39.0" on page 95 of International Publication No. 97/23478.
<Compound FPT39>

When FPT39 is used in combination therapy with the p53 expressing adenovirus of the invention for prostate tumor cells and breast tumor cells as described below, the combination is more effective at killing tumor cells than anything else used alone. .
Oligopeptides (usually also tetrapeptides or pentapeptides comprising the formula Cys-Xaa1-Xaa2-Xaa3; European Patent Publications 461,489; 520,823; 528,486 and International Publication 95/11917).
Peptido analog compounds, in particular Cys-Xaa-Xaa-Xaa analogs; European Patent Publication No. 535,730; US Pat. No. 535,731; US Pat. No. 618,221; International Publication No. 94/09766; International Publication No. 94/10138; International Publication No. 94/07966; US 5,326,773, 5,340,828, 5,420,245; International Publication No. 95/20396; U.S. Patent 5,439,918 and International Publication 95/20396.
Panesylated Peptide Analog Compounds—particularly panesylated Cys-Xaa-Xaa-Xaa analogs: German Patent Publication No. 2,276,618.
Other peptidom analog compounds: US Pat. No. 5,352,705, International Publication 94/00419; 95/00497; 95/09000; 95/09001; 91/12612; 95/25086; European Patent Publication 675,112 and French Patent Publication No. 2,718,149.
Fused ring tricyclic benzocycloheptapyridine: International Publication No. 95/10514; US 95/10515; US 95/10516; 96/30363; 96/30018; 96/30017; 96/30362; 96/31111; 96/31478; 96/31477; US9631505; PCT / US96 / 19603, International Publication No. 97/23478; US Patent Application No. 08/728104; No. 08 / 712,989 No. 08 / 713,326, No. 08 / 713,908, No. 08 / 713,705, No. 08 / 713,703, No. 08 / 710,225, No. 08 / 711,925, No. 08 / 712,924, 08 / 713,323, 08 / 713,297.
Farnesyl Derivatives: European Patent Publication 534,546, International Publication 94/19357, 95/08546, European Patent Publication 537,007 and International Publication 95/13059.
Natural products and derivatives: International Publication No. 94/18157; US Patent No. 5,430,055; German Patent Publication Nos. 2,261,373, 2,261,374 and 2,261,375; U.S. Patent 5,420,334, 5,436,263.
Other compounds: International Publication No. 94/26723; US 95/08542; US Patent No. 5,420,157; International Publication Nos. 95/21815 and 96/31501.
C) anti-angiogenic compounds
Tumor suppressor proteins or nucleic acids of the invention can also be administered in conjunction with anti-angiogenic compounds. Preferred anti-angiogenic compositions inhibit the formation or proliferation of blood vessels, more preferably the formation and / or proliferation of blood vessels into tumors.
Suitable anti-angiogenic compositions include galardine (GM6001 from Glycomed Inc., Alameda, CA), endocrine response inhibitors (eg, drugs such as interferon alpha, TNP-470 and ductal endocrine growth factor inhibitors), cells Drugs that promote and degrade the matrix (e.g., non-taxin (Ixsys Co., San Diego, Calif., Human LM-609 antibody) and drugs that directly affect blood vessel growth (e.g., Group A strepto CM-101, and thalidomide, which are derived from exotoxins of the Streptococcus antigen and bind to new blood vessels and induce a strong host inflammatory response.
Several types of steroids have also been shown to exhibit anti-angiogenic activity. In particular, a number of reports have demonstrated that synthetic progesterone, methoxyprogesterone acetate (MPA), strongly inhibits angiogenesis in rabbit corneal analysis (Oikawa (1988) Cancer Lett. 43:85) (Oikawa (1988) Cancer Lett. 43. 85). The 5FU prodrug 5'-deoxy-5-fluororidine (5'DFUR) can also be characterized as an anti-angiogenic compound, because 5'DFUR is a marker of PD-ECGF / TP positive tumors. This is because it is converted to 5-FU by thymidine phosphorylase activity. In this study, 5'DFUR could be selectively active against PD-ECGF / TP with high anti-angiogenic potential. Recent clinical studies suggest that 5'DFUR may be effective against PD-ECGF / TP positive tumors. A dramatic increase in the anticancer effect of 5'DFUR has been shown to be seen in cells transfected with PD-ECGF / TP compared to untransfected wild type cells (Haraguchi (1993) Cancer Res. 53: 5680-5682). In addition, the combination compound of 5'DFUR + MPA is also an effective anti-angiogenic agent (Yayoi (1994) Int J Oncol. 5: 27-32). The combination of 5'DFUR + MPA can be classified as a combination of angiogenesis inhibitors, endocrine growth factor inhibitors and protease inhibitors with different spectra. In vivo experiments with DMBA induced rat breast cancer also showed a combination effect with AGM-1470 (Yamamoto (1995) Oncol Reports 2: 793-796).
Other groups of anti-angiogenic compounds useful in the present invention include polysaccharides (eg, pentosan polysulfate) that can interfere with the function of heparin binding growth factors that promote angiogenesis.
Other modulators of angiogenesis include platelet factor IV and AGM 1470. Still other examples are natural sources of collagenase inhibitors, vitamin D3-analogues, fumigalin, herbimycin A, and isoflavones.
D) endocrine therapy
Endocrine therapy, a well-established representative cytostatic therapy, can make hormone-dependent cells inactive, reducing tumor cell numbers in vivo and inhibiting tumor growth in patients with hormone-dependent tumors. Such therapies are expected to increase the effect of tumor suppression on the treatment of excessively proliferating cells. Thus in another embodiment the invention provides for the use of, for example, tumor suppressor nucleic acids and / or polypeptides in combination with antiestrogens, antiandrogens or antiprogesterone. Endocrine therapies are well known to those skilled in the art and include, for example, tamoxifen, toremifene (see, eg, US Pat. No. 4,696,949), flutamide, megacene, and loopron (see, eg, International Publication No. 91/00732). , International Publication No. 93/10741, International Publication No. 96/26201 and Gauthier et al. J. Med. Chem. 40: 217-2122 (1997).
E) Delivery of Adjuvant Anticancer Agents: Pharmaceutical Compositions
Pharmaceutical composition
Adjuvant anticancer agents used in the methods of the present invention are typically combined with a pharmaceutically acceptable carrier (excipient) to form a pharmacological composition. The pharmaceutical composition of the present invention may comprise a tumor suppressor gene or polypeptide, eg, p53, or one or more adjuvant anticancer agents, with or without RB.
Pharmaceutically acceptable carriers may contain, for example, physiologically acceptable compounds which act to stabilize the composition or to increase or decrease the absorption of the drug and / or pharmaceutical composition. Examples of physiologically acceptable compounds include reducing carbohydrates (eg, glucose, sucrose, or dextran), antioxidants (eg, ascorbic acid or glutathione), chelating agents, low molecular weight proteins, secondary anticancer agents, or reducing hydrolysis. And excipients, or other stabilizers and / or buffers. Detergents may also be used to stabilize the composition or to increase or decrease the absorption of the pharmaceutical composition (especially detergents see below).
Other physiologically acceptable compounds include wetting agents, emulsifiers, dispersants, or preservatives that are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known in the art, for example phenol and ascorbic acid. Those skilled in the art will recognize that the choice of a pharmaceutically acceptable carrier, including pharmaceutically acceptable compounds, depends, for example, on the route of administration of the adjuvant anticancer agent and the specific physiological-chemical properties of the adjuvant anticancer agent.
Compositions for administration will typically comprise a solution of the secondary anticancer agent dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier for a water soluble secondary anticancer agent. Various carriers can be used, for example buffered saline and the like. This solution is a sterile solution and generally contains no undesirable substances. This composition can be sterilized by conventional sterilization techniques well known in the art. The composition may contain pH adjusting and buffering agents, toxicity adjusting agents and other pharmaceutically acceptable auxiliaries required for similar physiological conditions such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of adjuvant anticancer agents in this formulation can vary widely and will be selected based primarily on fluid volume, viscosity, weight, etc., depending on the particular mode of administration chosen and the requirements of the patient.
<Delivery path>
Adjuvant anticancer agents used in the methods of the invention may be used in any manner known in the art, for example systemically, topically, locally; Intraarterial, intratumoral, intravenous (IV), parenteral, pleural cavities, topical, oral or topical administration, subcutaneous, intratracheal (eg aerosol) or transmucosal (eg buccal, bladder) , Vaginal, uterine, rectal, nasal mucosa), intratumoral (eg, transdermal or topical infusion), can be delivered alone or as a pharmaceutical composition (with or without a tumor suppressor, eg p53). . Particularly preferred routes of administration include intraarterial infusion, which is particularly desirable if it has a local effect that is concentrated, for example, in certain organs (eg, brain, liver, spleen, lung). For example, hepatic arterial infusion is preferred if the anti-cancer local effect is needed in the liver, and the composition is one of the arteries of the brain (e.g., in the treatment of brain tumors), carotid arteries, or carotid arteries of the arterial system (laryngeal artery, heart artery, temporal bone Carotid artery infusion is preferred if needed to be delivered to arteries, central arteries, maxillary arteries, etc. (eg, in the treatment of brain tumors).
Paclitaxel and certain paclitaxel derivatives are only temporarily dissolved in aqueous solution. In a preferred embodiment, the composition is delivered directly to the tumor site (by direct application during injection, tubulation, or surgical operation), or solubilized in an acceptable excipient. Methods of administering paclitaxel and derivatives thereof are well known to those skilled in the art (see, eg, US Pat. Nos. 5,583,153, 5,565,478, 5,496,804, 45,484,809). Other paclitaxel derivatives are water soluble analogs and / or prodrugs (see, eg, US Pat. Nos. 5,411,984 and 5,422,364) and can be easily administered by the various methods described above.
Pharmaceutical compositions of the invention are particularly useful in topical administration, for example, surgical wounds for treating early tumors, neoplasms and metastatic cells and other processes. In another embodiment, the compositions of the present invention are useful for parenteral administration, eg, intravenous administration, or lumen of body cavity or organ.
Treatment Cure
Pharmaceutical compositions can be administered in a variety of unit dosage forms, depending on the method of administration. For example, unit dosage forms suitable for oral administration include powders, tablets, pills, capsules, and lozenges. It is recognized that adjuvant anticancer compounds (eg, paclitaxel and related compounds described) should be protected from digestion when administered orally. This can usually be accomplished by combining the adjuvant anticancer agent with the composition to render it resistant to acid and enzymatic hydrolysis, or by packing the adjuvant anticancer agent in a suitably resistant carrier such as liposomes. Methods of protecting compounds from digestion are well known in the art (see, eg, US Pat. No. 5,391,377, which describes lipid compositions for oral delivery of therapeutic agents).
Dosages for conventional chemotherapy are well known to those skilled in the art. In addition, such dosages are typically visibly enema and can be adjusted according to the particular treatment content, patient patience, and the like. That is, the dosage of a conventional pharmaceutical composition (eg, paclitaxel), for example for intravenous (IV) administration, is from 1 to 24 hours (typically 1, 3 or 6 hours, more preferably 3 hours). It will be administered at about 135 mg / m ² , more preferably repeating every 3 to 6 cycles every three weeks. In order to reduce the frequency and severity of hypersensitivity reactions, the patient also received about 20 mg of dexamethasone (Decadron and others), about 12 and 6 hours prior to treatment with paclitaxel, 50 mg of di Phenylhydramine (Benadryl® and others) + about 300 mg of cimetidine (Tagamet®) or 50 mg of lantidine (Zantac®) orally Can be awarded. Substantially large doses of up to about 350 mg / m ² per day may be used, particularly when the drug is administered to an isolated site other than the bloodstream, such as the body cavity or the lumen of the organ. Substantially higher doses may be administered, for example, by any route selected. Current methods for preparing compositions that can be administered parenterally are well known to those skilled in the art and are described in Remington's Pharmaceutical Science, 15th ed. Mack Publishing Company, Easton, Pennsylvania (1980) and US Pat. Nos. 5,583,153, 5,565,478, 5,496,804 and 5,484,809. Typical dosages will be, for example, 20 to 150 mg / m ² per week for intraperitoneal administration, or about 250 mg / m ² every three weeks.
Compositions containing adjuvant anticancer agents can be administered for therapy treatment. In therapeutic applications, the composition is administered to a patient suffering from a disease characterized by overproliferation of one or more cell types in an amount sufficient to treat or at least partially sufficient to stop the disease and / or complications. The amount necessary to achieve this is defined as "therapeutically effective dosage". The amount effective for this use will depend on the severity of the disease and the overall condition of the patient's health.
Single or multiple administrations of the compositions of the present invention can be administered differently depending on the dosage and frequency required and tolerated by the patient. In any case, the compositions of the present invention should provide a sufficient amount of adjuvant anticancer agent to effectively treat the patient.
II. Tumor Suppressor Genes and Gene Products
A) Tumor inhibitors known to be preferred
In one embodiment, as described above, the invention provides a method of inhibiting growth and / or proliferation of a cell by contacting the cell with a tumor suppressor nucleic acid and an adjuvant anticancer agent (eg, paclitaxel, paclitaxel derivative or paclitaxel like compound). To provide.
Tumor suppressor genes are well known to those skilled in the art and include, for example, RB, p53, APC, FHIT (eg, Siprashvili (1997) Proc. Natl. Acad. Sci. USA 94: 13771-13776). BRACA1, BRCA2, VHL, WT, DCC, FAP, NF, MEN, E-cadherin, nm23, MMACI and PTC. RB or retinoblastoma genes are circular tumor inhibitors and are well characterized (see, eg, Science 247: 712-715, Benedict (1980) Cancer Invest., 8: 535-540, Riley (1990) Ann Rev. Cell Biol. 10-1-29. And Wienberg (1992) Science 254: 1138-1146). The best characterized tumor suppressor is p53, which is included in the genetic constitution that develops into a variety of tumors in families with many neoplasms as well as Li-Fraumeni syndrome. (See, eg, Wills (1994) Hum. Gene Therao. 5: 1079-1088, US Pat. No. 5,532,220, International Publication No. 95/289048, and the cloning expression and use of p53 in gene therapy. Harris (1996) J. Nat. Canc. Inst. 88 (20): 1442). Other tumor inhibitors include WT (ie WT1 at 11p13), genetic properties of Williams' tumors (see, eg, Call et al. (1990) Cell. 60: 60: 509-520, Gessler (1990) Nature 343: 774-778 and Rose et al. (1990) Cell, 60: 495-508). The tumor suppressor gene, called FHIT, in the Fragile Histidine Triad, is located locally on chromosome 3 (3p14.2 and also 3p21), which are known to be prone to metastasis and cleavage. Gap is believed to cause esophageal, gastric and colon cancer (see, eg, Ohta et al. (1994) Cell, 84: 587-597, GenBank Accession No: U469227). Tumor suppressor genes DCC (18q21) and FAP are associated with colon cancer (see, eg, Hedrick et al. (1994) Genes Dev., 8 (10): 1174-1183. GenBank Accession No: X76132 for DCC, And Wienberg (1992) Science, 254: 1138-1146 for FAP]. NF tumor inhibitors (NF1 at 17q11 and NF2 at 22q12) are associated with neurological tumors (eg, neurofibromatosis for NF1 [Caivthon et al. (1990) Cell, 62: 193-201, Viskochil et al. (1990) Cell, 62: 187-192, Wallace et al. (1990) Science, 249: 181-186, and Xug et al. (1990) Cell, 62: 599-608 and meningioma and schwannoma for NF2) . MEN tumor inhibitors are associated with tumors of multiple endocrine neoplastic syndromes (see, eg, Wienberg Science, 254: 1138-1146, and Marshall (1991) Cell, 64: 313-326). VHL tumor inhibitors are associated with von Hippel-Landau (Lafit (1993) Science 260: 1317-1320, GenBank Accession No: L15409). The well known BRCA1 and BRCA2 genes are associated with breast cancer (see, eg, Skolnick (1994) Science, 266: 66-71, GenBank Accession No: U14680 for BRCA1, and Teng (1996) Nature Genet. 13: 241 -244, GenBank Accession No: U43746. In addition, the E-cadherin gene is associated with an invasive phenotype of prostate cancer [Umbas (1992) Cancer Res. 52: 5104-5109, Bussemakers (1992) Cancer Res. 52: 2916-2999, GenBank Accession No: 272397]. NM23 gene is associated with tumor metastasis [Dooley (1994) Hum. Genet., 93 (1): 63 66, GenBank Accession No: X75598]. Other tumor suppressors include DPC4 (as found in 18q21) associated with pancreatic cancer, hMLH1 (3p) and hMSH2 (2p) associated with colon cancer, and CDKN2 (p16) and (9p) associated with melanoma, pancreatic cancer and esophageal cancer. Finally, the human PTC gene (Drosophila fragment (ptc) gene) is associated with nevus basal cell cancer syndrome (NBCCS) and celiac basal cancer syndrome (see, for example, Hahn et al. (1996) Cell, 85: 841-851). Tumor suppressor genes in this list are not exhaustive and are not intended to be limiting, merely to illustrate various known tumor inhibitors.
B) Identification and screening of previously unknown tumor inhibitors
Methods of identifying or analyzing tumor suppressor genes are well known to those skilled in the art. Typically, hyperproliferative cells were screened for genes that had lost or mutated genes associated with the hyperproliferative state. The most stringent test for genes considered to be tumor suppressor genes (TSG) is the ability to inhibit the tumorigenic phenotype of tumors or cells derived from tumors. It is desirable to introduce tumor suppressor nucleic acids into tumor cells as cDNAs cleaned in an appropriate expression vector, or to introduce individual chromosomes containing candidate tumor suppressor genes into tumor cells by microcellular transfer technology. Alternatively, tumor suppressor gene products (eg, tumor suppressor polypeptides) are introduced into the cell (s) and the rate of proliferation of the cells is measured (eg, counting cells or measuring tumor volume, etc.). Complete or partial inhibition of proliferation, contact inhibition, loss of invasive phenotype, cell differentiation and cell death are measures of inhibition of all tumorigenic phenotypes (reduced response to neoplastic status).
Methods for screening tumors to identify modified or unexpressed nucleic acids are well known to those of skill in the art. Such methods include subtractive hybridization (see, eg, Hampson (1992) Nucleic Acids Res. 20: 2899), comparative genome hybridization (CGH) (see, eg, International Publication No. 93/18186). , Kallioniemi (1992) Science, 258: 818) and expression monitoring using high density arrayed nucleic acid probes (see, eg, Lockhart (1996 Nature Biotechnology, 14 (13): 1675-1680)). This is not restrictive.
C) Preparation of p53 and Other Tumor Suppressors
As described above, the present invention relates to contacting a tumor suppressor nucleic acid or a tumor suppressor gene product, such as a polypeptide, for example with ex vivo cells, cells in a physiological solution (eg blood), cells of tissue organs, or a polypeptide. Include. Tumor suppressor nucleic acids or polypeptides are described in the above RB, p53, h-NUC (Chen supra (1995)), APC, FHIT, BRACA1, BRCA2, VHL, WT, DCC, FAP, NF, MEN, E-cadherin, nm23 Or any known tumor suppressor nucleic acid or polypeptide, such as, MMACI, and PTC. In a preferred embodiment the tumor suppressor is an RB nucleic acid or RB polypeptide or p53 nucleic acid or p53 polypeptide, or active fragment (s) thereof.
In the most preferred embodiment the p53 or RB tumor suppressor nucleic acid is located in an expression cassette that expresses a tumor suppressor gene or cDNA under the control of a promoter when located in a target (eg tumor) cell. Methods of constructing expression cassettes and / or vectors encoding tumor suppressor genes are well known to those skilled in the art, as described below.
1. Preparation of Tumor Suppressing Nucleic Acids
The DNA encoding a tumor suppressor protein or protein sequence of the invention may be subjected to, for example, cloning and processing of the appropriate sequence with a restriction enzyme or direct chemical synthesis (e.g., using sequence information present as indicated above), eg See Narang, Meth. Enzymol. 68: 90-99 (1979); Brown et al. Meth. Enzymol. 68: 109-151 (1979); Beaucage et al., Tetra. Lett., 22: 1859-1862 (1981); diethylphosphoramidite method; And the solid phase support method of US Pat. No. 4,458,066.
Single stranded oligonucleotides were prepared by chemical synthesis. It can be converted to double stranded DNA by hybridizing with complementary sequences or polymerizing with DNA polymerase using a single strand as template. Those skilled in the art will appreciate that chemical synthesis of DNA is limited to about 100 base sequences, but longer sequences can be obtained by ligating shorter sequences.
Alternatively, the partial sequences can be cloned and the appropriate partial sequences can be cleaved using appropriate restriction enzymes. The fragment can then be ligated to the desired DNA sequence.
According to one embodiment, the tumor suppressor nucleic acid of the present invention can be cloned using DNA amplification methods such as polymerase chain reaction (PCR). As such, nucleic acid sequences or subsequences were amplified by PCR using sense primers carrying one restriction enzyme site (eg NdeI) and antisense primers carrying the other restriction enzyme site (eg HindIII). . This will produce a nucleic acid encoding the desired tumor suppressor sequence or subsequence and having a terminal restriction enzyme site. The nucleic acid can then be readily ligated to a vector comprising the nucleic acid encoding the second molecule and having the corresponding appropriate restriction enzyme site. Suitable PCR primers can be determined by one of skill in the art using well known sequence information for any particular tumor suppressor gene, cDNA, or protein known. Appropriate restriction enzyme sites can also be added to nucleic acids encoding tumor suppressor proteins or protein subsequences with site-directed mutations. Plasmids containing tumor suppressor sequences or subsequences were cut according to standard methods with appropriate restriction enzyme endonucleases and then ligated to the vector encoding the second molecule.
As described above, nucleic acid sequences of many tumor suppressor genes are known. Thus, for example, the nucleic acid sequence of p53 is described by Lamb et al., Mol. Cell Biol. 6: 1379-1385 (1986), GenBank Accession Number: M13111. Similarly, nucleic acid sequences of RBs are described in Lee et al., Nature, 329: 642-645 (1987), GenBank Accession Number: M28419). Nucleic acid sequences of other tumor suppressor materials are available as indicated in paragraph II (a) above. One skilled in the art can use available sequence information to clone tumor suppressor genes into a vector suitable for practicing in the present invention.
p53 and RB tumor suppressor are particularly preferred for use in the methods of the present invention. Methods of cloning p53 and RB into vectors suitable for the expression of each tumor suppressor protein or for application of gene therapy are well known to those skilled in the art. As such, cloning and use of p53 is described in Wills, supra (1994); It is described in detail in US Pat. No. 5,532,220, US Ser. No. 08 / 328,673, filed October 25, 1994, and WO 95/11984. Typically the expression cassette is operably linked to a promoter, more preferably a strong promoter (eg, Ad2 major late promoter (Ad2 MLP)), or human cytomegalovirus (CMV) early early gene promoter. It was constructed with tumor suppressor cDNA. According to a particularly preferred embodiment, this promoter is followed by the subdivided leader cDNA and behind the tumor suppressor cDNA is a polyadenylation site (e.g. Elb polyadenylation site) (e.g., pending USSN 08 / 328,673, WO 95/11984 and Wills, supra (1994)). It will be appreciated that several tissue specific promoters are also suitable. Thus, for example, the tyrosinase promoter can be used for targeted expression in melanoma (see, eg, Siders, Cancer Res. 56: 5638-5646 (1996)). In particularly preferred embodiments the tumor suppressor cDNA is expressed in a vector suitable for the following gene therapy.
2. Preparation of Tumor Suppressor Protein
a) De novo chemical synthesis
Known sequences of tumor suppressor polypeptides can be used to synthesize tumor suppressor proteins or subsequences thereof using standard chemical peptide synthesis techniques. If the desired subsequence is relatively short (eg, a particular antigenic determinant is desired), the molecule can be synthesized into a single continuous polypeptide. If larger molecules are desired, peptide bonds can be formed by synthesizing the subsequences individually (one or more units) and then condensing and fusing the amino terminus of one molecule with the carboxyl terminus of the other molecule.
Solid phase synthesis of attaching the C-terminal amino acid of the sequence to an insoluble support followed by continuous addition of the remaining amino acids of the sequence is a preferred method for chemical synthesis of the polypeptides of the invention. Solid phase synthesis techniques are described by Barany and Merrifield, Solid-Phase Peptide Synthesis in The Peptides, pages 3-284: Analysis, Synthesis, Biology, Vol. 2; Special Methods in Peptide Synthesis, Part a.], Merrifield et al. Am. Chem. Soc., 85: 2149-2156 (1963) and Stewart et al., Solid Phase Peptide Synthesis, 2nd Edition, Pierce Chem. Co., Rockford, III (1984).
b) recombinant expression
According to a preferred embodiment, the tumor suppressor protein or partial sequence thereof is synthesized using recombinant DNA method. In general, such methods include making a DNA sequence encoding a fusion protein and placing this DNA in an expression cassette under the control of a particular promoter to express the protein in the host, isolate the expressed protein and reduce it if necessary.
Methods of cloning tumor suppressor nucleic acids into specific vectors are described above. The nucleic acid sequence encoding the tumor suppressor protein or protein subsequence is then e.g. E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells, such as COS, CHO and HeLa cell lines and myeloma cell lines, can be expressed in a variety of host cells. Tumor suppressor proteins are usually found in eukaryotic cells and therefore eukaryotic cell hosts are preferred. Recombinant protein genes are operably linked to appropriate expression control sequences for each host. this. In the case of E. coli it comprises a promoter, for example a T7, trp, or lambda promoter, a ribosomal binding site and preferably a transcription termination signal. For eukaryotic cells the regulatory sequence will include a promoter and preferably an immunoglobulin gene, an enhancer derived from SV40, cytomegalovirus and the like, and a polyadenylation sequence and may include splice donor sequences and acceptor sequences.
The plasmid of the present invention is E. coli. E. coli can be transferred into selected host cells by well-known methods such as calcium chloride transformation, and mammalian cells by calcium phosphate treatment or electroporation. Cells transformed with plasmids can be selected for resistance to antibiotics conferred by genes contained on the plasmids, such as the amp, gpt, neo and hyg genes.
Once expressed, recombinant tumor suppressor proteins can be purified according to standard methods in the art, such as ammonium sulfate precipitation, affinity column, column chromatography, gel electrophoresis (generally by R. Scopes, Protein Purification, Springer-). Verlag, New York, USA (1982); see Deutcher, Methods in Enzymology, Volume 182: Guide to Protein Purification, Academic Press, Inc. New York, USA (1990). Pure compositions having substantially about 90-95% homogeneity are preferred, with 98-99% homogeneity being most preferred. The polypeptide can be purified once in part or as homogeneously as desired and then used, for example, as an immunogen for antibody production.
Those skilled in the art will appreciate that tumor suppressor protein (s) may retain a structure substantially different from the natural structure of the constitutive polypeptide after chemical synthesis, biological expression, or purification. In such cases it may be necessary to denature and reduce the polypeptide and then refold the polypeptide into the desired structure. Methods of reducing, denaturing and inducing refolding of proteins are well known to those skilled in the art (Debinski (1993) J. Biol. Chem. 268: 14065-14070; Kreitman (1993) Bioconjug. Chem. 4: 581-). 585 and Buchner (1992) Anal. Biochem. 205: 263-270). See, for example, Debinski (1993), which describes denaturing and reducing suture proteins in guanidine-DTE. This protein is then refolded in a redox buffer containing glutathione oxide and L-arginine.
Those skilled in the art will appreciate that several conservative modifications of the amino acids and polypeptides described herein result in functionally identical products. For example, degeneration of a genetic code indicates that "silent substitution" (ie, substitution of a nucleic acid sequence that does not change the encoded polypeptide) is an implicit characteristic of all nucleic acid sequences encoding amino acids. Similarly, “conservative amino acid substitutions” in one or several amino acids in which the amino acid sequence is replaced with other amino acids having very similar properties are also readily identified as being very similar to the disclosed amino acid sequence, or the disclosed nucleic acid sequence encoding the amino acid. Conservatively substituted modifications of such explicitly described sequences are a feature of the invention.
The skilled artisan will appreciate that tumor suppressor proteins can be altered without reducing their biological activity. Some may be altered to help clone, express or incorporate the target molecule into the fusion protein. Such alterations are well known to those skilled in the art and are conveniently located, for example, by adding methionine at the amino terminus to provide an initiation site or by placing an additional amino acid (eg poly His) at either terminus. Making a restriction enzyme site or stop codon or purified sequence.
Changes to nucleic acids and polypeptides can be assessed using conventional screening techniques in appropriate assays for the desired properties. For example, changes in the immunological properties of a polypeptide can be detected by appropriate immunological assays. Other properties such as nucleic acid hybridization to the target nucleic acid, redox or thermal stability of the protein, hydrophobicity, susceptibility to proteolysis, or alteration of aggregation tendency are all analyzed according to standard techniques.
D) transfer of tumor suppressor to target cells
Tumor inhibitors used in the methods of the invention can be introduced into cells as proteins or nucleic acids. When providing a tumor suppressor as a protein, a tumor suppressor gene expression product (e.g., a p53 or RB polypeptide or fragment thereof retaining tumor suppressor activity) is transferred to target cells using standard methods of protein transport (discussed below) See what happens). Alternatively, if the tumor suppressor is a tumor suppressor nucleic acid (eg, gene, cDNA, mRNA, etc.), the nucleic acid is introduced into the cell using conventional methods of transferring the nucleic acid to the cell. This method typically includes a method of transferring in vivo or ex vivo gene therapy as described below. Particularly preferred methods of transferring p53 or RB include the use of lipid or liposome transfer and / or retrovirus or adenovirus vectors.
1. In vivo gene therapy
In a more preferred embodiment a tumor suppressor nucleic acid (eg cDNA (s) encoding a tumor suppressor protein) can be transfected to a cell (eg a human or other mammalian cell) in vitro and / or in vivo. Cloned into a vector for gene therapy.
Several approaches have been used to introduce nucleic acids into cells in vivo, ex vivo and in vitro. These include gene transfer methods based on lipids or liposomes (WO 96/18372; WO 93/24640; Mannino (1988) BioTechniques 6 (7): 682-691; US Pat. No. 5,279,833 to Rose; WO 91/06309 And Felgner (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7414) and replication defective retroviral vectors with therapeutic polynucleotide sequences as part of the retroviral genome (eg Miller (1990) Mol); Cell. Biol. 10: 4239 (1990); see Kolberg (1992) J. NIH Res. 4: 43, and Cornetta (1991) Hum. Gene Ther. 2: 215).
For a review of gene therapy methods see Zhang (1996) Cancer Metastasis Rev. 15: 385-401; Anderson, Science (1992) 256: 808-813; Nabel (1993) TIBTECH 11: 211-217; Mitani (1993) TIBTECH 11: 162-166; Mulligan (1993) Science, 926-932; Dillon (1993) TIBTECH 11: 167-175; Miller (1992) Nature 357: 455-460; Van Brunt (1988) Biotechnology 6 (10): 1149-1154; Vigne (1995) Restorative Neurology and Neuroscience 8: 35-36; Kremer (1995) British Medical Bulletin 51 (1) 31-44; Haddada (1995) in Current Topics in Microbiology and Immunology, Doerfler and Bohm (edit) Springer-Verlag, Heidelberg, Germany; And Yu (1994) Gene Therapy, 1: 13-26.
Vectors useful in the practice of the present invention are generally derived from the viral genome. Vectors that can be used include DNA and RNA viruses with or without a recombinantly modified envelope, preferably Baculovirus, Parvovirus, Picornovirus, Herpesvirus, Poxvirus, Adenovirus or Picorna And one selected from the department of viruses. Chimeric vectors can also be used that utilize advantageous means of respective parental vector properties (see, eg, Feng (1997) Nature Biotechnology 15: 866-870). The viral genome can be altered with recombinant DNA technology to include tumor suppressor genes and engineered to be capable of replication defects, conditional replication or replication. In a preferred embodiment of the present invention the vector is defective or conditionally replicated. Preferred vectors are from adenoviruses, adeno satellite viruses and retroviral genomes. In the most preferred embodiment of the present invention the vector is a vector lacking the replication capacity derived from the human adenovirus genome.
Conditionally replicating viral vectors are used to allow selective expression in specific cell types while avoiding a broad spectrum of infections. Examples of conditionally replicating vectors are described in Bischoff et al., Science 274: 373-376 (1996); Pennisi, E. (1996) Science 274: 342-343; Russell, S. J. (1994) Eur. J. of Cancer 30A (8): 1165-1171. Additionally, the viral genome can be altered to include inducible promoters that replicate or express the transgene only under certain conditions. Examples of inducible promoters are known in the scientific literature (see for example Yoshida and Hamada (1997) Biochem. Biophys. Res. Comm. 230: 426-430; Iida et al. (1996) J. Virol. 70 (9): 6054-6059; Hwang et al. (1997) J. Virol 71 (9): 7128-7131; Lee et al. (1997) Mol. Cell. Biol. 17 (9): 5097-5105; and Dreher et al. (1997) J. Biol Chem 272 (46): 29364-29371). The transgene may be under the control of a tissue specific promoter region that allows the transgene to be expressed only in certain cell types.
Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon leukemia virus (GaLV), apes immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof. (E.g. Buchscher (1992) J. Virol. 66 (5) 2731-2739; Johann (1992) J. Virol. 66 (5): 1635-1640 (1992); Sommerfelt (1990) Virol. 176: 58 Wilson (1989) J. Virol. 63: 2374-2378; Miller (1991) J. Virol. 65: 2220-2224; Wong-Staal et al. PCT / US94 / 05700; and Rosenburg and Fauci (1993) in Fundamental Immunology, 3rd edition, Paul Compilation, Raven Press, Ltd., New York, and references in these documents and Yu (1994), supra). The vector is pseudotyping as necessary to extend the host range of the vector to cells that are not infected by the retrovirus corresponding to the vector. A vesicular stomatitis virus envelope glycoprotein (VSV-G) was used to construct a VSV-G- gastric HIV vector capable of infecting hematopoietic stem cells (Naldini et al. (1996) Science 272: 263, and Akkina (1996) J Virol). 70: 2581).
Vectors based on adeno satellite virus (AAV) are also used to transduce target nucleic acids into cells, for example in vitro production of nucleic acids and proteins, and in vivo and ex vivo gene therapy methods. For an overview of AAV vectors, see Okada (1996) Gene Ther. 3: 957-964; West (1987) Virology 160: 38-47; Carter (1989) US Pat. No. 4,797,368; WO 93/24641 (1993) to Carter et al .; Kotin (1994) Human Gene Therapy 5: 793-801; Muzyczka (1994) J. Clin. Invst. 94: 1351. Construction of recombinant AAV vectors is described in US Pat. No. 5,173,414 to Lebkowski; Tratschin (1985) Mol. Cell. Biol. 5 (11): 3251-3260; Tratschin (1984) Mol. Cell. Biol. 4: 2072-2081; Hermonat (1984) Proc. Natl. Acad. Sci. USA 81: 6466-6470; McLaughlin (1988) and Samulski (1989) J. Virol., 63: 03822-3828, et al. Cell lines that can be transformed with rAAV include Lebkowski's Mol. Cell. Biol., 8: 3988-3996 (1988). Other suitable viral vectors include herpes virus and vaccinia virus.
In a particularly preferred embodiment, the tumor suppressor gene is expressed in an adenovirus vector suitable for gene therapy. The use of adenovirus vectors in vivo and for gene therapy is well described in the patent and scientific literature (see, eg, Hermens (1997) J. Neurosci. Methods., January, 71 (1): 85). Zeiger (1996) Surgery 120: 921-925; Channon (1996) Cardiovasc Res. 32: 962-972; Huang (1996) Gene Ther. 3: 980-987; Zepeda (1996) Gene Ther. 3: 973 -979; Yang (1996) Hum. Mol. Genet. 5: 1703-1712; Caruso (1996) Proc. Natl. Acad. Sci. USA 93: 11302-11306; Rothmann (1996) Gene Ther. 3: 919-926 Haecker (1996) Hum. Gene Ther. 7: 1907-1914). The use of adenovirus vectors is described in detail in WO 96/25507. Particularly preferred adenovirus vectors are described in Wills, supra (1994); Co-pending USSN 08 / 328,673, and WO 95/11984.
Particularly preferred adenovirus vectors include those in which some or all of the Protein IX genes are deleted. In one embodiment, the adenovirus vector comprises a deletion of the E1a and / or E1b sequences. In the most preferred embodiment, the adenovirus construct is a p53 coding construct, eg A / C / N / 53 or A / M / N / 53 (eg USSN 08 / 328,673, and WO 95/11984 Reference).
Preferred vectors are also derived from human adenovirus type 2 or 5. Such a vector is preferably one having a replication defect due to alteration or deletion of the Ella and / or Elb coding regions. It is also desirable to modify the viral genome to achieve specific expression properties or to allow repeated administration or lower immune responses. More preferred are recombinant adenovirus vectors in which E4 coding zero is completely or partially deleted, and where necessary retains E4 ORF6 and ORF 6/7. The E3 coding sequence may be deleted but is preferably retained. In particular, it is desirable to alter the promoter operator region of E3 to increase the amount of expression of E3 to achieve a more favorable immunological profile of the therapeutic vector. Most preferred include a leader sequence consisting of a DNA sequence encoding p53 under the control of an amplification viral promoter region and a subdivision with E3 under the control of a CMV promoter, while the E4 coding region is deleted while the E4 ORF6 and ORF6 / 7 is the human adenovirus type 5 vector. In the most preferred embodiment of the invention exemplified herein, the vector is ACN53.
In a particularly preferred embodiment the tumor suppressor gene is p53 or RB. Cloning and use of p53 as described above is described in Wills, supra (1994); It is described in detail in USSN 08 / 328,673, and WO 95/11984, filed October 25, 1994, which is pending together.
2. In Vitro Gene Therapy
In one embodiment, the method of the present invention is used to hyperproliferate (e.g., mammals such as rats, rats, bovines, pigs, horses, dogs, cats, largomorphs, or humans) using a method of the present invention. Neovascular) cells. Pathological hyperproliferative cells are characteristic of disease states, including but not limited to Graves' disease, psoriasis, benign prostatic hyperplasia, Lee-Fraumeny syndrome, breast cancer, sarcoma, bladder cancer, colon cancer, lung cancer, various leukemias and lymphomas and other tumors to be.
In particular, the ex vivo application of the method of the present invention provides a means of depleting a suitable sample of pathological hyperproliferative cells. Thus, hyperproliferative cells, for example, contaminating hematopoietic precursors during bone marrow reconstitution, can be removed by ex vivo application of the method of the invention. Such methods generally involve obtaining a sample from a subject organism. Typically this sample is a heterologous cell preparation comprising both normal or pathogenic (hyperproliferative) cells with the phenotype. The sample is contacted with a tumor suppressor nucleic acid or protein according to the method of the present invention and an adjuvant anticancer agent. Tumor suppressor genes can be transferred, for example, to viral vectors such as retroviral vectors or adenovirus vectors. This treatment reduces the proliferation of pathogenic cells to provide a sample with a high ratio of normal cells to pathogenic cells, which can be reintroduced into the subject organism.
In vitro cell transformation (eg, via reinjection of transformed cells into a host organism) for diagnostic, research or gene therapy is well known to those skilled in the art. According to a preferred embodiment the cells are isolated from the subject organism, transfected with the tumor suppressor gene or cDNA of the invention and reinjected back into the subject organism (eg patient). Several cell types suitable for ex vivo transformation are well known to those skilled in the art. Particularly preferred cells are progenitor cells or hepatocytes (for example from Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, 3rd Edition, Wiley-Liss, New York, USA) and patients. See the references cited above for a discussion of how to isolate and culture cells. Transformed cells are cultured by methods well known in the art. Kuchler (1977) Biochemical Methods in Cell Culture and Virology, Kuchler, RJ, Dowden, Hutchinson and Ross, Inc., and Atlas (1993) CRC Handbook of Microbiological Media (edited by Parks), CRC press, Boca Raton, Fl. See Mammalian cell systems also use mammalian cell suspensions but are often in monolayer form of cells. Alternatively the cells may be derived from those stored in cell banks (eg blood banks). Exemplary mammalian cell lines include HEC-1-B cell lines, VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, W138, BHK, Cos-7 or MDCK cell lines (see, eg, Freshney, supra).
In one particularly preferred embodiment, hepatocytes are used in an ex vivo method for cell transformation and gene therapy. The advantage of using hepatocytes is that hepatocytes can be differentiated into other cell types in vitro or introduced into mammals (eg donors of cells) and fused to the bone marrow. Methods of differentiating hepatocytes (eg CD34 ⁺ ) into clinically important immune cell types in vitro using cytokines such as GM-CSF, INF-gamma and TNF-alpha are known (eg See, Inaba (1992) J. Exp. Med. 176: 1693-1702; Szabolcs (1995) 154: 5851-5861).
Instead of using hepatocytes, T cells or B cells are also used in some embodiments in an ex vivo method. Several techniques for isolating T cells and B cells are known. Expression of surface markers helps to identify and purify the cells. Methods for identifying and isolating cells include FACS, incubation in immobilized flasks with antibodies that bind specific cell types, and screening with magnetic beads.
Hepatocytes are isolated for transduction and differentiation using known methods. For example, in mice, bone marrow cells are isolated by sacrificing the mouse and cutting the leg bones with scissors. Hepatocytes use antibodies that bind to unwanted cells, such as CD4 ⁺ and CD8 ⁺ (T cells), CD45 ⁺ (panB cells), GR-1 (granulocytes), and Ia ^d (differentiated antigen presenting cells). Isolation from bone marrow cells by screening out bone marrow cells. See Inaba (1992), supra, as an example of this protocol.
In humans, the aspiration of bone marrow cells from the iliac crest is carried out under general anesthesia, for example in a working room. Bone marrow aspiration is about 1000 ml and collected from posterior iliac bone and crest. If the total number of cells collected is less than about 2 × 10 ⁸ / kg, a second aspiration is performed using the sternum and anterior iliac crest as well as the posterior crest. Irradiated packing erythrocytes are administered to replace the volume of bone marrow obtained by aspiration during the operation. A feature of human hematopoietic progenitor cells and hepatocytes is the presence of CD34 surface membrane antigens. This antigen is for example used for purification in an affinity column that binds to CD34. After harvesting the bone marrow, mononuclear cells are separated from other components by Ficoll gradient centrifugation. This can be done semi-automatically using a cell separator (eg Baxter Fenwal CS3000 + or Terumo machine). Light density cells consisting mainly of mononuclear cells are collected and incubated for about 1.5 hours at 37 ° C. in a plastic flask. Discard adsorbent cells (monocytes, macrophages and B cells). Non-adsorbent cells are then collected and incubated for 30 minutes at 4 ° C. with gentle rotation with monoclonal anti-CD34 antibody (eg murine antibody 9C5). The final concentration of the anti CD34 antibody is preferably about 10 μg / ml. After washing twice, paramagnetic microspheres coated with both anti-mouse IgG (Fc) antibodies (e.g., Dyna beads, supplied by Baxter Immunotherapy Group, Santa Ana, CA, USA) at a cell suspension of about 2 cells / beads Add to Cells rosette with magnetic beads are collected by magnet after incubating for an additional about 30 minutes at 4 ° C. Chemopapine (Baxter Immunotherapy Group, Santa Ana, Calif.) Can be added at a final concentration of 200 U / ml to allow beads to detach from CD34 + cells.
Alternatively, and preferably, isolation methods using affinity columns that bind to CD34, or antibodies bound to CD34, can be used (see, eg, Ho (1995) Stem Cells 13 (suppl. 3): 100-). 105 and Brenner (1993) Journal of Hematotherapy 2: 7-17).
In another embodiment, hematopoietic stem cells can be isolated from fetal umbilical cord blood. Yu, Proc. Natl. Acad. Sci., USA, 92: 699-703 (1995) describe preferred methods for transducing CD34 ⁺ from human fetal umbilical cord blood using retroviral vectors.
3. Administration of Nucleic Acids Expressing Tumor Suppressors: Vectors and Expression Cassettes
Route of administration
Expression cassettes and vectors (eg retroviruses, adenoviruses, liposomes, etc.) comprising a therapeutic nucleic acid expressing a tumor suppressor of the invention can be administered directly to an organism for transduction of cells in vivo. Administration is a route that is normally used to introduce the molecule into final contact with blood or tissue cells, such as systemic, local, or topical routes of administration, as discussed in detail above, for administration of adjuvant anticancer agents. Do this using any path. A "packaged" nucleic acid (tumor suppression coding sequence, at least including a promoter) is administered in any suitable manner, preferably with a pharmaceutically acceptable carrier, as discussed in the literature. Suitable methods of administering such bulk nucleic acids are available to those skilled in the art and are well known to those of skill in the art, and although one or more routes may be used for administration of a particular composition, certain routes are often more immediate than other routes and It can provide a more effective reaction.
For example, administration of a recombinant adenovirus vector engineered to express a tumor suppressor gene can result in an immune response to the adenovirus vector, specifically an antibody response. Some patients may have an anti-adenovirus reactive antibody already present. Thus, in some circumstances, local or topical administration of adenovirus vectors expressing tumor suppressor is optimal and most effective. For example, ovarian cancer limited to the abdominal cavity, as discussed below, is one clinical scenario where local p53 gene therapy, eg, intraperitoneal (IP) administration, should be considered a preferred treatment method. IP administration of recombinant adenoviruses also infects and absorbs adenovirus vectors into the peritoneal lining and enters the systemic circulation (other local methods of administration can also introduce adenovirus vectors and enter the systemic circulation). The degree of this effect may vary depending on the concentration and / or total amount of viral particles administered with IP. If a systemic effect is desired, it may be desirable to use high concentrations over several consecutive days. Local administration of tumor suppressor-expressing adenovirus vectors of the present invention is also in some cases, for example anti-adenovirus reactive antibodies in which the patient already exists. It is preferable to hold. Such “topical administration” may be, for example, by intratumoral injection if internal or by mucosal application if external. Alternatively the “local administration” effect can be brought about by targeting the adenovirus vector to the tumor, for example on liposomes, or using the tumor specific antigen recognition reagents (eg antibodies) of the adenovirus itself.
Formulation
Pharmaceutically acceptable carriers are determined in part by the particular composition being administered and the particular method used to administer the composition. Thus, a wide variety of suitable formulations of the pharmaceutical compositions of the present invention exist.
Formulations suitable for oral administration of a pharmaceutical composition containing a tumor suppressor expressing nucleic acid include (a) an effective amount of batch nucleic acid suspended in a liquid solution, such as a diluent (eg water, saline or PEG 400); (b) capsules, sachets or tablets each comprising a predetermined amount of the active ingredient in liquid, solid, granule or gelatin; (c) suspension in a suitable liquid; And (d) a suitable emulsion. Tablet formulations include one or more lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, tragacanth, microcrystalline cellulose, gum arabic, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate Rates, stearic acid and other excipients, colorants, fillers, binders, diluents, buffers, wetting agents, preservatives, flavoring agents, dyes, disintegrants, and carriers that can be used pharmaceutically. Lozenge formulations may contain the active ingredient in a flavourant, usually sucrose and gum arabic or tragacanth, and pastilles contain inert substrates (eg, gelatin and Active ingredients in glycerin or sucrose and gum arabic emulsions, gels, etc.).
Batch nucleic acids may be prepared as aerosol formulations that are administered inhaled (ie, nebulizable), alone or in combination with other suitable ingredients. Aerosol formulations may be formulated with pressurizable propellants (eg dichlorodifluoromethane, propane, nitrogen, etc.).
Suitable formulations for rectal administration include suppositories consisting of, for example, batch nucleic acids based on suppositories. Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons. It is also possible to use rectal gelatin capsules consisting of a combination of a batch nucleic acid and a substrate, such as liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.
Agents suitable for parenteral administration (eg, intra-articular, intravenous, intramuscular, intradermal, intraperitoneal and subcutaneous routes) include antioxidants, buffers, bacterial inhibitors, and blood of the intended receptor and the agent isotonic. Isotonic, aqueous and non-aqueous sterile injectable solutions that may contain solutes, and aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. In the practice of the present invention, the composition may be administered, for example, by intravenous infusion, oral administration, topical administration, intraperitoneal administration, intra Bladder administration or intrathecal administration. Batch nucleic acid preparations may be provided in unitized or multidose sealed containers, such as ampoules and vials.
The formulations of the present invention as injection solutions and suspensions can be prepared from the aforementioned types of sterile powders, granules, and tablets. The exact composition of the formulation, concentrations of reagents and nucleic acids in the formulation, pH, buffers, and other parameters may be determined by the dosage form and site of administration (e.g., systemic, topical or topical), storage of specific pharmaceutical compositions, It depends on the requirements for handling, transport and shelf life. Optimization of these parameters in accordance with the specific requirements of the present formulations can be done by conventional methods and any components and parameters of known injectable formulations can be used. One example of a suitable formulation is a recombinant wild-type p53 expressing adenovirus vector of the present invention, 0.42 mg, which is generally stored at a dose of 1.0 ml, eg, the concentration is about 7.5 × 10 ¹¹ to 7.5 × 10 ¹⁰ particles per ml. / ml sodium phosphate monohydrate, 2.48 mg / ml sodium diphosphate anhydride, sodium chloride, 5.8 mg / ml sodium phosphate monohydrate, 20.0 mg / ml sucrose, 0.40 mg / ml magnesium chloride hexahydrate.
Cells transduced with the batch nucleic acid in in vitro therapy can also be administered intravenously or parenterally as described above.
In the present invention, the dosage administered to a patient should be sufficient to cause a beneficial therapeutic response over time in the patient. Dosage will be determined by the efficacy of the particular vector used and the condition and weight or surface area of the patient to be treated. Dosage size will also be determined by the presence, nature, and extent of the harmful side effects associated with the administration of the particular vector in the particular patient, or the transduced cell type.
In determining the effective amount of the vector administered following treatment, the physician assesses the concentration of the vector in circulating plasma, vector toxicity, disease progression, and production of antivector antibodies. Typical dosages of nucleic acids are highly dependent on the route of administration and gene transfer system. Depending on the method of delivery, the dosage may easily range from about 1 μg to about 100 mg. In general, the dosage of naked nucleic acid from a vector is generally about 1 μg to 100 μg for a 70 kg patient and the dose of the vector comprising the viral particles is calculated to be the amount of therapeutic nucleic acid.
The transduced cells of the invention can be administered at a rate determined by the LD ₅₀ of the vector or transduced cell type, and the adverse effects of the vector or cell type at various concentrations, depending on the weight and overall health of the patient. Administration can be in single or divided doses as described below.
In a preferred embodiment, blood samples are obtained and stored for analysis before infusion. Monitor the vital signals and oxygen saturation in detail with a pulse oximeter. Blood samples are preferably taken 5 minutes and 1 hour after injection and stored for later analysis. In ex vivo therapy, leukocyte removal, transduction and reinjection may be repeated every 2-3 months. Infusion can be done to an outpatient at the discretion of the clinician after the first treatment. If infusion is done to an outpatient, the parties are monitored for at least 4 hours, preferably 8 hours after treatment.
As noted above, adenovirus constructs may be administered systemically (eg intravenously), topically (intraperitoneally) or topically (eg intratumorally or around the tumor, or in the bladder for the treatment of bladder cancer, for example). By injection). Particularly preferred dosage forms include intraarterial injection, more preferably intrahepatic injection (e.g. for the treatment of liver cancer), or when the composition needs to be transferred to brain cancer, such as a carotid or artery of the carotid artery system of the artery (e.g. For example, laryngeal arteries, atrial arteries, temporal arteries, cerebral arteries, maxillary arteries, etc.). Transfer for lung cancer treatment can be accomplished using, for example, bronchoscopes. Typically such dosage forms are in the form of a pharmacologically acceptable aqueous buffer as described above. However, in one preferred embodiment, the adenovirus construct or tumor suppressor expression cassette is combined with liposomes to make a liquid formulation, more specifically a lipid / nucleic acid complex (eg WO 93/24640 by Debs and Zhu). (1993); Mannino, supra (1988); Rose, U.S. Pat.No. 5,279,833; Brigham (1991) WO 91/06309; and Felgner (1987) supra), see liposomes, more preferably Is administered in a liquid formulation encapsulated in immunoliposomes for specific tumor markers. It will be appreciated that such liquid formulations may also be administered topically, systemically or by aerosol delivery.
4. Increased Tumor Suppressor Transfer Rate
Delivery of the tumor suppressor may be increased by using one or more "transport enhancers". "Transfer enhancer" means any agent that enhances the transfer of a therapeutic gene, such as a tumor suppressor gene, to cancer tissues or organs. This enhanced transport can be accomplished by several mechanisms. One such mechanism may include disrupting the protective glycosaminoglycan layer of the epithelial surface of an organ or tissue (eg bladder). Examples of such transport enhancers include detergents, alcohols, glycols, surfactants, bile salts, heparin antagonists, cyclooxygenase inhibitors, hypertonic salt solutions, and acetates. Alcohols include, for example, aliphatic alcohols such as ethanol, N-propanol, isopropanol, butyl alcohol, acetyl alcohol. Glycols include glycerin, propylene glycol, polyethylene glycol, and other low molecular weight glycols such as glycerol and thioglycerol. Other examples of transport enhancers are acetates such as acetic acid, gluconol acetate, and sodium acetate. Hypertonic salt solutions such as 1 M NaCl are also examples of transport enhancers. Examples of surfactants include sodium dodecyl sulfate (SDS) and lysocithin, polysorbate 80, nonylphenoxypolyoxyethylene, lysophosphatidylcholine, polyethylene glycol 400, polysorbate 80, polyoxyethylene ether, polyglycol ether surfactant And DMSO. Other astringents such as bile salts such as taurocholate, sodium tauro-deoxycholate, deoxycholate, kenodesoxycholate, glycocholic acid, glycokenodeoxycholic acid and silver nitrate can be used. Heparin-antagonists such as quaternary amines such as proramine sulfate can also be used. Cyclooxygenase inhibitors, such as sodium salicylate and salicylic acid, and nonsteroidal anti-inflammatory drugs (NSAIDS), such as indomethacin, naproxen, and diclofenac, can be used.
Detergents include anionic, cationic, zwitterionic, and nonionic detergents. Examples of detergents include taurocholate, deoxycholate, taurodeoxycholate cetylpyridium, benalconium chloride, ZWITTERGENT® 3-14 cleaner, CHAPS (3-[(3-colamidopropyl) dimethylammonol ] -1-propanesulfonate hydrate, from Aldrich), Big CHAP (USSN 08 / 889,355, filed Jul. 8, 1997; and international patent application WO 97/25072 (July 17, 1997). ), Deoxy Big CHAP (same document), Triton® X-100 cleaner, C12E8, Octyl-BD-glucopyranoside, PLURONIC®-F68 cleaner, Tween® 20 cleaner, and Tween 80 cleaners (available from CALBIOCHEM Biochemicals), but are not limited to these.
In one embodiment, the transfer enhancer is contained in a buffer in which the recombinant adenovirus vector transfer system is formulated. The transfer enhancer may be administered prior to or with the virus. In some embodiments, the delivery enhancer is provided with the virus by mixing with the delivery enhancer composition immediately prior to administration of the viral preparation to the patient. In another embodiment, the delivery enhancer and virus are provided in a single vial to the person caring for the patient for administration.
For pharmaceutical compositions containing a tumor suppressor gene included in a recombinant adenovirus vector delivery system formulated in a buffer further containing a transfer enhancer, the pharmaceutical composition preferably ranges from about 5 minutes to 3 hours, preferably about It is administered over a time ranging from 10 minutes to 120 minutes, most preferably from about 15 minutes to 90 minutes. In other embodiments, the transfer enhancer can be administered prior to administration of the recombinant adenovirus vector transfer system comprising the tumor suppressor gene. A delivery enhancer may be administered about 30 seconds to 1 hour, preferably 1 minute to 10 minutes, most preferably about 1 minute to 5 minutes before administration of the adenovirus vector delivery system comprising the tumor suppressor gene. .
The concentration of the transfer enhancer will depend on a number of factors known to those skilled in the art, such as the transfer enhancer, buffer, pH, target tissue or organ, and dosage form used. The concentration of the transfer enhancer is in the range of 1 to 50% (v / v), preferably 10 to 40% (v / v) and most preferably 15 to 30% (v / v). Preferably the detergent concentration in the final formulation administered to the patient is a critical micelle concentration (CMC) of about 0.5 to 2 times. Preferred concentrations of Big CHAP are about 2-20 mM, more preferably about 3.5-7 mM.
Buffers containing transport enhancers are described in any pharmaceutical buffer, such as phosphate buffered saline or sodium phosphate / sodium sulfate, Tris buffer, glycine buffer, sterile water and Good et al., Biochemistry 5: 467 (1966). As may be other buffers known to those skilled in the art. The pH of the buffer in the pharmaceutical composition containing the tumor suppressor gene included in the adenovirus vector transfer system may be in the range of 6.4 to 8.4, preferably 7 to 7.5, most preferably 7.2 to 7.4.
Preferred formulations for administration of recombinant adenoviruses are about 10 ⁹ to 10 ¹¹ PN, about 2 to 10 mM Big CHAP or about 0.1 to 1.0 mM TRITON per ml of virus in phosphate buffered saline (PBS) having a pH of about 6.4 to 8.4. In addition to (TM) -X-100 detergent, about 2-3% sucrose (w / v) and about 1-3 mM MgCl ₂ . The use of the transfer enhancer is described in detail in co-pending USSN 08 / 779,627, filed January 7, 1997.
To help improve gene transfer of nucleic acid compositions containing commercially available Big-CHAP agents, the concentration of Big-CHAP will vary based on commercial sources. When Big CHAP is purchased from CALBIOCHEM its concentration is preferably in the range of 2-10 mmol. The concentration of Big CHAP is more preferably 4 to 8 mmol, most preferably about 7 mmol.
When Big CHAP is purchased from Sigma its concentration is 15 to 35 mmol, more preferably 20 to 30 mmol, most preferably about 2.5 mmol.
According to another embodiment of the present invention there is provided a transfer enhancer of formula (I).
Wherein n is an integer of 2 to 8, X ₁ is a cholic acid group or a deoxycholic acid group, and X ₂ and X ₃ are each independently selected from the group consisting of a cholic acid group, a deoxycholic acid group, and a saccharide group do. At least one X ₂ and X ₃ is a saccharide group. The saccharide groups are pentose monosaccharide groups, hexose monosaccharide groups, pentose-pentose disaccharide groups, hexose-hexose disaccharide groups, pentose-hexose disaccharide groups, and hexose -Can be selected from the group consisting of pentose disaccharide groups. In one preferred embodiment, the compounds of the present invention have the formula
Wherein X ₁ and X ₂ are selected from the group consisting of a cholic acid group and a deoxycholic acid group and X ₃ is a saccharide group.
These compounds are preferably used in the range of about 0.002 to 2 mg / ml, more preferably about 0.02 to 2 mg / ml, most preferably about 0.2 to 2 mg / ml in the formulations of the present invention. Most preferably about 2 mg / ml.
Phosphoric acid buffered saline (PBS) is the preferred solubilizer of the compound. However, those skilled in the art will further appreciate that certain excipients and additives may be desirable to achieve solubility of these agents in various pharmaceutical compositions. For example, well-known solubilizers such as detergents, fatty acid esters, and surfactants can be added at appropriate concentrations to help dissolve the compounds in the various solvents used. If the solvent is PBS, the preferred solubilizer is Tween 80 at a concentration of about 0.15%.
5. Administration of Tumor Suppressor Proteins
Tumor suppressor proteins (polypeptides) can be delivered directly to the tumor site by infusion or systemically administered as described above. According to a preferred embodiment, the tumor suppressor protein is combined with a pharmaceutically acceptable carrier (excipient) to prepare a pharmacological composition as described above. Tumor suppressor polypeptides are administered in a therapeutically effective amount. Thus, the composition is administered in an amount sufficient to treat or at least partially stop the disease and / or its complications. Effective amounts in these applications depend on the severity of the disease and the overall state of patient health.
It will be appreciated that oral administration of a tumor suppressor polypeptide should protect the polypeptide from digestion. This is generally accomplished by complexing the polypeptide with the composition to resist acid hydrolysis and enzymatic hydrolysis or packaging the polypeptide in a carrier having suitable resistance, for example the liposomes described above. Methods of protecting polypeptides in oral delivery are well known in the art (see, eg, US Pat. No. 5,391,377 which discloses a lipid composition for oral delivery of therapeutic agents).
III. Combination constraint
Tumor inhibitors and adjuvant anticancer agents may be administered separately a tumor inhibitor nucleic acid or polypeptide to be administered before adjuvant anticancer agents (tumor inhibitor pretreatment) or adjuvant anticancer agents to be administered prior to tumor inhibitor nucleic acid and / or polypeptide (cancer drug pretreatment). ). Of course, tumor suppressor nucleic acids and / or polypeptides and adjuvant anticancer agents may be administered simultaneously.
In one embodiment the tumor suppressor nucleic acid and / or polypeptide and adjuvant anticancer agent are administered as a single pharmacological composition. In such embodiments the tumor suppressor nucleic acid and / or polypeptide and adjuvant anticancer agent may be suspended or dissolved in a single uniform delivery vehicle. Alternatively, the tumor suppressor nucleic acid and / or polypeptide and adjuvant anticancer agent may be suspended or dissolved in different delivery vehicles that are suspended (dispersed) upon administration or in succession in a single excipient. Thus, for example, an adjuvant anticancer agent may be dissolved in a polar solvent (eg, paclitaxel in ethanol), and tumor suppressor nucleic acids may be combined with lipids and then stored together in suspension or alternatively administered when administered. Various suitable delivery vehicles, excipients, and the like are described above.
IV. Treatment regimens: combination and individual treatments
A) Tumor Suppressor Therapy
It has been found by the present invention that tumor suppressor nucleic acids or polypeptides, more particularly tumor suppressor nucleic acids, have greater efficacy in inhibiting tumor growth when administered in multiple doses rather than in a single dose. The present invention therefore provides a therapeutic regimen for tumor suppressor genes or polypeptides comprising multiple administrations of a tumor suppressor nucleic acid or polypeptide.
Tumor inhibitor protein or tumor suppressor nucleic acid is the total dose in a single dose, the total dose divided over 5 days or administered daily for 5 days, the total dose divided over 15 days or administered daily for 15 days, and A therapeutic regimen selected from the group consisting of total dosages divided over 30 days or administered daily for 30 days, wherein the total dose of adenovirus particles is from about 1 × 10 ⁹ to about 1 × 10 ¹⁴ , about 1 × 10 ⁹ to about 7.5 x 10 ¹⁵ , preferably from about 1 x 10 ¹¹ to about 7.5 x 10 ¹³ (with or without the adjuvant anticancer agent). The method of administration can be repeated for two or more cycles (more preferably three cycles) and the two or more cycles can be separated by three or four weeks. Treatment can include a single dose cycle or a dose cycle that can vary from about 2 to about 12, more preferably from about 2 to about 6 cycles.
Particularly preferred treatment regimens include the total dose divided over five days or administered daily, the total dose divided over 15 days or administered daily and the total dose divided over 30 days or administered daily.
In some preferred embodiments a daily dose of 7.5 x 10 ⁹ to about 7.5 x 10 ¹⁵ , preferably about 1 x 10 ¹² to about 7.5 x 10 ¹³ , of the adenovirus particles may be administered daily for up to 30 days (eg , 2 days, 2 to 5 days, 7 days, 14 days, or 30 days of therapy, in which the same dose is administered daily). Multiple therapies can be repeated in cycles of 21 to 28 days.
In some embodiments, different routes of administration will result in different and preferred dosage ranges. For example, a preferred range for intrahepatic arterial delivery is typically from 7.5 x 10 ⁹ to about 1 x 10 ¹⁵ , more preferably from about 1 x 10 ¹¹ to about 7.5 x 10, per day for 5 to 14 days. Will be ¹³ The therapy is either adjuvant anticancer agent FUDR or 5'-deoxy-5-fluorouridine (5'-DFUR), or irinotecan hydrochloride (CPT-11; 7-ethyl-10- [4- (1-piperi) Dino) -1-piperidino] carbonyloxycamptothecin). Preferred ranges for intratumoral delivery will typically be from 7.5 x 10 ⁹ to about 1 x 10 ¹³ , more preferably from about 1 x 10 ¹¹ to about 7.5 x 10 ¹² , per day. Preferred ranges for intraperitoneal delivery will be from 7.5 x 10 ⁹ to about 1 x 10 ¹⁵ , more preferably from about 1 x 10 ¹¹ to about 7.5 x 10 ¹³ , per day for 5 to 10 days.
B) Combination Therapies
When a tumor suppressor is used in combination with an adjuvant anticancer agent, the tumor suppressor nucleic acid is administered at a total dosage as described above. In combination the adjuvant anticancer agents are administered in a total dosage depending on the reagent used. For example, paclitaxen or paclitaxel derivatives can be administered in a single dose, daily doses on days 1 and 2, daily doses on days 1, 2 and 3, daily doses over 15 days, 75 to 350 mg over 1 hour, 3 hours, 6 hours or 24 hours with a treatment regimen selected from the group consisting of daily doses for 30 days, daily continuous infusions for 15 days, daily continuous infusions for 30 days Is administered at a total dose of / m 2. Preferred dosages are 100 to 250 mg / m 2 for 24 hours.
Pretreatment of adjuvant anticancer agents (eg, paclitaxel) prior to treatment of tumor inhibitor nucleic acids improves the efficacy of tumor inhibitors. Thus, in one particularly preferred embodiment the cells, tissues or organisms are treated with adjuvant anticancer agents before tumor suppressor nucleic acids. Adjuvant anticancer agents are preferably tolerated longer or shorter time but are treated about 24 hours prior to tumor suppressor nucleic acid.
Pretreatment is particularly effective when the adjuvant anticancer agent is a paclitaxel type compound, more preferably paclitaxel or paclitaxel derivatives (eg Taxol or Taxotere). Particularly preferred tumor inhibitors are RB and p53, with p53 being the most preferred, particularly p53 (eg A / C / N / 53) of adenovirus vectors.
V. Treatment and Prevention of Metastases
As illustrated in Examples 2 and 3, tumor inhibitor (eg, p53) gene replacement therapy has efficacy on human tumor cells in vitro, human tumor xenografts in immunocompromised hosts, and human lung tumors (in vivo) Indicates. Surgical debulking of primary tumors in a patient causes tumor regrowth at the primary site and tumor metastasis from that site due to microscopic “nests” of tumor cells often missed by the surgeon. . Alternatively, to ensure that all tumors are removed from the primary site, the patient may be subjected to dicfiguring surgery to remove large amounts of normal tissue including the primary tumor site.
In another embodiment the invention provides a method of inhibiting growth and / or proliferation of metastases (metastatic cells). The method generally comprises systemic or topical administration of the tumor suppressor, more preferably topical administration of p53 or RB.
A) systemic treatment
As described in Examples 2 and 3, systemic treatment of tumor suppressor vectors (eg, A / C / N / 53) (eg, intravenous injection) inhibited the progression of metastasis in vivo. Thus, in one embodiment the present invention provides a method of inhibiting the development of metastatic disease by administering a tumor suppressor nucleic acid and / or a tumor suppressor polypeptide to an organism as described above. Tumor inhibitors are preferably tumor inhibitor nucleic acids, more preferably p53 tumor inhibitor nucleic acids and most preferably p53 nucleic acids (eg A / C / N / 53) in adenovirus vectors. In another preferred embodiment the tumor suppressor nucleic acid is encapsulated in intracellular fat particles or provided in combination with lipids (see, eg, Debs and Zhu (1993) WO 93/24640; Mannino and Gould-Fogerite (1988) BioTechniques 6 (7): 682-691; Rose US Pat. Nos. 5,279, 833; Brigham (1991) WO 91/06309; and Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7414.
B) topical treatment
In another embodiment topical application of a tumor suppressor protein or tumor suppressor nucleic acid is preferred in connection with a surgical operation. In this embodiment the tumor suppressor, preferably in the form of an infection vector, is applied along the surface of the wound cavity after tumor removal. Infected particles carry p53 from any wound site to any remaining tumor cells, inducing their apoptosis (programmed cell death). The treatment will affect the long-term survival of the patient and / or reduce the amount of normal tissue that includes the tumor site that needs to be removed during surgical operation.
Tumor inhibitors are preferably combined with one of a number of agents known to those skilled in the art, suitable for topical application. Thus, for example, an infectious agent of a human p53 tumor suppressor gene (eg A / C / N / 53) is suspended in a vehicle (eg petroleum jelly or other cream or ointment) suitable for spraying along the surface of the wound cavity. Alternatively, tumor suppressors may be prepared with an erosol vehicle for application as a spray inside the wound cavity. In another embodiment, the tumor suppressor may be made of a degradable (resorbable) material, such as a resorbable sponge, that can fill the wound cavity and release the tumor suppressor protein or vector in a time dependent manner.
Preferred embodiments for the application of recombinant adenovirus vectors to certain restricted local areas, such as the cornea, gastrointestinal tract, tumor ablation site, use solid carriers to support longer incubation times and facilitate viral infection. . The carrier may be a gauze or ointment immersed in a recombinant adenovirus solution. The virus can be applied to the cornea through a gauze scaffold to obtain improved transgene effects. Drained gauze can also be applied prophylactically to resected tumor areas to avoid recurrence. Ointments can be applied topically to the gastrointestinal tract or locally to the area of the pancreas for tumor suppressor gene therapy.
Exemplary ointment carriers are petroleum based Puralube ^(R ) or water soluble KJ-Jelly ^(R) . In a representative method, a sterile gauze pad (5 × 5 cm) or tear flow test strip may be immersed in an adenovirus vector solution (eg, 1 × 10 ⁹ PN / ml) until fully wetted. The pad or strip is placed on top of the target tissue and incubated at 37 ° C. for 30 minutes. Those skilled in the art will recognize that there are other foams, gelatin or ointments that can be absorbed or mixed with water. In addition, other excipients may be added to improve gene transfer as described above.
VI. Combination treatment with other chemotherapeutic agents
A) Tumor inhibitors administered in combination with multiple chemotherapeutic combinations
It will be appreciated that the methods of the present invention are not limited to the combination of a single adjuvant anticancer agent with a tumor suppressor. While the methods typically involve contacting the cells with tumor inhibitors (eg, p53) and adjuvant anticancer agents such as paclitaxel, the methods of the present invention also provide for tumor suppressor genes or polypeptides and double or triple or multiple adjuvant anticancer agents and Contact with any other combination of chemotherapeutic agents. Those skilled in the art will also recognize that chemotherapeutic agent (s) may be used with tumor suppressor proteins or genes in the absence of adjuvant anticancer agent (s).
Many chemotherapeutic agents are well known in the scientific and patent literature, and representative drugs that may be used in the methods of the present invention include DNA damaging agents (including DNA alkylating agents) such as cisplatin, carboplatin (Duffull ( 1997) Clin Pharmacokinet. 33 161-183); Droz (1996) Ann Oncol. 7: 997-1003), navelvin (vinorelbine), asaley, AZQ, BCNU, busulfan, carboxyphthalatoplatinum, CBDCA, CCNU, CHIP, chlorambucil, chlorozo Toxin, cis-platinum, clomezone, cyanomorpholino-doxorubicin, cyclodizone, cytoxane, dianhydrogalactitol, fluorodopan, hepsulpam, hycanton, melphalan, methyl CCNU, mitomycin C, mitosol Mead, nitrogen mustard, PCNU, piperazine alkylating agent, piperazinedione, piperobroman, porphyromycin, spirohydantoin mustard, theeroxylone, tetraplatin, thio-tepa, triethylenemelamine, uracil nitrogen mustard ( Yoshi-864); Topoisomerase I inhibitors (eg, topotecan hydrochloride, irinotecan hydrochloride (CPT 11), camptothecin, camptothecin Na salt, aminocamptothecin, CPT 11 and other camptothecin derivatives); Topoisomerase II inhibitors (including doxorubicin encapsulated in intracellular fat particles (see US Pat. Nos. 5,013,556 and 5,213,804), amonafides, m-AMSA, anthrapyrazole derivatives, pyrazoloacridins , Bisantrene HCL, daunorubicin, deoxydoxorubicin, mitoxantrone, menogaryl, N, N-dibenzyl daunomycin, oxanthrazole, rubidazone, VM-26 and VP-16); RNA / DNA antimetabolic products (e.g., L-alanosine, 5-azacytidine, 5-fluorouracil, abyssinine, aminopterin, aminopterin derivatives, antipol, Baker soluble antipol, di Chloralyl Lawson, Brequinar, Protopura (Prodrug), 5,6-dihydro-5-azacytidine, methotrexate, methotrexate derivative, N- (phosphonoacetyl) -L-aspartate (PALA) , Pyrazopurin, and trimetrexate); And DNA anti metabolites (eg, 3-HP, 2'-deoxy-5-fluorouridine, 5-HP, alpha-TGDR, apidicholine glycinate, al-C, 5-aza-2 ' -Deoxycytidine, beta-TGDR, cyclocytidine, guanazole, hydroxyurea, inosine, glycodialdehyde, macbecin II, pyrazoloimidazole, thioguanine and thiopurine). Tumor inhibitor nucleic acids and / or polypeptides may also be combined with chemotherapeutic agents such as vincristine, temozolomide (see US Pat. No. 5,260,291) and toremifene (see, eg, US Pat. No. 4,696,949 for information about toremifene). Can be administered.
Preclinical studies in related animal models showed that p53 adenovirus combined with cisplatin, carboplatin, nabelbin, doxorubicin, 5-fluorouracil, methotrexate, or etoposide tumors: SSC-9 head and neck, SSC-15 head and neck , SSC-25 head and neck, SK-OV-3 ovary, DU-145 prostate, MDA-MB-468 breast and MDA-MB-231 breast tumor cells inhibited cell proliferation much more efficiently than chemotherapy alone It is shown. In another embodiment, the improved antitumor efficacy is shown using a mixture of three drugs of the p53 gene (e.g. expressed in adenovirus vector), adjuvant anticancer agent (e.g. paclitaxel) and DNA damaging agent (e.g. cisplatin). . The combination of p53, paclitaxel and cisplatin has been shown to be efficient for ovarian tumor models. The data support a combination of chemotherapy and p53 gene therapy in clinical trials.
Such other chemotherapeutic agents may be used in combination with tumor suppressor nucleic acids and / or polypeptides in the presence or absence of an adjuvant anticancer agent. The present invention also contemplates the use of radiation therapy in combination with any of the above described tumor inhibitors or in combination with adjuvant anticancer agents.
It will also be appreciated that any chemotherapeutic agent may be used separately in combination with a tumor suppressor nucleic acid or polypeptide according to the methods of the present invention.
When a tumor suppressor nucleic acid (e.g., p53) is administered in an adenovirus vector in combination with an adjuvant anticancer agent (e.g. paclitaxel) and a DNA damaging agent (e.g. cisplatin, carboplatin or navelbine) The particles are topically administered from about 7.5 × 10 ¹² to about 7.5 × 10 ¹³ for 5-14 days. For example, a daily dose of about 7.5 × 10 ¹³ of adenovirus particles in combination with carboplatin can be used. In one embodiment a daily dose of about 7.5 × 10 ¹² of adenovirus particles may be used for administration to the lungs. In another embodiment p53 is administered with topotecan.
Typically, DNA damaging agents will be administered at recommended dosages (see Physician's Desk Reference, 51st Edition, Medical Economics, Montvale, NJ 1997). For example, carboplatin is administered at about 6-7.5 mg / ml / min AUC (area under the curve).
Protease Inhibitors
In another embodiment the invention provides for the combined use of tumor inhibitor nucleic acids and / or polypeptides and protease inhibitors. Particularly preferred protease inhibitors include, but are not limited to, collagenase inhibitors, matrix metalloproteinase (MMP) inhibitors. For example, Chambers (1997) J. Natl. Cancer Inst. 89: 1260-1270]. In a preferred embodiment the method comprises administering an effective amount of a protease inhibitor and an effective amount of a tumor suppressor polypeptide and / or nucleic acid simultaneously or sequentially. Examples of compounds that are protease inhibitors are well known in the scientific and patent literature.
<Immunomodulators>
Tumor inhibitor proteins and nucleic acids of the present invention upregulate an immune response that is hyperproliferating or responsive to cancer cells (e.g., an immune response that is responsive to tumor specific antigens), or tumor suppressor proteins, tumor inhibitor nucleic acids, tumor inhibitor vectors (e.g., Anti-adenovirus response) and / or immunomodulators that downregulate immune responses that are combined with chemotherapeutic agents.
Thus, for example, the present invention provides for combined sequential or simultaneous administration of an effective amount of an immunomodulator and an effective amount of a tumor suppressor nucleic acid and / or a tumor suppressor polypeptide. Immunomodulators include IL-2, IL-4, IL-10 (US Pat. No. 5,231,012; Lalani (1997) Ann.Allergy Asthma Immunol. 79: 469-483: Geissler (1996) Curr. Opin. Hematol. 3: 203-208), IL-12 [e.g., Branson (1996) Human Gene Ther. 1: 1995-2002, and gamma-interferon, but are not limited thereto.
Immunomodulators that act as immunosuppressants can be used to mitigate the immune response targeted to a therapeutic agent (eg, a tumor suppressor protein or nucleic acid or a secondary anticancer agent, etc.). Immunosuppressants are well known to those skilled in the art. Suitable immunosuppressants include, but are not limited to, cyclo-phosphamide, dexamethasone, cyclosporin, FK506 (Takchlorimus) (Lochmuller (1996) Gene Therapy 3: 706-716), IL-10, and the like. No. Antibodies to cell surface receptors that modulate an immune response can also be used, for example antibodies that interfere with ligands that bind to cell receptors for B cells, T cells, NK cells, macrophages and tumor cells. Examples of such methods are described in Yang (1996) Gene Therapy 3: 412-420; Lel (1996) Human Gene Therapy 7: 2273-2279; Yang (1996) Science 275: 1862-1867. See also.
VII. Treatment kit
In another embodiment the invention provides a treatment kit. Such kits include, but are not limited to, tumor suppressor nucleic acids or polypeptides or pharmaceutical compositions thereof. The kit also includes an adjuvant anticancer agent or a pharmaceutical composition thereof. Various compositions may be provided in separate containers for individual administration or for combination prior to administration. Alternatively, the various compositions can be provided in a single container. Kits may also include various devices, buffers, assay reagents, and the like, for carrying out the methods of the present invention. In addition, the kit may contain indicators that direct the use of the kit in various methods of the invention (eg, treating a tumor, preventing and / or treating metastases, etc.).
Optionally the kit may comprise one or more immunomodulators (eg, immunosuppressants). Particularly preferred immunomodulators include any of the immunomodulators described herein.
VIII. Heterologous Tumor Suppressor Nucleic Acids or Polypeptides, and Other Reagent-Containing Cells
Further provided are primitive or mature nucleus host cells, such as heterologous tumor suppressor nucleic acids and / or tumor suppressor polypeptide-containing animal cells (eg, mammalian cells) transfected or otherwise treated by the present invention. Such cells may optionally further contain adjuvant anticancer agents such as paclitaxel or other microtubule affecting agents.
Suitable primitive nucleus cells include, but are not limited to, bacterial cells such as E. coli cells. Suitable animal cells preferably include mammalian, more preferably human cells. Host cells include, but are not limited to, any mammalian cell, more preferably any neoplastic or tumor cell as described above.
Transfected host cells described herein are useful as diagnostic or therapeutic compositions. When used in pharmaceuticals, it can be combined with various pharmaceutically acceptable carriers as described above for ex vivo gene therapy. The cells may be administered therapeutically or prophylactically in the effective amounts detailed above. In diagnosis, cells can be used for teaching or other reference purposes and provide a suitable model for the identification of transfected and / or treated cells.
IX. Preclinical and Clinical Efficacy of p53 Adenovirus Gene Therapy
Adenovirus-mediated p53 gene therapy is concurrently running phase I / II clinical trials in several countries. Pharmaceutical compositions used in such clinical trials are representative wild type p53 of the invention comprising a replication lacking type 5 adenovirus vector expressing a human tumor suppressor gene under the control of a cytomegalovirus promoter (“rAd5 / p53) as described herein. Expression adenoviruses (see Willis (1994)).
<Local administration>
Ovarian cancer confined to the abdominal cavity is one clinical scenario where local p53 gene therapy, ie, intraperitoneal administration, should be considered as a treatment regimen recommended.
The following examples are intended to illustrate and do not limit the invention.
<Example 1>
Combination treatment of p53 and Taxol ^(R)
The present invention provides a combined administration of a tumor suppressor polypeptide and a nucleic acid expressing paclitaxel in the treatment of neoplasia. The following example details the performance of the p53 expressing adenovirus of the present invention in combination with Taxol to treat neoplasia, and the combination treatment was more efficient at killing tumor cells than the single agent.
In vitro combination therapy
Cells were treated in one of three treatment regimens. In Treatment 1 cells were pretreated with Taxol 24 hours prior to exposure to p53 adenovirus construct A / C / N / 53. In Treatment 2, cells were pretreated with p53 adenovirus constructs and then contacted with Taxol. In Treatment 3, the cells were contacted simultaneously with Taxol and p53 adenovirus. Thus, p53 Ad and Taxol can be administered within the same 24 hours or simultaneously.
Cultures (0.4 μg / ml Cortisol and DNEM + Ham F12 Culture 1: 1 Mix and Head and Neck Cell Lines SCC-9, SCC-15 and SCC-25, Eagles Essential Cultures and 10% FBS in 10% FBS and 1% Essential Amino Acids) Approximately 1.5 × 10 ⁴ cells in medium prostate PU-145 and ovarian SK-OV-3) were added to each well on a 96 well microtiter plate and incubated at 37 ° C. and 5% CO ₂ for 4 hours. Drug (taxol), p53 adenovirus, or appropriate vehicle / buffer was added to each well. Since paclitaxel is not water soluble, the drug was dissolved in ethyl alcohol prior to administration. The cells were then incubated overnight at 37 ° C. and 5% CO ₂ . p53 adenovirus was administered in phosphate buffer (20 mM NaH ₂ PO ₄ , pH 8.0, 130 mM NaCl, 2 mM MgCl ₂ , 2% sucrose).
Cell death was then quantified according to the Mosmann (1983, J. Immunol. Meth., 65: 55-63) method. Briefly, approximately 25 μl of 5 mg / ml MTT bio dye [3- (4,5 dimethylthiazol-2yl) -2,5-diphenyltetrazolium bromide] was added to each well and 3-4 Incubate at 37 ° C. and 5% CO ₂ for hours. 100 μl of 10% SDS cleaner was then added to each well and incubated overnight at 37 ° C. and 5% CO ₂ . Signals from each well were then quantified using a molecular apparatus microtiter plate reader (TermoMax). The specific cell lines used and the results obtained are listed in Table 1 below.
In Vivo Evaluation of Adjuvant Anticancer Taxols in Combination with Tumor Suppressor Nucleic Acids Cell line cancerTaxol dose (μg / ml)A / C / N / 53 Dose (m.o.i.)process Taxol full measurep53 pretreatmentThe same time SK-OV-3 Ovarian Cancer0.3740Nougat p ≤ 0.0001No effect p> 0.2000Nougat p ≤ 0.0001 SCC-25 Head and Neck Cancer0.10 or 0.012.5 or 5.0Nougat p ≤ 0.0001Very small effect p = 0.0606Nougat p ≤ 0.0001 SCC-15 Head and Neck Cancer0.10 or 0.012.5 or 5.0Negative action p ≤ 0.002Nougat p ≤ 0.0001Nougat p ≤ 0.0001 DU-145 prostate cancer0.36 or 0.036 or 0.00362.5 or 5.0Nougat p ≤ 0.03Nougat p ≤ 0.0001Nougat p ≤ 0.0001 SCC-9 Head and Neck Cancer0.12 or 0.012 or 0.00122.5 or 5.0Negative action p ≤ 0.01Nougat p ≤ 0.0001Nougat p ≤ 0.0001
In general, p53 adenovirus was more efficient when added simultaneously with or after Taxol than when added first. The results suggest a synergy between A / C / N / 53 and Taxol.
<Synergy effect established by isobologram analysis>
SK-OV-3 (no p53) ovarian tumor cells were treated with a combination of Taxol and p53 / adenovirus (A / C / N / 53) as illustrated in Table 2 below. Administration was performed as described above. Cell death was quantified at day 3 using the MTT assay as described above. In addition, a dose response curve for p53 Ad alone (using the doses listed in Table 2) was obtained (after two days of cell exposure to the drug), and the dose response curve for Taxol alone was used using the doses described above. (3 days of cell exposure to drug).
Treatment group for combined Taxol and p53 Ad (A / C / N / 53) treatment groupTaxol (μg / ml)p53 AD (m.o.i.)groupTaxol (μg / ml)p53 AD (m.o.i.) One0.0010.5250.00110 20.010.5260.0110 30.10.5270.110 40.50.5280.510 5One0.529One10 650.530510 7100.5311010 8200.5322010 90.0010.5330.00125 100.01One340.0125 110.1One350.125 120.5One360.525 13OneOne37One25 145One38525 1510One391025 1620One402025 170.0015410.00150 180.015420.0150 190.15430.150 200.55440.550 21One545One50 225546550 23105471050 24205482050
1 illustrates inhibition of cell proliferation as a function of treatment (as compared to buffer control). In general, increasing doses of Taxol or p53 reduce the rate of cell proliferation and the combination of p53 and Taxol has a much greater effect than with the drug alone.
2 is described in Berenbaum (1989) Pharmacol. Rev. 93-141 illustrates isobologram analysis of this data using an Isobole method such as. Synergy between Taxol and p53 (A / C / N / 53) was observed when cells were pretreated with Taxol 24 hours before p53 (A / C / N / 53) treatment. The straight line in FIG. 2 (isobols for ED ₃₀ ) shows the effect on cell proliferation expected when treatment with the two drugs is only additive. Indeed, the observed effect corresponds to the lower left side of the isoboles, indicating that a concentration below the expected concentration of each drug was needed and a synergy between the two drugs occurred.
<Example 2>
P53 adenovirus-mediated gene therapy for metastasis
The present invention provides administration of nucleic acids expressing tumor suppressor polypeptides in the treatment of metastasis. The following examples detail the ability of the p53 expressing adenoviruses of the present invention to infect and treat metastases in the body.
Seed mouse females [homogenous mice for SCID mutations lacking T and B cells due to defects in V (D) J recombination] to 5 x 10 ⁶ MDA-MB-231 breast carcinoma cells with their mammary fat pad Injection. Primary tumors were surgically removed (day 11) after the primary tumor had been well achieved and metastasized to the lung. Mice were treated with 4 × 10 ⁸ CIU / with control buffer, control buffer or A / C / N / 53 (p53 in adenovirus) on days of intravenous A / C / N / 53 or 23, 30, 37, 44 (1qW) Treatment was by injection. On day 49 lungs were harvested, fixed, stained and examined under the microscope.
The results are illustrated in Table 3 below.
Inhibition of MDA-MB-231 Pulmonary Metastasis Using A / C / N / 53 processNo transitionTransition ≤ 6Transition ≥ 84 Buffer n = 1711 (65%)1 (6%)5 (29%) A / C / N / 53 n = 105 (50%)4 (40%)1 (10%)
A / C / N / 53 treatment reduced the number of metastases in mice with them.
In a second experiment 231 tumors in the mammary fat pad of the seed or seed-beige mice were injected around the tumor by A / C / N / 53. The total dose of 2 to 4 × 10 ⁹ CIUs by 10 injections reduced the number of mice that had metastasized lung to 80% of seed mice and 60% of seed-beige mice. In addition, the number of metastases per mouse was significantly reduced in mice with any lung tumor. As indicated above, intravenous administration by A / C / N / 53 showed efficacy against lung metastasis in seed mice. The data indicate that cancer gene therapy with A / C / N / 53 may not only affect the severity of metastatic disease but may also reduce primary tumors.
In another embodiment female seed mice were injected with 5 × 10 ⁶ MDA-MB-231 breast tumor cells / mouse with a breast fat pad on day 0. Primary (breast) tumors were surgically removed on day 18. Mice were treated with intravenous injection of buffer, beta-gal AD, or p53 Ad (A / C / N / 53) at 21, 24, 32, 39 and 36 days. Virus dose per injection was 4 × 10 ⁸ CIU (A / C / N / 53) (PN / CIU = 23.3) and 9.3 × 10 ⁹ particle beta-gal Ad (PN / CIU = 55.6; 1.7 × 10 ⁸ CIU) It was.
Lungs and livers were harvested on day 51 and fixed in formalin. Tissue sites were evaluated for lung tumors and liver damage. Main organs from 2 buffer and 2 beta-gal Ad mice were frozen for cold incision and analysis for B-galactosidase enzyme activity.
Inhibition of MDA-MB-231 Pulmonary Metastasis Using A / C / N / 53 Lung Metastasis Per MouseBuffer Gp ^* Beta-gal Ad Gp. ^* p53 Ad Grp. ≤ 2011% (1)8% (1)21% (3) > 20 and ≤ 10011% (1)33% (4)79% (11) > 100 and ≤ 20033% (3)33% (4)0% (0) > 200 and ≤ 30033% (3)17% (2)0% (0) > 30011% (1)8% (1)0% (0) Total number evaluated91214 Regrowth of Primary Tumors82% (9/11)88% (14/16)100% (14/14)^* The number of metastases was underestimated. Multiple tumors grew with the lungs.
The number of metastases per lung in the buffer and beta-gal Ad groups was not significantly different. (p = 0.268, see Table 4).
p53 Ad treatment significantly reduced the number of metastases per lung when compared to either of the buffers of the beta-gal Ad group (p <0.001 and p <0.002, respectively). Not only was the number of metastases reduced, but also the magnitude of lung metastases in the p53 Ad group was greatly reduced. As a comparison, tissue sites from most lungs had more than 50% neoplastic tissue, so individual tumors could no longer be recognized over large areas of the lungs. In contrast, lung metastasis in most p53 Ad groups was small and distinguished as individual tumors.
<Adenovirus tissue distribution>
Liver tissue had the highest number of infected cells (about 50%) and beta galactosidase activity was potent. The lungs distributed patches of infected cells uniformly distributed throughout the tissue. The intestine and stomach had periodic infection of cells with external smooth muscle around the organs. There was also beta-galactosidase activity in the microvilli dispersed along the lumen. The upper smooth muscle wall around the uterus had a periodic infection of cells similar to that seen in the intestine. Most stromal cells in the ovary have been infected. The spleen dispersed beta-galactosidase activity in the smooth muscle component of the organ. There were very few (less than 1%) infected cells inside the main bulk of the rhabdomyomyocardium. There were almost no infected cells in the primary tumor either in the mammary fat pad or the corresponding rhabdomyo. There were no infected cells in the kidneys.
Liver Pathology
All livers were overall normal at necropsy. There was no apparent necrosis in either liver. However, mice treated with adenovirus were hepatocellular abnormalities (not present in the buffer group), including increased numbers of cells in mitosis, cell inclusion, and changes in hepatocyte size and shape.
Example 3 p53 Adenovirus-Mediated Gene Therapy for Human Breast Cancer Xenografts
The present invention provides methods for the treatment of various cancers by administration of nucleic acids expressing tumor suppressor polypeptides. The following example details the performance of the p53 expressing adenovirus of the invention to treat human breast cancer.
Introduction of wild-type p53 into tumors with or without mutant p53 provides a new method of controlling tumor growth. Casey (1991) Oncogene 6: 1791-1797 introduced wild-type p53 into breast cancer cells in vitro via plasmid DNA vectors. The number of MDA-MB-468 (p53 ^mut ) and T47D (p53 ^mut ) colonies that occurred after plasmid transfection was reduced by 50% by wild type p53. In addition, none of the resulting colonies expressed wild-type p53 transfectants. In contrast, the number of MCF-7 (p53wt) colonies was not affected. Negrini (1994) Cancer Res. 54: 1818-1824, performed a similar study using MDA-MB-231 cells. Colon formation was reduced by 50% by transfection with wild type p53 containing plasmids and any resulting colonies did not express wild type p53. Paradoxically, similar results were observed for MCF-7 cells in this study.
In the studies described in the above examples, the recombinant EI region removed p53 adenovirus lacking replication [53 Ad; (A / C / N / 53) Wills (1994)] were tested for mutant p53 expressing three human breast cancer cell lines, MDA-MB-231, MDA-MB-468, and MDA-MB-435. MDA-MB-231 cells contained an Arg-to-Lys mutation in codon 280 of the p53 gene (Bartek (1990) Oncogene 5: 893-899). MDA-MB-468 cells contained an Arg-to-His mutation in codon 273. MDA-MB-435 cells contained a Gly-to-Glu mutation in codon 266 of the p53 gene [Lesoon-Wood (1995) Hum. Gene Ther. 6: 395-405.
Previous studies have shown high levels of wild-type p53 expression in tumor cells from human breast, ovary, lung, colorectal, liver, brain and bladder after infection with p53 Ad [Wills (1994), Harris et al. (1996) Cancer Gene Therapy 3: 121-130. Adenovirus mediated p53 expression ultimately altered cell morphology and induced apoptosis in the absence of p53 or in mutant p53 tumor cells. Infection of 468 breast cancer cells with p53 Ad at 10 moi (multiple infections) resulted in nearly 100% inhibition of DNA synthesis for 72 hours after infection. In addition, infection with p53 Ad in vitro inhibited MDA-MB-468 and MDA-MB-231 cell proliferation with ED ₅₀ values of 3 ± 2 and 12 ± 10 mou, respectively. In addition, proliferation of three different p53 mutant breast carcinoma strains was inhibited even at low concentrations of p53 Ad. ED ₅₀ values were 16 ± 4 moi for SK-BR-3 cells, 3 ± 3 moi for T-47D cells, and 2 ± 2 moi for BT-549 cells. Infection of MDA-MB-468 and MDA-MB-231 cells with the same amount of recombinant adenovirus expressing E. Coli beta-galactosidase (beta-gal) 30 moi instead of p53 was greater than 67% beta-gal positive MDA-MB-468 cells and 34-66% beta-gal positive MDA-MB-231 cells were obtained. By correlating the p53 antiproliferative effect with the percentage of beta-gal positive cells in a large panel of tumor cells with altered p53, Harris et al. Showed a strong positive relationship between p53 induction inhibition and adenovirus transduction. . In contrast, cell lines expressing normal levels of wild type p53 were minimally affected by p53 transduction independent of adenovirus transduction ratio.
Proliferation of two human breast cell lines, MCF7 and HBL-100 cells containing wild type p53, was relatively unaffected by p53 Ad concentrations of 99 moi or higher in vitro. In other words, growth inhibition of MCF-7 and HBL-100 cells required a p53 Ad concentration of 8 and 33 times higher than the ED ₅₀ value for -231 alc -468 cells individually. Using similar recombinant p53 Ad, Katayose (1995), Clin. Cancer Res. 1: 889-897 show increased p53 protein expression, decreased cell proliferation, and increased apoptosis cell death in in vitro transduced -231 cells. The study extends these results in vitro with -468 and -231 cells for in vivo breast cancer xenografts. The efficacy of adenovirus-mediated p53 gene therapy is assessed in another breast cancer cell line (MDA-MB-435) that is resistant to in vitro adenovirus transduction.
<Materials and Methods>
In vitro cell line and adenovirus infection
Human breast cancer cell lines MDA-MB-231, -468 and -435 are commercially available from ATCC (Rockville, Maryland, USA). -231 cells were cultured in DMEM (Life Technologies, Grand Island, NY) with 10% fetal calf serum (FCS; Hyclone, Logan, Utah) at 37 ° C. and 5% CO ₂ . -468 cells were cultured in Leibovitz L-15 culture (Life Technologies) containing 10% FCS at 37 ° C. -435 cells were cultured in Leibobits L-15 culture with 15% FCS and 10 μg / ml bovine insulin (Sigma Chem. Co., St. Louis, Missouri) at 37 ° C.
The structure and reproduction of human wild-type p53 expression and E. Coli beta-galactosidase (beta-gal) expressing recombinant adenovirus (rAd) in which the transgene expression is performed by human cytomegalovirus promoters has been described above [Wills ( 1994). Adenovirus was administered in phosphate buffer (20 mM NaH ₂ PO ₄ , pH 8.0, 130 mM NaCl, 2 mM MgCl ₂ , 2% sucrose). CIU was defined as cell infection unit. The concentration of infectious virus particles was determined by measuring viral hexon protein positive 293 cells after a 48 hour infection period (Huyghe (1995)).
Cells were placed at a density of 1-5 × 10 ⁴ cells / well in 12 well tissue culture dishes for in vitro infection studies with p53 Ad (Becton Dickinson, Lincoln Park, New Jersey, USA). Cells were transduced with 0, 10 or 50 moi (multiple infections = CIU / cells) p53 Ad as described above and incubated for 72 hours [Wills (1994)]. Cells were placed at a density of 1 × 10 ⁵ cells / well for in vitro infection studies with beta-gal Ads. Cells were transduced with 0, 10, 50 or 100 moi beta-gal Ads. After 48 hours the cells were fixed with 0.2% glutaraldehyde (Sigma Chemical Co.) and washed three times with PBS (Life Technologies). The cells were then subjected to X-Gal solution [1.3 mM MgCl ₂ , 15 mM NaCl, 44 mM Hepes buffer, pH 7.4, 3 mM potassium ferricyanide in N, N-dimethylformamide, and 1 mg / ml X-Gal ( 10% final concentration)] in 1 ml. X-Gal is commercially available from Boehringer Mannheim Corp., Indianapolis, Indiana. All other chemicals are commercially available from Sigma.
Five microscopic fields were counted from each culture well to determine the percentage of transduced cells, and the average percentage of beta-galactosidase expressing was calculated for three wells in each m.o.i.
In vivo adenovirus treatment
Athymic nude mouse females were purchased from Charles River laboratory (Wilmington, Mass.). All mice were maintained in a VAF-barrier facility and all animal procedures were described in N.I.H. It was carried out according to the rules described in Guide for the Care and Use of Laboratory Animals. Tumor cells were injected subcutaneously or in breast fat pads.
Cell incubations were 5 × 10 ⁶ -231 cells / mouse, 1 × 10 ⁷ MDA-MB-468 cells / mouse or 1 × 10 ⁷ MDA-MB-435 cells / mouse. Dosing was started except for one -468 experiment in which tumors were grown in vivo for 10-11 days and tumors grew for 33 days prior to initiation of treatment. Tumor volume was calculated as the product of the measurements at 3 dimensions. Tumor volumes for different treatment groups were compared daily by Starview II software (Abacus Conceps, Berkeley, California) Student t-test. Average percent inhibition for groups administered on days 0-4 and 7-11 was calculated using the significance level (p <0.05) from day 14 to the last day of the study.
Specific effects of p53 were distinguished from adenovirus vector effects by subtracting the average tumor growth inhibition induced by beta-gal Ad from the growth inhibition induced by p53 Ad. All viral infections were marginal / intratumoral. In general, two tumor treatment 5-day courses (ie 5 injections) were performed on each mouse and separated by 2 days “rest”. In some cases, the dosing regimen has been extended for more than two weeks and / or viral for several injections with a buffer vehicle. Tumor growth curves showed mean tumor volume ± s.e.m.
Histology and apoptag immunohistochemistry
Tissue samples were fixed in 10% buffered formalin and run overnight in a Miles VIP tissue process and then impregnated in paraffin. Five micron tissue sites were cut with a Leitz microtome. Slides were stained with conventional Harris hematoxylin and eogen dyes (Luna et al. (1968), Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. New York; McGraw Hill Book Co.]
Apoptotic in situ apoptosis detection kits are commercially available from Oncor, Gaithersburg, Maryland, U.S.A. Samples were analyzed according to kit instructions. Briefly, deparaffinized and rehydrated tissue sites were treated with oncoprotein digestive enzymes, incubated with TdT, avidin-peroxidase kit (rabbit IgG-Sigma Chem, Co. EXTRA-3) and DAB (Vector Lab.SK4100). ) To develop. Slides were back stained with methyl green.
β-galactosidase assay
Tumors were impregnated in TBS (Triangle Biomedical Sciences, Durham, North Carolina, USA) and flash frozen in a 2-methylbutane / dry ice bath. Frozen tissue sections (8 μm thick) were fixed in 0.5% glutaraldehyde at 4 ° C. for 5 minutes and then analyzed for β-galactosidase expression as described above.
Integrin FACS Analysis
Cells were suspended by treatment with 0.02% EDTA, pelleted and washed twice with PBS. Cells were then resuspended at a concentration of 1 × 10 ⁶ cells / ml and incubated with primary antibody (final concentration 1: 250 / ml) for 1 hour at 4 ° C. The cell suspension was washed twice with PBS to remove excess primary antibody. Cells were then washed twice with FITC-conjugated rabbit antimouse accessory antibody (final concentration 1: 250 / ml, Zymed) at 4 ° C. for 1 hour. Cells were washed with PBS as before and analyzed immediately. Fluorescence was measured with a FACS Vantage flow cytometer (Becton Dickinson, Mauveview, CA, USA). Lateral divergence and forward divergence were measured simultaneously and all data was collected on a Hewlett Packard computer with FACS study software (Becton Dickinson). Primary antibodies used for detection of integrin receptors were obtained from the following suppliers. Anti-alpha _v (12084-018, Gibco BRL), anti-beta ₃ (550036, Becton Dickinson), anti-alpha _v beta ₃ (MAP1976, Chemicon), anti-alpha ₁ (550034, Becton Dickinson), anti-alpha _v Beta ₅ (MAB 1961, Chemicon).
result
In vitro adenovirus transduction efficiency and p53 growth inhibition
Both -231 and -468 cells were highly transduced at 10 m.o.i. In contrast, -435 cells were rarely transfected even at 100 m.o.i. For -231 cells 8% (10 m.o.i.), 46% (50 m.o.i.) and 62% (100 m.o.i.) cells were transduced with β-galactosidase adenovirus. For 468 cells 78% (10 m.o.i.), 84% (50 m.o.i.), and 97% (100 m.o.i.) cells were transfected with β-galactosidase adenovirus. For 435 cells 0.5% (10 m.o.i.), 1% (50 m.o.i.) and 1.3% (100 m.o.i.) cells were transduced with β-galactosidase adenovirus.
50 m.o.i. Infection with p53 Ad almost completely killed the cells in 231 and 468 cell cultures. In contrast, p53 Ad had no detectable effect on the growth of 435 cells.
P53 Ad Efficacy on Human Breast Cancer Xenografts
Adenovirus mediated p53 gene therapy was very effective against -231 and -468 xenografts (FIGS. 3A and 3B). In 231 experiments, one mouse in the β-gal Ad group and three mice in the p53 Ad group had no tumors at the end of the study and all tumors regressed during p53 Ad treatment. The inhibition rate of -231 tumor growth was on average 86% (p ≦ 0.01). The growth inhibition fraction due to p53 was 37% on average, whereas the adenovirus specific inhibition rate was 49% (p ≦ 0.01). The inhibition rate of -468 tumor proliferation was on average 74% (p ≦ 0.001). One mouse in the p53 Ad group had no tumors at the end of the study and all tumors regressed during p53 Ad treatment. The growth inhibition fraction by p53 was 45% on average (p <0.001), while the adenovirus specific inhibition was on average 28% (p ≦ 0.05). None of the experiments showed any side effects. ED ₅₀ values for -231 and -468 tumor growth inhibition were 3 × 10 ⁸ CIUs (cell infection units) and 2 × 10 ⁸ CIUs, respectively (FIG. 4). -435 tumors were almost completely resistant to p53 Ad treatment (FIG. 3C). Growth inhibition was minimal in the 435 tumor group treated with adenovirus.
FIG. 5 shows a comparison of the efficacy of two p53 Ad administration regimens for -231 tumors. All mice were injected per tumor five times per week. All mice treated with the therapeutic agent (p53 Ad) received a total of 2.2 × 10 ⁹ CIUs per week per mouse. One group was given a bolus containing a full weekly dose of adenovirus. The remaining four times of the week were injected with the buffer vehicle (group 1X). The other Ad group received the same Ad dose divided five times per week (5X group). This dosage regimen was given for 1 and 3 weeks (0-4 days, 14-18 days). Growth inhibition rate was on average 73% in the 5X group (p <0.01), but only 44% in the 1X group (p <0.05) during the first 3 weeks of the study, but insignificant after 21 days. The first cycle of p53 gene therapy was more effective than the second cycle. After the first treatment cycle, four in the 1X group, five in the 5X group and one in the vehicle control group were tumor free. One in the 5X group had a very small tumor recurrence by day 21. No further treatment traces were observed after the second cycle of treatment.
FIG. 6 shows an experiment treated with adenovirus 10-fold low dose using 468 tumors that were initially four times larger than the 468 tumors shown in FIG. 3b. A total of 2.2 × 10 ⁸ CIU p53 Ad doses were administered per week per mouse. One group received the virus in one bolus infusion, followed by four buffers per week (1X group). The other groups treated were given the same virus dose divided five times a week (5X group). These dosing schedules were for 6 weeks. The total virus dose administered over six weeks was approximately half of the dose used in FIG. 3. This dosage system produced a cytostatic effect on tumor volume in mice treated with p53 Ad (p ≦ 0.05). Treatment done at the beginning of the study appeared to be more effective than the treatment provided later in the study. One of the 5X groups had no tumors until day 21. However, when comparing tumor growth inhibition in all mice, the 1X dosing regimen (60%) was slightly more effective than the 5X group (55%), but not a significant difference. One week after the dosing, tumor growth rate in the 5X group began to increase. One month after starting the study, the vehicle control tumor began to necrosis and growth remained stagnant.
In Vivo Infection After Repeated Adenovirus Exposure
At the end of the studies shown in FIGS. 5 and 6, some tumors were injected with β-gal Ad. These tumors were harvested 24 hours later and frozen tissue sections were analyzed for β-galactosidase expression. Tumors treated with p53 Ad for 2 or 6 weeks were transduced with β-gal Ad, but transduction was lowest for 468 tumors treated with p53 Ad for 6 weeks 5 times a week. Sections obtained from only one of the 3-468 tumors injected in the 5X group had cells expressing β-galactosidase.
Induction of apoptosis in vivo by p53 Ad
Nude mice were harvested 48-72 after injection of 1-5 × 10 ⁸ CIU p53 Ad or buffer into MDA-MB-231 and MDA-MB-468 breast cancer xenografts. The incidence of cell death by p53 Ad was analyzed for tissue sections using Apoptag immunohistochemistry. Tumors injected with p53 Ad showed large area of apoptosis along the needle course of the intratumorally injected tumor and along the outer periphery of the peritumorally injected tumor. In contrast, tumors injected with buffer showed only a few sporadic cell deaths, as expected.
Comparison of Integrin Expression in MDA-MB-231 and MDA-MB-435 Cells
FACS analysis of integrin expression in MDA-MB-231 and MDA-MB-435 cells was performed to ensure that low Ad transduction of MDA-MB-435 cells resulted in Ad2, 3, and 4 internalization alpha. _v Determined due to lack of integrins (Wickham et al. (1993) Cell, 73: 309-319; Wickham et al. (1994) J. Cell Biol., 127: 257-264; and Mathias et al. 1994) J. Viol. 68: 6811-6814). Both cells expressed moderately identical levels of alpha _v , alpha _v beta ₃ , alpha _v beta ₅ and beta ₁ integrins. Integrin alpha _v beta ₃ , and beta ₃ expression were higher in MDA-MB-435 cells than in MDA-MB-231 cells.
Discussion: Tumor growth inhibition rate averaged 76% for MDA-MB-468 tumors and 86% for MDA-MB-231 tumors with 10 injections of a total of 2.2 x 10 ⁹ CIU p53 Ad. -MB-435 tumors were insignificant. In MDA-MB-468 tumors, 61% of the total responses were specific for p53, and in MDA-MB-231 tumors, 43% of the total responses were specific for p53. The ability of β-gal Ad to transduce in vitro into MDA-MB-231, MDA-MB-468 and MDA-MB-435 cells was usually estimated in vivo. At the same virus concentration -468 cells showed slightly higher transduction rate than MDA-MB-231 cells, and MDA-MB-435 cells were resistant to adenovirus transduction. MDA-MB-435 has been shown in vitro results related to very poor response in vivo.
Systemic treatment of nude mice with MDA-MB-435 tumors with the p53-liposomal vector has been observed to cause tumor growth inhibition and in some cases the tumor has been killed (Lesoon-Wood et al. (1995) Hum. Gene Ther., 6: 395-405). p53-liposomal treatment also reduced the number of lung metastases. This result demonstrates that the lack of response to p53 Ad treatment of MDA-MB-435 tumors in this study was not due to p53's inability to inhibit the growth and metastasis of MDA-MB-435 tumors. Rather, this result is due to the low adenovirus transduction of these cells resulting in non-responsiveness of MDA-MB-435 cells to p53 Ad treatment.
Alpha _v integrins were understood to be cellular elements required for effective introgression of type 2, 3 and 4 adenoviruses (books before Wickham (1993); books before Wickham (1994); and books before Mathias (1994)). Alpha _v integrins appear to play the same role as type 5 Ad. Literature (Wickham et al. (1994) in front of the book) in the internal launch of the alpha _v recombinant type 5 adenovirus in cells transfected with _beta-5 cells as compared to cells transfected with either not express alpha _v or _{alpha-v beta-3} cells 5 to 10 times larger was observed. Human embryonic kidney -293 cells used herein for the production of p53 Ad express alpha _v beta ₁ but not alpha _v beta ₃ integrin (Bodary (1990) J. Biol. Chem. 265: 5838-5941) . Thus, it was considered economical to measure alpha _v , beta ₁ , beta ₃ and alpha ₅ integrin subunit expression of -435 cells. MDA-MB-231 and MDA-MB-435 cells both expressed approximately the same level of integrin family molecules. Thus, the lack of Ad transduction of MDA-MB-435 cells is not due to a lack of alpha ₅ integrin expression. No document currently describes the identification of cellular receptors required for Ad binding to target cells. MDA-MB-435 cells may be defective in this receptor or may be defective in some other component necessary for viral binding, introgression or gene expression.
The efficacy of continued p53 Ad on the cycle treatment was tested in the MDA-MB-231 and MDA-MB-468 tumor models. The efficacy has been shown to decrease with continued dosing, but this effect needs to be tested in more detail. It was a strong theory that adenovirus infections produce rapid inflammatory and cytolytic responses mediated by cytotoxic T cells in hosts with a fully functional immune system (Wilson (1995) Nature Med. 4: 887-889). This T cell response is stimulated by the adenovirus antigen produced in the host cell and provided with the MHC moiety at the cell surface. Neutralization of antibodies specific for cells transduced by adenoviruses occurs later in the immune response and is thought to be responsible for a decrease in the ability to re-infect host cells with adenoviruses after initial inoculation. Athymic nude mice used in this study are defective in T cell immune responses to foreign antigens but can cause B cell mediated antibody responses (Boven (1991) The Nude Mouse in Oncology Research. Boston: CRC Press). The incidence of neutralization of anti-adenovirus antibodies could explain the decrease in efficacy of p53 adenovirus (p53 Ad) treatment over time in this study. Partial efficacy of p53 Ad, which is impaired immune function and poor blood supply into the tumor xenografts in 6 weeks after dosing, and the ability to infect several tumor cells with β-gal Ad even after repeated p53 Ad infusions Could explain.
In addition to breast cancer, many other cancers were treated with recombinant adenoviruses expressing wild type p53. Among these reports are cervical cancer (Hamada (1996) Cancer Res. 56: 3047-3054), prostate cancer (Eastham (1995) Cancer Res. 55: 5151-5155), head and neck cancer (Clayman (1995) Cancer Res. 55 : 1-6), lung cancer (a book before Wills (1994)), ovarian cancer (13), glioblastomas (27, 28), and colorectal cancer (13, 29). These data collectively support ongoing clinical studies evaluating the effectiveness of adenovirus mediated p53 gene therapy. This result demonstrates the ability of wild type p53 to reduce cancer cell growth in vivo in breast cancer xenografts expressing p53 mutants. The study also confirms that adenovirus is an effective carrier for p53 when the target cell expresses the appropriate viral “receptor (s)”.
<Example 4>
Further study of the treatment system on tumor suppression rate
The present invention provides methods for treating various cancers by administering nucleic acids expressing tumor suppressor polypeptides using various dosage systems. The following examples detail the increased efficacy of split administration of p53 expressing adenovirus of the present invention.
To compare the effects of single-dose and split-dose administration over a period of time, the total dose per mouse of severe combined immunodeficiency (SCID) mice injected with MDA-MB-468 and MDA-MB-231 tumors 1 × 10 ⁹ CIU p53 Ad (A / C / N / 53) was injected into one bolus or divided three or five times once daily over a week (indicated by arrows in FIG. 7).
The results obtained with the MDA-MB-468 tumors were similar to those obtained with the MDA-MB-23 tumors and are shown in Figures 7a, 7b and 7c. In general, divided doses inhibited tumor growth better than single bolus infusions, and the five infusion dosing regimens were much improved over the three infusion dosing regimens.
Example 5
Dexamethasone silences the inhibition of tumor growth accompanied by NK cell mediated anti-adenovirus immune responses.
It has been demonstrated that repeated administration of adenovirus vectors can elicit an anti-adenovirus immune response. Immunosuppressor Properties of Low Dose Dexamethasone (Dex) To study whether it can inhibit the anti-adenovirus immune response (e.g., NK cell response), MDA-MB-231 tumor in Severe Immunodeficiency Mice Was treated with and without the dexamethasone recombinant virus of the present invention.
Approximately 5 × 10 ⁶ MDA-MB-231 cells per mouse were injected on day 0 into the mammary fat pad of female Severe Immunodeficiency Mice. Dexamethasone or placebo pellets were implanted subcutaneously on day 11. 5 mg pellets were designed to release dexamethasone continuously for 8 days at 83.3 μg per day (Innovative Research of America, Sarasota, FL, USA). All mice received a total of 10 peritumoral infusions (0.1 ml per injection) at 14-18 and 21-25 days once daily. Total virus dose was 2 × 10 ⁹ CIU p53 Ad (A / C / N / 53 or β-galactosidase Ad) per mouse. The therapeutic agents are listed in Table 5.
Treatment of MDA-MB-231 Tumors in Severe Complex Immunodeficiency Mice grouphormoneGene therapy OnePlaceboBuffer 2Placeboβ-gal Ad 3Placebop53 Ad 4DexamethasoneBuffer 5Dexamethasoneβ-gal Ad 6Dexamethasonep53 Ad
Treatment with low doses of dexamethasone had minimal effect on the growth rate of MDA-MB-231 tumors in severe combined immunodeficiency mice (p> 0.05). No adverse side effects of dexamethasone were observed. Treatment of tumors with β-galactosidase adenovirus significantly inhibited tumor growth in placebo-controlled tumors (p ≦ 0.001, 21-30 days), but not in dexamethasone treated tumors (p> 0.05, see FIG. 8). . Placebo and β-gal Ad treated tumors grew more slowly than placebo and dexamethasone treated tumors (p ≦ 0.01, 23-30 days).
P53-specific inhibition of tumor growth in placebo-controlled tumors was insignificant (p> 0.05). In contrast, tumors treated with dexamethasone and p53 Ad grew much slower than tumors treated with dexamethasone and β-gal Ad (p <0.02, days 21-30) or placebo and p53 Ad (p <0.04, days 21-30). did.
Thus, low dose dexamethasone treatment silenced tumor growth inhibition accompanied by anti-adenovirus immune responses (eg, NK cell responses) in severe combined immunodeficiency mice without adverse side effects. This data means that low dose dexamethasone treatment can stimulate trans gene (eg p53) expression induced by the CMV promoter in recombinant adenovirus. Conversely, because dexamethasone can increase adenovirus transduction efficacy, it can increase tumor cell mortality.
The MDA-MB-231 breast cancer model was then used to assess the antitumor efficacy of Ad with or without p53 in mice with differing ability to elicit an immune response to foreign antigens. Nude mice, in which T cells have lost their function, severe combined immunodeficiency mice in which T and B cells have lost their function, and severe complex immunodeficiency malignant mice in which T, B, and NK cells have lost their function were studied.
To study the efficacy of rAd5 / p53 (described above) on MDA-MD-231 xenografts, nude mice received a total dose of 2.2 × 10 ⁹ CIU Ad per mouse from 10 to 0 to 4 days and 7 to 11 days. The dose was divided into two doses. Severe combined immunodeficiency mice received a total virus dose of 4 × 10 ⁹ CIU divided into ten doses on days 0-4 and 7-11. Severe complex immunodeficiency malignant mice received a total virus dose of 1.6 × 10 ⁹ CIU divided into ten doses on days 0-4 and 7-11. All mice were treated with p53 Ad, β-gal Ad, or vehicle only.
Intratumoral administration of a control Ad vector (no p53 insert) in nude mice (loss of T cells) or severely complex immunodeficiency mice (loss of T and B; NK cell activity) inhibited tumor growth to some extent. Ads expressing p53 (rAd5 / p53) significantly improved antitumor efficacy compared to control Ad. Conversely, in severe multiplex immunodeficiency malignant mice (loss of function in both T, B and NK cells), antitumor efficacy was entirely due to p53 expression and the Ad vector did not contribute to tumor growth inhibition at all. These data demonstrate a previously unrecognized role for NK cells in Ad mediated tumor growth inhibition. This data also means that inhibition of the immune system can discard some vector specific, NK cell mediated side effects.
<Example 6>
Combination of p53 adenovirus and chemotherapy treatment
The present invention provides a combination administration of nucleic acids and chemotherapeutic agents that express tumor suppressor polypeptides in the treatment of neoplasia. The following examples detail the neoplastic capacity of the p53 expressing adenovirus of the present invention in combination with various anticancer agents, cisplatin, doxorubicin, 5-fluorouracil (5-FU), methotrexate and etoposide, Combination therapy was more effective (ie, synergistic) in killing tumor cells than when treating each agent alone.
Combination administration of chemotherapeutic agent and p53 in vitro
Combination of p53 with Cisplatin, Doxorubicin, 5-Fluorouracil (5-FU), Methotrexate, and Etoposide
Clinically suitable anticancer agents cisplatin, doxorubicin, 5-fluorouracil (5-FU), methotrexate, and etoposide were tested for the effect of combining the tumor suppression vector (A / C / N / 53) of the present invention. Study in the hall. Three treatment regimes were applied to SCC-9 head and neck squamous cell carcinoma, SCC-15 head and neck plate cell carcinoma, SCC-25 head and neck plate cell carcinoma, and DU-145 prostate carcinoma cells . In Treatment 1, cells were pretreated with chemotherapy 24 hours before exposure to p53 adenovirus construct A / C / N / 53. In Treatment 2, cells were pretreated with p53 adenovirus constructs and then contacted with anticancer chemotherapeutic agents. In Treatment 3, the cells were contacted with an anticancer chemotherapeutic agent and p53 adenovirus simultaneously.
All cell lines were obtained from ATCC (Rockville, MD). SCC-9, SCC-15, and SCC-25 head and neck tumor cells (p53 ^null ) were treated at 37 ° C. and 5% CO ₂ with 10% fetal calf serum (FCS; Hyclone, Logan, Utah), 0.4 μg / ml Hydrocortisone (Sigma Chem. Co., St. Louis, MO), and Ham's F-12 (Gibco / Life Technologies, Grand Island, NY, USA) containing 1% non-essential amino acids (Gibco) and DMEM: 1 was incubated in the mixture. SK-OV-3 human ovarian tumor cells (p53 ^null ) and DU-145 human prostate tumor cells (p53 ^mut ) were incubated in Eagles MEM + 10% FCS at 37 ° C. and 5% CO ₂ . MDA-MB-231 human breast tumor cells (p53 ^mut ) were incubated in DMEM (Gibco) containing 10% fetal calf serum (Hyclone) at 37 ° C., 5% CO ₂ . MDA-MB-468 human breast tumor cells (p53 ^mut ) were incubated in Leibovitz's L-15 medium (Gibco) containing 10% FCS under CO ₂ at 37 ° C.
MDA-MB-231 breast tumor cells carry an Arg versus Lys mutation in the 280 codon of the p53 gene, thus expressing the mutation p53 (book before Barteck (1990)). DU-145 prostate tumor cells carry two p53 mutations on different chromosomes, ie Pro to Leu mutations at 223 codons and Val to Phe mutations at 274 codons (Isaacs (1991) Cancer Res. 51: 4716-4720), Expresses mutant p53. SK-OV-3 ovarian tumor cells are p53-null (p53-null) (Yaginuma (1992) Cancer Res. 52: 4196-4199). SCC-9 cells have a deletion between 274 codons and 285 codons, so that no immunoreactive p53 protein is detected in the SCC-9 nucleus because a frame shift mutation is induced (Jung (1992) Cancer Res. 52: 6390-6393 Caamano (1993) Am. J. Pathol. 142: 1131-1139; Min (1994) Eur. J. Cancer 30B: 338-345). SCC-15 cells have five base pairs inserted between the 224 codons and the 225 codons, which produce small amounts of p53 mRNA, but no p53 protein is detected at all (Min et al., 1994). SCC-25 lost heterozygosity (LOH) on chromosome 17 and two base pairs in the remaining allele were deleted at codon 209. p53 mRNA is not detected in SCC-25 cells, and no immunoreactive p53 protein is observed in these nuclei (book before Caimano (1993)). Approximately 1.5 × 10 ⁴ cells (described in Example 1) in the culture medium were added to each well of a 96 well microtiter plate and incubated at 37 ° C., 5% CO ₂ for about 4 hours.
Construction and propagation of human wild type p53 and Escherichia coli galactosidase (β-gal) adenovirus (Ad) are as described above (book before Wills (1994)). The concentration of infectious viral particles was determined by measuring the concentration of viral hexon protein positive 293 cells after a 48-hour infection period (book before Huyghe (1995)). CIU is defined as a cell infection unit. p53 expressing adenovirus was administered in phosphate buffer (20 mM NaH ₂ PO ₄ (pH 8.0), 130 mM NaCl, 2 mM MgCl ₂ , 2% sucrose). Phenol, p53 adenovirus or appropriate vehicle / buffer was added for each well. For in vitro studies with p53 Ad, cells were plated at a density of 1.5 × 10 ⁴ cells per well in 96 well plates and incubated at 37 ° C., 5% CO ₂ for 4 hours. About, p53 Ad or a suitable vehicle was added for each well and the cells were incubated overnight. About p53 Ad or appropriate vehicle was then added to each well. Cell culture continued for an additional 2 days.
Cell mortality was quantified according to the MTT assay as described in Mosmann (1983) J. Immunol. Meth., 65: 55-63. In short, approximately 25 μl of 5 mg / ml MTT biodye [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] is added for each well, Incubate at 37 ° C., 5% CO ₂ for 3-4 hours. 100 μl of 10% SDS cleaner was then added to each well and incubated overnight at 37 ° C., 5% CO ₂ . The signal in each well was then quantified using a Molecular device microtiter plate reader.
In all cases, combination treatment with cisplatin (see Table 6 for a summary of results), doxorubicin (see Table 7 for a summary of results), 5-FU, methotrexate, and etoposide resulted in a tumor than the use of either agent alone. It was more effective at killing cells. Combining methotrexate and p53 Ad was tested on one cell line. SCC-15 cells were treated with 0.7 μM methotrexate for 24 m. 5 m.o.i. The combination antiproliferative effect of the two drugs with p53 Ad was only 5% greater than that with p53 Ad alone, but this difference was statistically significant (p ≤ 0.003). 5 or 10 m.o.i. 24 h after DU-145 cells were pretreated with 2.6 μM etoposide. Treatment with p53 Ad resulted in greater combination efficacy compared to treatment with either drug alone (p ≦ 0.001). SCC-15 cells were treated with 0.3 μM etoposide 24 h after 5 m.o.i. When treated with p53 Ad, the combined anti-proliferative effect of the two drugs was only 5% greater than that of p53 Ad alone, but this difference was statistically significant (p ≤ 0.003). The combination of tumor suppressor gene therapy and anti-neoplastic agents did not show antagonistic effects.
In the second experiment, the therapeutic efficacy of normal cells (MRC-9 cells) was compared with tumor cells (FIG. 9). In this experiment, growth was analyzed by ³ H-thymidine incorporation rather than MTT analysis. Normal cells (ploidy fibroblast MRC-9 cells) did not show a more pronounced effect following the combination treatment. As expected, the effect of tumor suppressor alone was negligible on normal cells and very large on tumor cells. In contrast, anticancer chemotherapeutic agents alone (eg, cisplatin, doxorubicin, 5-FU, methotrexate, and etoposide) were more effective in normal cells than in cancer cells (see FIG. 9).
Antiproliferative effect combining cisplatin and p53 Ad Cell linep53 proteinOrganization typeIs there a greater combination Cisplatin firstp53 Ad firstAt the same time SK-OV-3invalidityovaryYes (p≤0.001)Yes (p≤0.001)Yes (p≤0.001) SCC-9invalidityTofu and neckYes (p≤0.001)Yes (p≤0.001)Yes (p≤0.001) SCC-15invalidityTofu and neckYes (p≤0.001)NDND SCC-25invalidityTofu and neckYes (p≤0.001)Yes (p≤0.001)Yes (p≤0.001) MDA-MB-468MutationbreastYes (p≤0.001)NDYes (p≤0.001) MDA-MB-231MutationbreastYes (p≤0.002)Yes (p≤0.001)Yes (p≤0.001) ND = none
Antiproliferative Effects of Combining Doxorubicin and p53 Ad Cell linep53 proteinOrganization typeIs there a greater combination Cisplatin firstp53 Ad firstAt the same time SK-OV-3invalidityovaryYes (p≤0.001)Yes (p≤0.001)Yes (p≤0.001) SCC-9invalidityTofu and neckYes (p≤0.001)Yes (p≤0.001)Yes (p≤0.001) SCC-15invalidityTofu and neckYes (p≤0.001)Yes (p≤0.001)Yes (p≤0.001) SCC-25invalidityTofu and neckYes (p≤0.001)Yes (p≤0.001)Yes (p≤0.001) DU-145MutationprostateYes (p≤0.001)Yes (p≤0.001)Yes (p≤0.001) MB-231MutationbreastYes (p≤0.001)Yes (p≤0.001)Yes (p≤0.001)
Doxorubicin and p53 effects on human hepatocellular carcinoma
The following examples show the ability of the p53 expressing adenoviruses of the invention to treat neoplasms in combination with doxorubicin, and that the combination treatment is more effective in killing tumor cells than using each agent alone, ie a synergistic effect. It is explained in detail. The results show a synergistic relationship between p53 expression vector (ACN 53) and doxorubicin of the present invention.
Doxorubicin (Adriamycin) and p53 (ACN53, a recombinant adenovirus construct that expresses the human wild-type p53 trans gene) were transferred to the human hepatocellular carcinoma cell line HLE (Hsu (1993) Carcinogenesis 14: 987-992; Farshid (1992) J. with mutant p53. Med. Virol. 38: 235-239; Dor (1975) Gann. 66: 385-392), human hepatocellular carcinoma cell line HLF with mutant p53 (same book), human hepatocellular carcinoma Hep 3B (1995) with invalid p53 ) In Vitro Cell Dev Biol Anim. 31: 55-61); Human hepatocellular carcinoma with p53 wild type Hep G2 (same book) and human liver adenocarcinoma with p53 wild type SK-HEP-1 (Lee (1995) FEBS Lett. 368: 348-352). Cell viability was measured by hepatocyte probe calcinen AM (Molecular Probes) (eg Poole (1993) J. Cell Sci. 106: 685-691). When the substrate calciene AM is cleaved by intracellular esterases, fluorescence is generated.
Cells were plated (5 × 10 ³ cells / well) in 96 well plates, allowed to attach overnight, and treated with ACN53 dilution and doxorubicin dilution three times on day 0, yielding a dose response curve for doxorubicin treatment. Obtained for each dose. Media was aspirated on day 3 and calciene AM in PBS was added to the cells. Fluorescence intensity in each well was measured using a fluorescence plate reader. The fluorescence values of wells without cells were subtracted and the data expressed as% survival (fluorescence intensity) compared to untreated control wells. Isobolograms were made using ED ₅₀ values and the correlation between ACN53 and doxorubicin was evaluated.
Isobologram analysis for each cell line showed a synergistic relationship between p53 expression vector (ACN53) and doxorubicin of the present invention, and this synergy was independent of the p53 status of the cell line. However, in the absence of doxorubicin, ED ₅₀ for ACN53 was higher in p53 wild type cell line than in p53 modified cell line.
In another similar experiment, HLE cells were plated in 96 well plates (5 × 10 ³ cells / well), attached overnight, and subjected to three replicates of dilutions of ACN53 and dilutions of doxorubicin to obtain dose response curves for doxorubicin. Obtained for each ACN53 dose. Three groups were used to test the effect on the order of administration on the correlation between ACN53 and doxorubicin.
group0 days1 day2 days3 days The same timeACN53, doxorubicin Collection ACN53 firstACN53Doxorubicin Collection Doxorubicin firstDoxorubicinACN53 Collection
Cells were incubated for 3 days after the first treatment. The medium was aspirated and calcinen AM in PBS was added to the cells. Fluorescence intensity in each well was measured using a fluorescence plate reader. The fluorescence values of wells without cells were subtracted and the data expressed as% survival (fluorescence intensity) compared to untreated control wells. Isobolograms were made using ED ₅₀ values and the correlation between ACN53 and doxorubicin was evaluated. Isobologram analysis for each dosing regime demonstrated similar correlations between ACN53 and doxorubicin in HLE cells that were synonymous with synergistic effects, irrespective of the dosing order of treatment.
Use of chemotherapy and p53 in the body
The effects of the clinically relevant anticancer drugs cisplatin, doxorubicin and 5-fluorouracil (5-FU) used in combination with the tumor suppression vector (A / C / N / 53) of the present invention were further studied in the body. C.B. 17 / ICR-SCID mice were purchased from Taconic Farms (Germantown, NY) or Charles River Laboratories (Wilmington, Mass.). Athymic nu / nu mice were purchased from Charles River Laboratories. All mice were maintained in VAF-barrier devices, and all animal procedures were maintained in N.I.H. This was done according to the rules set out in the guide. Tumor volumes for different treatment groups were compared daily by Student test using Statview II software (Abacus Concepts, Berkeley, CA). Tumor growth curves were prepared to show mean tumor volume ± s.e.m. 10 mice per group were used.
SK-OV-3-ovarian tumor model:
Established intraperitoneal SK-OV-3 tumors were treated with intraperitoneal administration of vehicle, p53 Ad, cisplatin or both drugs. Mice were injected six times with p53 Ad over two weeks. Total virus dose was 1.5 × 10 ⁹ CIU (3.1 × 10 ¹⁰ virus particles).
Cisplatin Efficacy: SCID mouse females were injected intraperitoneally with 5 × 10 ⁶ SK-OV-3 ovarian tumor cells on day 0. Mice were injected intraperitoneally on days 6, 8, 10, 13, 15 and 17 (only p53 Ad was injected on day 17). A total of 0.2 ml volume (0.1 ml of cisplatin vehicle or cisplatin + Ad buffer or 0.1 ml of p53 Ad) was administered to mice. The p53 Ad dose was 2.5 × 10 ⁸ CIU / mouse / day (5.2 × 10 ⁹ virus particles). Cisplatin dose was 2 mg / kg / day. Tumors were harvested and weighed on day 20.
Mice in one treatment group received five doses of cisplatin at the same time as the first five doses of p53 Ad. This intraperitoneal p53 Ad administration reduced tumors in mice by only 17% on day 20 (p ≦ 0.01). However, in combination with cisplatin, p53 Ad reduced tumors by 38% compared to cisplatin alone (p ≦ 0.0008). Mice treated with drug vehicle or p53 Ad alone developed hemorrhagic ascites and invasive tumor nodules of the diaphragm muscle. These symptoms did not occur in mice treated with cisplatin alone or with cisplatin and p53 Ad.
Cisplatin / Paclitaxel Efficacy: SCID mouse females were injected intraperitoneally with 2.5 × 10 ⁶ SK-OV-3 ovarian tumor cells on day 0. Mice were injected intraperitoneally on days 7, 9, 11, 16 and 18. A total of 0.3 ml volume (0.1 ml of cisplatin vehicle or cisplatin + paclitaxel vehicle or 0.1 ml of paclitaxel + Ad buffer or 0.1 ml of p53 Ad) was administered to mice. The p53 Ad dose was 2.5 × 10 ⁸ CIU / mouse / day (5.2 × 10 ⁹ virus particles). Cisplatin dose was 0.5 mg / kg / day. Paclitaxel dose was 1 mg / kg / day. Tumors were harvested and weighed on day 30. 7-10 mice per group were used.
In this second study, SK-OV-3 ovarian tumors were treated by concurrent intraperitoneal administration of vehicle, p53 Ad, cisplatin + paclitaxel, or all three drugs. The combination of all three drugs reduced tumors by 34% more than the combination of cisplatin plus paclitaxel, demonstrating the increased efficacy of the combination of three drugs (p ≦ 0.0006).
DU-145 prostate tumor model
Cisplatin Efficacy: Intraperitoneal DU-145 tumors were treated with intraperitoneal administration of vehicle, p53 Ad, cisplatin or both drugs. SCID mouse males were intraperitoneally injected with 2.5 × 10 ⁶ DU-145 cells on day 0. Mice were injected intraperitoneally on days 7, 9, 11, 14 and 16. A total of 0.2 ml volume (0.1 ml of cisplatin vehicle or cisplatin + Ad buffer or 0.1 ml of p53 Ad) was administered to mice. The p53 Ad dose was 8.3 × 10 ⁸ CIU / mouse / day (2.9 × 10 ¹⁰ PN). Cisplatin dose was 1 mg / kg / day. Tumors were harvested and weighed on day 22. The combination of p53 Ad and cisplatin significantly increased antitumor efficacy compared to either drug treatment alone (p ≦ 0.0004).
MDA-MB-468-breast tumor model:
Cisplatin Benefits:
Established MDA-MB-468 tumors were treated with intraperitoneal administration of vehicle, p53 Ad, cisplatin or both drugs. SCID mouse females were injected with 1 × 10 ⁷ MDA-MB-468 cells into the mammary fat pad 11 days before the start of Day 0 administration. Cisplatin dose was 1 mg / kg / day. Intratumoral p53 Ad dose was 8.3 × 10 ⁸ CIU / mouse / day (2.9 × 10 ¹⁰ virus particles) administered concurrently on days 0-4. p53 Ad showed increased potency when used in combination with cisplatin (8-31 days, p ≦ 0.0004).
Doxorubicin Efficacy:
In a second experiment, MDA-MB-468 tumors were treated with vehicle, p53 Ad, doxorubicin or both drugs. Nude mouse females were injected subcutaneously with 1 × 10 ⁷ MDA-MB-468 cells 12 days before the start of Day 0 dosing. Intraperitoneal doxorubicin doses were 4 mg / kg / day on days 0, 2, 7 and 9. Intratumoral p53 Ad doses were 5 × 10 ⁸ CIU / mouse / day (1.03 × 10 ¹⁰ virus particles) at days 0-4 and 7-11. p53 Ad retained greater efficacy when administered in combination with doxorubicin (14-24 days, p ≦ 0.05).
SCC-15 Head and Neck Tumor Model
5-Fluorouracil Efficacy:
Subcutaneous SCC-15 tumors were treated with vehicle, p53 Ad, 5-fluorouracil or both drugs. SCID mice were injected subcutaneously with 5 × 10 ⁶ SCC-15 cells 7 days before the start of Day 0 dosing. Intraperitoneal 5-Fluorouracil Dosages were administered at 0, 7 and 14 days (once per week for 3 weeks) 40% hydroxypropyl-beta-cyclodextran (Cerestar Inc., Hammond, Indiana) 50 mg / kg / day. p53 Ad doses were 2 × 10 ⁸ CIU / mouse / day (4 × 10 ⁹ virus particles) at 0, 1, 7, 8, 14 and 15 days (6 intratumoral injections for 3 weeks). The 5-FU dose was 50 mg / kg. Combination of p53 Ad with 5-FU increased antitumor efficacy compared to either drug treatment alone (p ≦ 0.004).
P53 in combination with FPT inhibitor
The effect of the farnesyl protein transferase inhibitor in combination with a tumor suppression vector named A / C / N / 53 was investigated in vitro. The following example is combined with an FPT inhibitor designated "FPT39", as described in International Application Publication No. WO97 / 23478, filed Dec. 19, 1996, wherein FPT39 is designated as compound "39.0", see page 95. The combination of p53 expressing adenovirus of the invention of the present invention in combination with neoplastic capacity and prostate tumor cells and breast tumor cells has a greater tumor cell lethal effect than when used alone.
Antiproliferative Efficacy of rAd5 / p53 and FPT39 on SK-OV-3 Ovarian Tumors
Methods: SK-OV-3 human ovarian tumor cells (p53 ^null ) were aliquoted into 96 well plates at a density of 250 cells per well in Eagle MEM + 10% fetal bovine serum. Cells were incubated at 37 ° C. and 5% CO ₂ for 4 hours. FPT39 or drug vehicle was added to each well and cell culture continued for 3 days. After 3 days, untreated cells in some wells were counted to calculate the amount of rAd5 / p53 addition. RAd5 / p53 or drug vehicle was then added to each well and cell culture continued for an additional 3 days. Cell proliferation was measured using MTT assay. 25 μl of 5 mg / ml MTT biodye [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] was added to each well, followed by 37 ° C. and 3-4 h. Incubate at 5% CO ₂ . 100 μl of 10% SDS detergent was then added to each well and incubated overnight. Fluorescence of each well was quantified using a Molecular Devices microtiter plate reader. Cell proliferation data are described in O'Connell and Wolfinger (1997) J. Comp. Graph. Stat. 6: 224-241] using the Thin Plate Spline method.
Results: rAd5 / p53 and FPT39 retained additional efficacy in inhibiting cell growth. Synergistic effects (p> 0.05) or antagonism (p> 0.05) were not seen in this experiment.
Antiproliferative and Synergistic Effects of rAd5 / p53 and FPT39 (FPT39 Inhibitors) on DU-145 Prostate Tumor Cells
Methods: DU-145 human prostate tumor cells (p53 ^mut ) were treated with FPT39 or drug vehicle and rAd5 / p53 and cell cultures were analyzed as described for SK-OV-3 human ovarian tumor cells. The experiment was repeated twice.
Results: Experiment 1: rAd5 / p53 and FPT39 retained additional efficacy in inhibiting cell growth. Synergistic effects (p> 0.05) or antagonism (p> 0.05) were not seen in this experiment.
Experiment 2: rAd5 / p53 and FPT39 showed synergistic efficacy (p = 0.0192). This result demonstrates that rAd5 / p53 and FPT39 can interact and have a synergistic effect on prostate tumor cell proliferation.
Antiproliferative and Synergistic Effects of rAd5 / p53 and FPT39 (FPT39 Inhibitors) on MDA-MB-231 Breast Tumor Cells
Methods: MDA-MB-231 human breast cancer cells (p53 ^mut ) were treated with FPT39 or drug vehicle and rAd5 / p53 and cell cultures were analyzed as described for SK-OV-3 human ovarian tumor cells. The experiment was repeated twice.
Results: Experiment 1: rAd5 / p53 and FPT39 retained additive efficacy. Synergy (p> 0.05) was not seen in the experiment.
Experiment 2: rAd5 / p53 and FPT39 showed additive efficacy on most response surfaces. However, synergistic effects were evident in isoboles above 70 (ie less than 30% cell mortality, p = 0.0001). These results demonstrate that rAd5 / p53 and FPT39 can interact to have a synergistic effect on human breast cancer cell proliferation.
Example 7
Immune response profile in patients with metastatic liver carcinoma treated with adenovirus vectors with p53
The present invention provides in vivo combination administration of p53 expressing nucleic acids and other chemotherapeutic agents in the treatment of neoplasia. The following example details the ability of the p53 expressing adenovirus of the invention to increase the level of tumor lethal lymphocytes found in human liver carcinoma.
The purpose of this study was to characterize genotypes and phenotypes of tumor-infiltrating lymphocytes (TILs) of metastatic hepatocellular carcinoma from p53 mutations present in the colon (see for example, Wang (1997) Mol. Med. 3: 716). -731; Marrogi (1997) Int. J. Cancer 74: 492-501). A total of 16 patients were treated with 10 ⁹ -10 ¹¹ particles in an incremental manner through the hepatic artery using adenovirus vectors carrying the wild type p53 gene. A total of four biological samples from each patient were obtained 3-7 days after adenovirus vector administration. Immunohistochemical analysis was performed on frozen tissue obtained from normal liver and tumor-host tissue covalent sites. Computer image analysis was performed to quantify the immune responsiveness to the monoclonal antibodies CD3, CD4, CD8, CD25, CD28, CD56, HLA-DR, IFN-gamma, TNF-alpha and IL-2. The maximum increase in TIL (CD3 ⁺ and CD4 ⁺ ) was observed in 7.5 × 10 ¹⁰ particles. At high doses, a decrease in CD3 ⁺ and CD4 ⁺ populations was observed. The opposite relationship was observed for CD8 ⁺ cells. At high doses (2.5 × 10 ¹¹ ), an increase in CD3 ⁺ , CD4 ⁺ and CD8 ⁺ populations was observed in tumor cells compared to normal cells. This result demonstrates that high dose delivery of adenovirus particles increased TIL consisting of CD4 ⁺ and CD8 ⁺ populations.
The examples and embodiments described herein are merely intended to illustrate the invention, and those skilled in the art will understand that different variations thereof are included within the spirit and scope of the present application and the appended claims. All documents, patents and patent applications are incorporated herein by reference.

权利要求:
Claims (77)
[1" claim-type="Currently amended] A method of treating a mammalian cancer cell or hyperproliferative cell comprising contacting the mammalian cancer cell or hyperproliferative cell with a tumor suppressor protein or tumor suppressor nucleic acid and contacting the cell with one or more accessory anticancer agents.
[2" claim-type="Currently amended] The method of claim 1, wherein the adjuvant anticancer agent is a microtubule blocking agent.
[3" claim-type="Currently amended] The method of claim 2, wherein the microtubule blocker is paclitaxel or a paclitaxel derivative.
[4" claim-type="Currently amended] The method of claim 1, wherein the method further comprises contacting the cell with a chemotherapeutic agent.
[5" claim-type="Currently amended] The method of claim 4, wherein the chemotherapeutic agent is cisplatin, carboplatin or navelvin.
[6" claim-type="Currently amended] The method of claim 1, wherein the tumor suppressor nucleic acid is a nucleic acid encoding a tumor suppressor protein selected from the group consisting of wild type p53 protein and retinoblastoma (RB) protein.
[7" claim-type="Currently amended] The method of claim 6, wherein the tumor suppressor nucleic acid encodes a wild type p53 protein.
[8" claim-type="Currently amended] The method of claim 6, wherein the retinoblastoma protein is p110 ^RB or p56 ^RB .
[9" claim-type="Currently amended] The method of claim 1, wherein the nucleic acid is delivered by a vector selected from the group consisting of naked DNA plasmids, plasmids in liposomes, plasmids bound to lipids, viral vectors, AAV vectors, and recombinant adenovirus vectors. Way.
[10" claim-type="Currently amended] The method of claim 1, wherein the nucleic acid is delivered by a recombinant adenovirus vector.
[11" claim-type="Currently amended] The method of claim 10, wherein said nucleic acid is delivered by a recombinant adenovirus vector comprising a partial or complete deletion of Protein IX DNA and comprising a nucleic acid encoding a wild type p53 protein.
[12" claim-type="Currently amended] The method of claim 11, wherein the deletion of the Protein IX gene sequence ranges from about 3,500 bp from the 5 'virus end to about 4,000 bp from the 5' virus end.
[13" claim-type="Currently amended] 13. The method of claim 12, further comprising the deletion of non-essential DNA sequences of adenovirus initial region 3.
[14" claim-type="Currently amended] The method of claim 11, further comprising the deletion of non-essential DNA sequences of adenovirus initial region 4.
[15" claim-type="Currently amended] The method of claim 11, further comprising the deletion of the DNA sequences designated E1a and E1b.
[16" claim-type="Currently amended] The method of claim 10, wherein the recombinant adenovirus vector comprises an adenovirus type 2 late main promoter or human CMV promoter, adenovirus type 2 tripartite leader cDNA and human p53 cDNA.
[17" claim-type="Currently amended] The method of claim 16, wherein the vector is A / C / N / 53.
[18" claim-type="Currently amended] The method of claim 2, wherein the microtubule blocker is selected from the group consisting of paclitaxel and Taxotere®.
[19" claim-type="Currently amended] 19. The method of claim 18, wherein the microtubule blocker is Taxol®.
[20" claim-type="Currently amended] The method of claim 3, wherein the cells are first contacted with the tumor suppressor nucleic acid or tumor suppressor protein followed by the paclitaxel or paclitaxel derivative.
[21" claim-type="Currently amended] The method of claim 3, wherein the cells are first contacted with the paclitaxel or paclitaxel derivatives followed by the tumor suppressor protein or tumor suppressor nucleic acid.
[22" claim-type="Currently amended] The method of claim 2, wherein the cells are contacted simultaneously with the paclitaxel or paclitaxel derivatives and the tumor suppressor protein or tumor suppressor nucleic acid.
[23" claim-type="Currently amended] The method of claim 1, wherein said cells are neoplastic cells.
[24" claim-type="Currently amended] The method of claim 23, wherein the neoplastic cells are ovarian cancer, pancreatic cancer, non-small cell lung cancer, small cell lung cancer, liver cancer, melanoma, retinoblastoma, breast tumor, colorectal cancer, leukemia, lymphoma, brain tumor, cervical cancer, sarcoma, Prostate tumor, bladder tumor, tumor of retinal endothelial tissue, Wilm's tumor, astrocytoma, glioblastoma, neuroblastoma, osteosarcoma, kidney cancer, and cancer selected from the group consisting of head and neck cancer .
[25" claim-type="Currently amended] The method of claim 1, wherein the tumor suppressor protein or tumor suppressor nucleic acid is dispersed in a pharmaceutically acceptable excipient.
[26" claim-type="Currently amended] The method of claim 3, wherein the paclitaxel or paclitaxel derivative is dispersed in a pharmaceutically acceptable excipient.
[27" claim-type="Currently amended] The method of claim 2, wherein the tumor suppressor protein or tumor suppressor nucleic acid and the paclitaxel or paclitaxel derivative are dispersed in a single composition.
[28" claim-type="Currently amended] The method of claim 1, wherein said contacting step comprises injecting said tumor suppressor protein or tumor suppressor nucleic acid into a tumor.
[29" claim-type="Currently amended] The method of claim 1, wherein said contacting comprises intraarterial injection of said tumor suppressor protein or tumor suppressor nucleic acid.
[30" claim-type="Currently amended] The method of claim 29, wherein the contacting step is selected from the group consisting of intrahepatic injection of the tumor suppressor protein or tumor suppressor nucleic acid for treating liver cancer and intraperitoneal administration of the tumor suppressor protein or tumor suppressor nucleic acid for treating ovarian cancer. How.
[31" claim-type="Currently amended] The method of claim 3, wherein said contacting comprises intratumoral injection of said paclitaxel or paclitaxel derivatives.
[32" claim-type="Currently amended] The method of claim 3, wherein said contacting comprises intravenous injection of said paclitaxel or paclitaxel derivative.
[33" claim-type="Currently amended] The method of claim 1, wherein the contacting step comprises systemic, local, local, topical, intraperitoneal, intrathoracic, oral, buccal, sublingual, intratracheal, transmucosal, bladder, vaginal, uterine, rectal or intranasal administration. How.
[34" claim-type="Currently amended] The method of claim 2, comprising contacting the cells with A / C / N / 53 and paclitaxel.
[35" claim-type="Currently amended] The method of claim 1, wherein contacting the cell with a tumor suppressor protein or a tumor suppressor nucleic acid comprises contacting the cell with the tumor suppressor protein or a tumor suppressor nucleic acid by multiple treatment at intervals of at least about 6 hours each. How to be.
[36" claim-type="Currently amended] The method of claim 1, wherein the method comprises three or more treatments at intervals of about 24 hours.
[37" claim-type="Currently amended] The method of claim 1, wherein the tumor suppressor protein or tumor suppressor nucleic acid is administered once in a total dose of about 1 × 10 ⁹ to about 7.5 × 10 ¹⁵ adenovirus particles, daily divided doses for 5 days, daily for 15 days Administered in a therapeutic regimen selected from the group consisting of divided doses and daily divided doses for 30 days, wherein the paclitaxel or paclitaxel derivative is administered once, for a total of about 75 to about 350 mg / m ² Administered as a treatment regimen selected from daily administration, daily administration for 3 days, daily administration for 30 days, daily continuous infusion for 15 days, and daily continuous infusion for 30 days.
[38" claim-type="Currently amended] 38. The method of claim 37, wherein the method is repeated in two or more cycles.
[39" claim-type="Currently amended] The method of claim 38, wherein the two or more cycles are conducted at three or four week intervals.
[40" claim-type="Currently amended] The method of claim 38, wherein the method is repeated in three cycles.
[41" claim-type="Currently amended] A mammal comprising a first container comprising a tumor suppressor protein or nucleic acid selected from the group consisting of wild type p53 protein or nucleic acid, or retinoblastoma (RB) protein or nucleic acid, and a second container comprising one or more accessory anticancer agents Animal cancer cell or hyperproliferative cell therapy kit.
[42" claim-type="Currently amended] 42. The kit of claim 41, wherein the tumor suppressor nucleic acid encodes a wild type p53 protein.
[43" claim-type="Currently amended] 42. The kit of claim 41, wherein said adjuvant anticancer agent is paclitaxel or a paclitaxel derivative.
[44" claim-type="Currently amended] 42. The kit of claim 41, further comprising instructions describing administration of said tumor suppressor protein or nucleic acid and said adjuvant anticancer agent to inhibit growth or proliferation of said cells.
[45" claim-type="Currently amended] 42. The kit of claim 41, wherein the tumor suppressor protein or tumor suppressor nucleic acid is selected from the group consisting of p53, p110 ^RB and p56 ^RB .
[46" claim-type="Currently amended] 42. The kit of claim 41, wherein said first container contains nucleic acid contained in a recombinant adenovirus vector.
[47" claim-type="Currently amended] 47. The kit of claim 46, wherein said nucleic acid is contained in a recombinant adenovirus vector comprising a partial or complete deletion of Protein IX DNA and comprising a nucleic acid encoding a p53 protein.
[48" claim-type="Currently amended] 48. The kit of claim 47, wherein the deletion of the Protein IX gene sequence ranges from about 3,500 bp from the 5 'virus end to about 4,000 bp from the 5' virus end.
[49" claim-type="Currently amended] The kit of claim 48 further comprising a deletion of the DNA sequences designated E1a and E1b.
[50" claim-type="Currently amended] 47. The kit of claim 46, wherein said recombinant adenovirus vector comprises an adenovirus type 2 late main promoter or human CMV promoter, adenovirus type 2 partial leader cDNA and human p53 cDNA.
[51" claim-type="Currently amended] 47. The kit of claim 46, wherein said vector is A / C / N / 53.
[52" claim-type="Currently amended] A pharmaceutical composition comprising a tumor suppressor protein or tumor suppressor nucleic acid and at least one adjuvant anticancer agent.
[53" claim-type="Currently amended] The composition of claim 52, wherein the adjuvant anticancer agent is paclitaxel or a paclitaxel derivative.
[54" claim-type="Currently amended] The method of claim 52, wherein the tumor suppressor protein or tumor suppressor nucleic acid is selected from the group consisting of a nucleic acid encoding a wild type p53 protein, a nucleic acid encoding a retinoblastoma (RB) protein, a wild type p53 protein, and a retinoblastoma (RB) protein. Composition.
[55" claim-type="Currently amended] The composition of claim 52, wherein the nucleic acid encodes a wild type p53 protein.
[56" claim-type="Currently amended] The composition of claim 52, wherein the nucleic acid encodes the retinoblastoma p110 ^RB or p56 ^RB .
[57" claim-type="Currently amended] The composition of claim 52, wherein the nucleic acid is contained in a recombinant adenovirus vector.
[58" claim-type="Currently amended] 59. The composition of claim 57, wherein said nucleic acid is contained in a recombinant adenovirus vector comprising a partial or complete deletion of Protein IX DNA and comprising a nucleic acid encoding a wild type p53 protein.
[59" claim-type="Currently amended] 59. The composition of claim 58, wherein the deletion of the Protein IX gene sequence ranges from about 3,500 bp from the 5 'virus end to about 4,000 bp from the 5' virus end.
[60" claim-type="Currently amended] 60. The composition of claim 59, further comprising a deletion of the DNA sequences designated E1a and E1b.
[61" claim-type="Currently amended] 58. The composition of claim 57, wherein said recombinant adenovirus vector comprises an adenovirus type 2 late main promoter or human CMV promoter, adenovirus type 2 tripartite leader cDNA and human p53 cDNA.
[62" claim-type="Currently amended] 58. The composition of claim 57, wherein said vector is A / C / N / 53.
[63" claim-type="Currently amended] The composition of claim 53, wherein the paclitaxel or paclitaxel derivative is paclitaxel.
[64" claim-type="Currently amended] A composition comprising a mammalian cancer cell or hyperproliferative cell containing an exogenous tumor suppressor nucleic acid or tumor suppressor protein and paclitaxel or paclitaxel derivatives.
[65" claim-type="Currently amended] 65. The composition of claim 64, wherein said tumor suppressor nucleic acid is a nucleic acid encoding a tumor suppressor protein selected from the group consisting of wild type p53 protein and retinoblastoma (RB) protein.
[66" claim-type="Currently amended] 65. The composition of claim 64, wherein the tumor suppressor nucleic acid encodes a wild type p53 protein.
[67" claim-type="Currently amended] 66. The composition of claim 65, wherein the retinoblastoma protein is p110 ^RB or p56 ^RB .
[68" claim-type="Currently amended] 65. The composition of claim 64, wherein said cells are in a mammal.
[69" claim-type="Currently amended] 65. The composition of claim 64, wherein said cells are neoplastic cells.
[70" claim-type="Currently amended] 70. The method of claim 69, wherein the neoplastic cells are ovarian cancer, pancreatic cancer, non-small cell lung cancer, small cell lung cancer, liver cancer, melanoma, retinoblastoma, breast tumor, colorectal cancer, leukemia, lymphoma, brain tumor, cervical cancer, sarcoma, A composition comprising a cancer selected from the group consisting of prostate tumors, bladder tumors, tumors of retinal endothelial tissue, Wilm's tumor, astrocytoma, glioblastoma, neuroblastoma, osteosarcoma, kidney cancer, and head and neck cancer .
[71" claim-type="Currently amended] Contacting the metastatic cell with a tumor suppressor nucleic acid or a tumor suppressor polypeptide.
[72" claim-type="Currently amended] The method of claim 71, wherein said contacting comprises topical administration of tumor suppressor nucleic acid to trauma.
[73" claim-type="Currently amended] The method of claim 71, wherein the cell is further contacted with one or more accessory anticancer agents.
[74" claim-type="Currently amended] The method of claim 73, wherein said adjuvant anticancer agent is paclitaxel.
[75" claim-type="Currently amended] 74. The method of claim 73, wherein said adjuvant anticancer agent is a microtubule blocker.
[76" claim-type="Currently amended] The method of claim 71, further comprising contacting the cell with a chemotherapeutic agent.
[77" claim-type="Currently amended] 77. The method of claim 76, wherein the chemotherapeutic agent is cisplatin, carboplatin or navelvin.

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同族专利:

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公开号 | 公开日

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CA2282683A1|1998-08-20|

---

AT361668T|2007-06-15|

---

ES2287974T3|2007-12-16|

---

SK285969B6|2007-12-06|

---

WO1998035554A2|1998-08-20|

---

NO993943L|1999-10-15|

---

CN1248731C|2006-04-05|

---

SK112299A3|2000-12-11|

---

WO1998035554A3|1998-11-26|

---

CN1252689A|2000-05-10|

---

JP2001511815A|2001-08-14|

---

HU0004326A3|2003-07-28|

---

NO993943D0|1999-08-17|

---

IL195605D0|2011-08-01|

---

AU6438098A|1998-09-08|

---

DE69837754D1|2007-06-21|

---

EP0969720A4|2004-05-12|

---

EP0969720A2|2000-01-12|

---

KR100620939B1|2006-09-06|

---

NZ337283A|2001-02-23|

---

PL193767B1|2007-03-30|

---

BR9807418A|2002-01-22|

---

PL335334A1|2000-04-25|

---

KR100688409B1|2007-03-02|

---

HU0004326A2|2001-02-28|

---

CZ293399A3|2000-03-15|

---

AU737621B2|2001-08-23|

---

HK1026579A1|2008-02-22|

---

IL131447D0|2001-01-28|

---

DE69837754T2|2008-02-07|

---

EP0969720B1|2007-05-09|

---

KR20050113287A|2005-12-01|

---

CZ298488B6|2007-10-17|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
1997-02-18|Priority to US8/801,285

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1997-02-18|Priority to US8/801,681

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1997-02-18|Priority to US80176597A

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1997-02-18|Priority to US80175597A

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1997-02-18|Priority to US80128597A

---

1997-02-18|Priority to US80168197A

---

1997-02-18|Priority to US3806597P

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1997-02-18|Priority to US08/801,285

---

1997-02-18|Priority to US60/038,065

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1997-02-18|Priority to US8/801,765

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1997-02-18|Priority to US8/801,755

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1997-02-18|Priority to US08/801,681

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1997-02-18|Priority to US08/801,755

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1997-02-18|Priority to US08/801,765

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1997-05-28|Priority to US60/047,834

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1997-05-28|Priority to US4783497P

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1998-02-17|Application filed by 이. 엘. 나가부샨, 리차드 비. 머피, 캔지, 인크.

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1998-02-17|Priority to PCT/US1998/003514

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2000-11-25|Publication of KR20000071185A

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2006-09-06|Application granted

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2006-09-06|Publication of KR100620939B1

优先权:

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申请号 | 申请日 | 专利标题

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US80176597A| true| 1997-02-18|1997-02-18|

---

US80175597A| true| 1997-02-18|1997-02-18|

---

US80128597A| true| 1997-02-18|1997-02-18|

---

US80168197A| true| 1997-02-18|1997-02-18|

---

US3806597P| true| 1997-02-18|1997-02-18|

---

US08/801,285|1997-02-18|

---

US60/038,065|1997-02-18|

---

US8/801,765|1997-02-18|

---

US8/801,755|1997-02-18|

---

US08/801,681|1997-02-18|

---

US08/801,755|1997-02-18|

---

US08/801,765|1997-02-18|

---

US8/801,681|1997-02-18|

---

US8/801,285|1997-02-18|

---

US4783497P| true| 1997-05-28|1997-05-28|

---

US60/047,834|1997-05-28|

---

PCT/US1998/003514|WO1998035554A2|1997-02-18|1998-02-17|Combined tumor suppressor gene therapy and chemotherapy in the treatment of neoplasms|