WO2004050112A1

WO2004050112A1 - Intratumor administration of heat shock protein and its combination tumor therapy with cell toxin

Info

Publication number: WO2004050112A1
Application number: PCT/CN2002/000867
Authority: WO
Inventors: Xuefen Huang
Original assignee: Shanghai Bioworld Information Co., Ltd.
Priority date: 2002-12-04
Filing date: 2002-12-04
Publication date: 2004-06-17
Also published as: AU2002349478A1; CN1717248A

Abstract

The present invention discloses a composition, kit and method for tumor therapy. The method includes administering a heat shock protein (Hsp) or a construct which expresses Hsp into tumor to express Hsp, and administering a cell toxin simultaneously, to kill tumor and induce systemic tumor-specific response.

Description

Combined tumor therapy of intra-tumor administration of heat shock protein and cytotoxic agent Technical field

The present invention generally relates to a method and a kit for applying an Hsp protein, or a construct comprising HSP or co-expressing HSP and a suicide gene, into a tumor, and co-administering a cytotoxic agent to kill the tumor, thereby inducing a systemic anti-tumor response And composition. Background technique

Usually, solid tumors are treated by a combination of surgery, chemotherapy, and radiation. However, these treatments are not ideal because radiotherapy and chemotherapy can cause adverse side effects, and some tumors such as brain tumors cannot be treated surgically. Immunotherapy, including tumor vaccines, is a promising tumor treatment. Because tumors are composed of a population of cloned cells with a high degree of random genetic mutation (estimated 11,000 mutations per tumor cell), antigenic peptides from mutant and shared tumor antigens can potentially induce specific anti-tumor Immunity. However, despite this immunogenicity, tumor cells often cannot stimulate an immune response. Inadequate antigen presentation cells (APC) present antigens, which prevents the immune system from producing an effective antitumor immune response. Therefore, improving the presentation of APCs on tumor antigens is an attractive method to improve the efficacy of tumor vaccines.

Heat shock protein (HSP) can accompany and present a broad spectrum of tumor resistance, and play a key role in the induction of anti-tumor immune responses. HSP forms a complex with the antigen peptide, and through its receptors such as CD91-mediated uptake, it can enter dendritic cells (DC) class I and class II MHC antigen processing pathways, thereby triggering CD8 + and CD4 + T cells Response [Binder, RJ, Blacere, N .., and Srivastava, PK, Heat shock protein-chaperoned peptides but not free peptides introduced into the cytosol are presented efficiently by major histocompatibility complex I molecules, Journal of Biological Chemistry- 276: 17163- 71, 2001; Srivastava, PK Immunotherapy of human cancer: lessons from mice, Nature Immunology. 1: 363-6, 2000.]. HSP also provides innate immunity, which is characterized by the release of cytokines such as IL-12 by dendritic cells, thereby activating natural killer (NK) cells and other immune cells [Multhoff, G., Mizzen, L., Winchester, CC, Milner, CM, Wenk, S., Eissner, G., Kampinga, HH, Laumbacher, B., and Johnson, J. Heat shock protein 70 (Hsp70) stimulates proliferation and cytolytic activity of natural killer cells, Experimental Hematology. 27: 1627-36, 1999]. In addition to directing immune cells to tumors as a cytokine, HSP also confirms the cost Presentation molecules, including B7-1, B7-2 and class II MHC molecules, which pass the maturation signal to DC [A _Sea , A., Kraeft, SK, Kurt-Jones, EA, Stevenson, MA, Chen, LB, Finberg, RW, Koo, GC, and Calderwood, SK HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine, Nature Medicine. 6: 435-42, 2000.].

Several HSP-based vaccines have been developed to enhance anti-tumor responses, including HSP protein vaccines for individual tumors, or antigen-HSP fusion DNA or proteins [Chen, CHT-LW, Chien-Fu Hung, Yanqin Yang, Richard A. Young, Drew M. Pardoll, and TC. Wu Enhancement of DNA vaccine potency by linkage of antigen gene to an HSP70 gene, Cancer Research. 60: 1035-42, 2000]. The HSP protein vaccine has been extensively tested in animals and has recently been tested in clinical trials [Janetzki, S., Schaed, S., Panageas, KS, Wang, S., Williams, L., Meyers, M., Butterworth, L., Livingston, P. 0., Chapman, PB, and Houghton, AN Immunization of cancer patients with autologous cancer- derived heat shock protein gp96 preparations: a pilot study, International Journal of Cancer. 87: 391-8, 2000] Where HSP is purified from tumor cells and then administered back to the same patient. However, this method has many limitations. Because HSP elicits specific immunity against isolated tumors, and because of the unique nature of mutant antigen peptides, they must be generated separately from patient tumor samples, making it difficult for HSP vaccines to establish reproducible treatment standards. In addition, HSP vaccines have difficulty destroying large tumors. Antigen-HSP fusion DNA or protein vaccines can overcome certain limitations, but these methods are not effective because they can only increase the immune response against one or a few common tumor antigens.

Therefore, there is an urgent need in the art to develop new and effective methods for treating tumors. Summary of invention

The object of the present invention is to provide a new method, a kit and a composition for effectively treating tumors. In a first aspect of the present invention, a kit is provided, which contains:

(a) a first composition comprising a pharmaceutically acceptable carrier and an Hsp protein and / or a construct expressing Hsp, said construct comprising a coding sequence of Hsp operably linked to an expression control sequence ;

(b) a second composition comprising a cytotoxic agent.

In a preferred example, the second composition is applied before, at the same time or after the first composition is applied. In another preferred example, the construct is selected from the group consisting of a replication-deficient virus, an expression vector, an RNA molecule, DNA molecules, and combinations thereof.

In another preferred example, the replication-deficient virus is selected from the group consisting of replication-deficient adenovirus, herpes simplex virus (HSV), vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), and combinations thereof.

In another preferred example, the construct further contains a suicide gene operably linked to an expression control sequence. More preferably, the suicide gene is selected from the group consisting of HSV thymine kinase (TK) and cytosine deaminase (CD) genes.

In another preferred example, the HSP is an Hsp selected from the group consisting of: human, Mycobacterium tuberculosis, Mycobacterium leprosy 0¾ Ze rae human trypanosoma (ja; 3osd¾z73 cii / z human malignant

falciparum), non-human primates, mice, and combinations thereof. More preferably, the HSP is selected from the group consisting of HSP70, HSP96, HSP90, HSP20, and combinations thereof.

In another preferred example, the cytotoxic agent is selected from the group consisting of-i). Chemotherapeutic agent: cisplatin, antimetabolite, fluorouracil, methotrexate, aminopterin; ii). Suicide gene toxin agent: GCV , Cyclophosphamide, cytarabine, 5-fluorocytosine; iii). Cytotoxins from plants, fungi and bacteria: Pseudomonas exotoxin, saporin, ricin, white tree Gelonin, diphtheria toxin, ribonuclease, or an expression vector expressing the above cytotoxin;

iv) radioactive isotopes;

V). Inhibitors of hormones / steroids: hydroxyflutamide, letrozole;

vi) a combination of (i) _ (v) above.

In a second aspect of the invention, a composition is provided, which contains

(: 0. · Hsp protein and / or a construct expressing Hsp, said construct containing a coding sequence of Hsp operably linked to an expression control sequence;

(ii) a cytotoxic agent; and

(iii) a pharmaceutically acceptable carrier.

In a third aspect of the present invention, a method for treating a tumor is provided, including steps:

(a) administering a first composition to an individual, the composition comprising a pharmaceutically acceptable carrier, and an Hsp protein and / or a construct expressing Hsp, said construct comprising Hsp operably linked to an expression control sequence Encoding sequence

(b) administering a second composition to an individual in situ or systemically, the composition comprising a cytotoxic agent and a pharmaceutically acceptable carrier, wherein the second composition is before, simultaneously with, or after the first composition Apply.

In a preferred example, the method of applying the first and second compositions includes topical application (such as tumor Intramuscular), mucosal, intravenous or intraperitoneal.

In a fourth aspect of the present invention, there is provided an Hsp protein or an Hsp-expressing construct for use in preparing a medicament for treating tumors, said medicament being administered to a subject intratumorally, and in combination with a cytotoxic agent. Brief description of the drawings

Figure 1. Schematic representation of a plasmid expressing HSP and a plasmid expressing HSV-TK. Among them, the plasmid pCMV-HSP contains HSP70 under the control of the CMV promoter and the SV40 polyadenylation sequence (SV40 poly (A)). The plasmid pCMV-TK contains the HSV-TK gene under the control of the CMV promoter and the SV40 polyadenylation sequence (SV40 poly (A)).

Figure 2. Schematic representation of vectors co-expressing Hsp and TK. The plasmid pCMV-Hsp-TK contains HSP70 and HSV-TK under the control of the CMV promoter and SV40 polyadenylation sequence (SV40 poly (A)), where HSP70 and HSV-TK are linked by IERS sequences.

Figure 3. Schematic representation of replication-deficient adenovirus expressing Hsp70. Replication-deficient (E1 and E3 deletions) Ad-HSP70 contains the human Hsp70 gene under the control of the CMV promoter and the SV40 polyadenylation sequence (SV40 poly (A)) in the E1 region.

Figure 4. Schematic representation of replication-deficient adenoviruses that co-express Hsp70 and TK. Replication-deficient (E1 and E3 deletions) Ad-HSP70 contains the human Hsp70 gene and the HSV-TK gene linked to the IERS sequence under the control of the CMV promoter and SV40 poly (A) in the E1 region.

Figure 5. Schematic representation of a fragment of a replication defective adenovirus insertion gene that co-expresses Hsp70 and TK. Figure 6. Hsp70 and TK protein expression. Hep3B cancer cells were infected with an adenovirus (Ad-Hsp_TK) or an empty vector (Ad-null) that co-expressed Hsp70 and TK. After 48 hours, labeled with ³⁵ S-methionine and immunoprecipitated, SDS-PAGE gel showed high levels of TK and Hsp70 (lanes B and C) protein expression. Ad-null infected cells (lane A) showed no expression of Hsp70 and TK.

Figure 7. Local tumor injection of Ad-Hsp-TK virus inhibits distant tumor growth. CT26 (colon cancer) tumor cells (2xl0 ⁶ ) were subcutaneously inoculated on the right side of the mice. When the tumor grew to a diameter of 3-5 cm, Ad Hsp-TK, Ad-TK, and PBS (5xl0 ⁸ pfu / small Rat) Injected into the tumor once a day for 3 consecutive days. Mice treated with Ad-Hsp-TK and Ad-TK were injected intraperitoneally with GCV (10 mg / g mouse body weight) twice daily for 5 days. After 2 weeks, with the left lxl0 ⁶ CT26 cells inoculated mice. The figure shows that after the mice received Ad-Hsp-TK intratumoral treatment, the growth of re-inoculated tumors was inhibited. Detailed description of the invention

After extensive and intensive research, the inventors discovered that the genetic structure of Hsp protein and / or Hsp expression The combination of the building and the cytotoxic agent can elicit an anti-tumor immune response that suppresses local and metastatic tumors in individuals in need of tumor treatment, thereby effectively treating tumors. The present invention has been completed on this basis. As used herein, the terms "construct" and "genetic construct" are used interchangeably and refer to a nucleic acid molecule containing an Hsp coding sequence, which includes expression vectors, plasmids, viruses (preferably replication defective viruses), RA molecules, DNA molecules . The construct also contains expression control sequences for expressing Hsp and / or suicide genes operatively linked to it. Generally, a construct may contain one or more (e.g., 1-5) Hsp coding sequences and suicide genes. -When multiple Hsp or suicide genes are contained, each Hsp may be the same or different, and the respective killing genes may also be the same or different.

As used herein, the term "replication-deficient virus" is defined as a virus that cannot replicate in tumor cells because of a deletion or mutation in a gene necessary for the replication of certain viruses. Examples of replication-deficient viruses include replication-defective herpes simplex virus-1 (HSV), vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), and adenovirus (Ad).

As used herein, the term "expression vector" may be double-stranded or single-stranded, and may be RNA or DNA. Generally, a nucleic acid contains only one sequence encoding a polypeptide as defined herein, but a nucleic acid may also contain two, three, four, or more sequences each encoding a polypeptide as defined herein.

As used herein, the term "heat shock protein" is defined as a family of highly conserved molecules with ATPase activity. They are found in the compartments of all prokaryotes and most eukaryotic cells. Under stress and non-stress conditions, Hsp expression plays an important role in protein metabolism, including in protein refolding, membrane transport, and degradation of misfolded expression building blocks. Hsp protein preparations, including hsp70 and grp94 / gp96 derived from tumor cells and virus-infected cells, can trigger cellular immunity. HSP proteins can be purified from HSP-expressing transduced cells and tumor cells.

The immunogenicity of Hsp protein preparations is due to the immunogenic peptides that bind to hsp. Hsp accompanies the antigen peptide into the antigen-presenting cells, which may allow the peptide to enter the class I and class II MHC pathways and thus be loaded on the class I and class II MHC molecules, where they are presented to CD8 + cytotoxic T cells and CD4 + T-helper cells. In addition, HSP also provides a "dangerous" signal for the maturation of dendritic cells (DCs), a crucial step in T-cell activation.

The heat shock protein used in the present invention is not particularly limited and may be derived from various sources. Representative examples include human-derived HSPs (including human HSP70, HSP96, HSP90, HSP20, and other human HSPs :), pathogen-derived Psp, including Mycobacterium tuberculosis (^ berci ^^ is into Mycobacterium leprae (He rae ^ Trypanosoma cruzi, Plasmodium falciparum (^ a ^ Oi / iw / H fkZci araW and other pathogens; or HSP derived from other species (including non-human primates, mice, etc.). US Patent 6 , 399,069 (Srivastava et al.) And many other patents have descriptions of heat shock proteins such as hsp70, hsp90, and gp96. In the description, Hsp proteins include not only naturally occurring or wild-type Hsp, but also Hsp variants, especially variants that differ only in conservative substitutions and / or modifications.

As used herein, a "suicide gene" refers to any gene that expresses a cytotoxic molecule or a precursor thereof, and the expression of a suicide gene results in cell death. Representative examples of suicide genes include (but are not limited to): HSV thymine kinase (TK), cytochrome P450 (Manome et al. (1996) Gene Therapy 3: 513-520), human deoxycytidine kinase (Manome et al. (1996) Nature Medicine 2 (5): 567-573) and cytosine deaminase (CD) genes (Dong et al. (1996) Human Gene Therapy 7: 713-720). Cells expressing these genes become sensitive to relatively non-toxic substances such as GCV (HSV-TK), cyclophosphamide (cytochrome P450 2B1), cytarabine (human deoxycytidine kinase), or 5-fluorocytidine Pyrimidine (cytosine deaminase) ".

For the DNA sequences of various Hsp and suicide genes that can be used in the present invention, primers can be synthesized based on the disclosed sequences, and DNA or RNA extracted by conventional methods can be used as a template, or a commercially available plasmid or library containing the relevant gene can be used. As a template, it is obtained by conventional techniques such as PCR or RT-PCR. These genes can also be synthesized directly by artificial methods. In addition, mutations such as conservative substitutions can be introduced into each gene by conventional techniques such as site-directed mutagenesis.

Take HSV-TK as an example, it has been located, cloned and sequenced (EMBL HEHSVLTK, Accession

X03764, EMBL HEHS07, Accession V00466). The HSV-TK gene can be obtained from natural sources, such as the genome of type I or type II herpes simplex virus. HSV-TK can also be obtained from the HSV-TK plasmid HSBV-106 (commercially available from Gibco, Gaithersburg, Md). In addition, the oligonucleotides of HSV-TK can also be artificially synthesized.

As used herein, the definition of the term "Hsp inducing agent" for the induction of the expression of Hsp molecules, for example 15-deoxy-_Δ ¹² ^'14 - or any prostaglandin J Hsp expression inducer.

As used herein, the term "cytotoxic agent" is defined as any chemical agent, biological agent or radioactive substance capable of killing tumor cells. The role of cytotoxic agents is to kill some tumor cells and release tumor antigens. These antigens form complexes with heat shock proteins, effectively stimulating the immune response. Cytotoxic agents include the following types of substances:

i). Chemotherapy agent: cisplatin, antimetabolite, fluorouracil, methotrexate, aminopterin; ii). Suicide gene toxin agent: 9-1, 3-dihydroxy-2-propoxymethylguanine (6 (^), cyclophosphamide, cytarabine, 5-fluorocytosine;

iii). Derived from plant, fungal and bacterial cytotoxins: Pseudomonas exotoxin, saponin, ricin, lutein, diphtheria toxin, and ribonuclease. Or expression vectors (viral vectors, RNA or DNA) expressing the above cytotoxins;

iv) radioactive isotopes: such as _X- rays, γ-rays, and radioisotopes injected into tumors; v). Inhibitors of hormones and steroids: Hormone-dependent cancers can be killed with substances that inhibit hormone function. Because androgens are needed to maintain prostate cancer cells, detached androgens are used to treat prostate cancer patients. Hydroxyflutamide (HF) is used as an antiandrogenic drug to block prostate cancer growth. Letrozole (Letrozole or Femara), which inhibits estrogen biosynthesis, can be used to treat estrogen-dependent breast cancer in women.

vi) a combination of (i)-(V) above.

As used herein, the term "antigen" is defined as a molecule that elicits an immune response. The immune response can involve the production of antibodies, the activation of specific immune-active cells, or both. Antigens can be derived from organisms, protein / antigen subunits, inactivated or inactive whole cells or lysates.

As used herein, the term "cancer" is defined as a malignant cell tumor (tumor) that invades other cells. Examples include

(But not limited to): breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, and lung cancer.

As used herein, the term "expression" is defined as the transcription and translation of a certain nucleotide sequence, driven by a promoter. As used herein, the term "expression control sequence" is defined as the nucleotide sequence required to achieve the expression of a foreign gene, and mainly refers to a promoter.

As used herein, "operably linked to" refers to a condition where certain parts of a linear DNA sequence can affect the activity of other parts of the same linear DNA sequence. For example, if the signal peptide DNA is expressed as a precursor and is involved in the secretion of the polypeptide, then the signal peptide (secret leader sequence) DNA is operably linked to the polypeptide DNA; if the promoter controls the transcription of the sequence, it is operably linked to Coding sequence; if the ribosome binding site is placed in a position that enables it to translate, it is operably linked to the coding sequence. In general, "operably linked" means adjacent, while for a secretion leader sequence it means adjacent in the reading frame.

As used herein, the term "promoter" is defined as a region of a nucleotide sequence that regulates the transcription of a particular nucleotide sequence. The term promoter includes enhancers, silencers, and other cis-acting regulatory elements.

As used herein, the term "T cell" is defined as a cell derived from the thymus that participates in the respective cell-mediated immune response. In short, the present invention provides methods, kits, and compositions that express HSP constructs in tumors or directly inject Hsp proteins and then elicit a systemic anti-tumor response in individuals in need of tumor treatment. In the present invention, by administering (preferably intratumoral injection) a Hsp protein and / or a replication defective adenovirus or expression vector expressing HSP (more preferably, co-expressing HSP and suicide genes) and co-administering a cytotoxic agent, HSP is expressed in local tumors and elicits antitumor immune responses in patients that inhibit local and metastatic tumors.

In a preferred example, a replication defective adenovirus (Ad) expressing human HSP70 is produced. In the practice of this invention In this case, the therapy of directly applying HSP-expressing adenovirus to tumors (with cytotoxic agents) can induce specific anti-tumor immunity, resulting in the destruction of local and metastatic tumors.

In another preferred embodiment of the present invention, a therapy is also provided in which an adenovirus co-expressing HSP and a suicide gene (such as TK) is directly administered to a tumor, and then GCV is administered. The therapy can also induce specific killing effects on infected tumor cells and immunity against tumor antigens, leading to the destruction of local and metastatic tumors.

In the present invention, HSP can also be expressed using viral or non-viral vectors or DNA or RNA, or HSP and suicide genes can be co-expressed. The therapy of the present invention can be used not only in combination with other forms of immunotherapy, such as the administration of cytokines and traditional Chinese medicine, but also in combination with other therapies such as radiation therapy.

In the present invention, it is also Hsp expression construct / Hsp protein inducer and Hsp (e.g., 15-deoxy - prostaglandin J - Δ ^12, ¹⁴⁾ administered together.

In a preferred example, the suicide gene HSV-TK is used to prepare adenoviruses that co-express HSP and TK to enhance immune response. A cytotoxic agent, 9-1,3-dihydroxy-2-propoxymethylguanine (GCV) was used to kill TK-expressing cells.

In a preferred example, the cytotoxic agent cisplatin is used to induce the killing of local tumor cells to enhance the release of tumor antigens and the immune response. Alternatively, any chemical and biological molecule capable of killing tumor cells can be used in the therapy.

The kit or composition of the present invention generally comprises a Hsp protein and / or a construct expressing HSP or co-expression and suicide genes, and a cytotoxic agent, which can destroy tumors and release tumor antigens, and induce a systemic anti-tumor immune response.

In a specific example, the replication defective virus is an adenovirus. Replication-deficient adenoviruses, such as those lacking the E1 and E3 regions, have been widely used in animal models and clinical trials to express genes related to tumor therapy. In a specific example, the replication-deficient adenovirus contains a nucleic acid encoding human hsp70, or human Hsp70 and HSV-TK, which is linked to the IRES sequence and under the transcriptional control of a promoter in the replication-deficient adenovirus.

In a specific example, encoding human hsp70 is under the transcriptional control of a promoter in an expression plasmid. A "promoter" refers to a DNA sequence that is recognized by a cell's synthetic mechanism or an introduced synthetic mechanism and is required to initiate specific transcription of a gene. Most of the knowledge of promoter tissue structure comes from the analysis of several viral promoters, including the promoters of CMV, HSV thymine kinase (tk) and SV40 early transcription units. These studies, coupled with recent work, have shown that promoters consist of discrete functional modules, each consisting of approximately 7-20 bp of DNA and containing one or more recognition sites for transcriptional activator or repressor proteins. Other promoter elements, enhancers, regulate the frequency of transcription initiation. Generally, they are located in the region of 30-110 bp upstream of the start site, although some promoters have recently been shown to contain functional elements located downstream of the start site. The spacing between promoter elements is flexible, so the promoter function is retained when each element is reversed or moved relative to the other. The tk promoter In this case, the interval between promoter elements can be increased to within 50 bp without any decrease in activity. Depending on the promoter, it has been shown that the elements can work together or independently to activate transcription.

The promoter may be a promoter that is naturally linked to a gene or a sequence, such as a promoter that can be obtained by isolating a 5 'non-coding region located upstream of a coding region and / or an exon. Such promoters are called "endogenous". Similarly, an enhancer may be an enhancer naturally associated with a nucleic acid sequence, located downstream or upstream of the sequence. Alternatively, certain benefits can be obtained by placing a nucleic acid coding region under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally linked to a nucleic acid sequence in its natural environment. Recombinant or heterologous enhancers also refer to enhancers that are not normally linked to a nucleic acid sequence in the natural environment. These promoters or enhancers may include promoters or enhancers of other genes, promoters or enhancers isolated from other prokaryotic, viral or eukaryotic cells, and promoters or enhancers that are not "naturally occurring" (I.e. containing different elements of different transcriptional regulatory regions, and / or mutant forms that alter expression). In addition to the synthetically generated promoter and enhancer nucleic acid sequences, recombinant cloning and / or nucleic acid amplification techniques (including PCR) can be used to generate these sequences. In addition, control sequences for transcription and / or expression of sequences found in nonnuclear organelles such as mitochondria and chloroplasts can also be used, which is also within the concept of the present invention.

A representative promoter sequence in the present invention is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that is capable of driving high-level expression of any nucleic acid sequence to which it is operatively linked. However, other constitutive promoter sequences can also be used, including (but not limited to): simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat Sequence (LTR) promoter, Moloney virus promoter, avian leukocyte virus promoter, Epstein Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoter, including (but not limited to): actin Promoter, myosin promoter, hemoglobin promoter, and muscle creatine promoter. Furthermore, the invention is not limited to using only constitutive promoters. Inducible promoters are also part of the inventive concept. The use of an inducible promoter in the present invention provides a molecular switch that can turn on the expression of a nucleic acid sequence that is operatively connected to it when expression is needed, or turn off the expression when expression is not needed. Examples of inducible promoters include (but are not limited to): metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter. In addition, the invention includes the use of a tissue-specific promoter that is active only in the desired tissue. Tissue-specific promoters are well known in the art and include, but are not limited to, HER-2 promoter, E2F promoter, insulin promoter, and PSA-related promoter sequences.

For Hsp expression, a polyadenylation signal is usually included in order to properly polyadenylate the transcript. The nature of the polyadenylation signal is believed to be not critical to the successful implementation of the invention, and any such sequence can be employed. Preferred examples include SV polyadenylation signal, LTR polyadenylation signal, and / or bovine growth factor Daughter polyadenylation signals, these signals are convenient and can work well in different target cells. Also useful as an expression cassette element are transcription termination sites. These elements can be used to enhance the level of information and / or reduce the number of readings beyond the expression cassette into other sequences.

It is worth noting that in the present invention, for correct protein expression, it is not necessary to integrate a replication-deficient viral vector into the host cell's genome. Instead, the expression vector may be present in the desired cell as an episome. For example, there are certain cell types in which expression vectors do not require replication to express the desired protein. These cells are those that normally do not replicate, such as muscle cells, but are still fully capable of expressing genes. An expression vector can be introduced into non-dividing cells and express the encoded protein without the expression vector replicating.

In a specific example, a method of killing a local tumor and inducing an immune response includes the steps of: directly or indirectly administering a replication-deficient virus expressing Hsp to a tumor of an organism. Numerous studies have shown that the anti-tumor immune response can be enhanced by co-administration of cytokines or vectors expressing cytokines. Those skilled in the art will readily recognize that the nucleotide sequence of cytokines and the nucleotide sequence of HSP can be integrated into one expression vector, so that it is not necessary to use two independent vectors. '

Those skilled in the art will recognize that the replication-deficient virus of the present invention is useful for tumor treatment. For tumor treatment, the skilled artisan will know that the replication defective virus used must contain a Hsp gene operably linked to a promoter. If appropriate, the carriers can be formulated as solid, semi-solid, liquid or gaseous preparations by methods known in the art for each route of administration. Means known in the art can be used to prevent the composition from being released and absorbed before it reaches the target organ, or to ensure a timed release of the composition. The pharmaceutically acceptable form chosen should not render the composition of the invention ineffective. In the pharmaceutical dosage form, the composition may be used alone, or suitably combined or combined with other pharmaceutically active compounds. A sufficient amount of virus containing a therapeutic nucleic acid sequence must be administered to provide a pharmacologically effective amount of a gene product. Replication-deficient viruses expressing heat shock proteins can be administered alone or formulated with a variety of other adjuvants. Replication-deficient viruses expressing heat shock proteins can be administered topically, mucosally, intravenously or intraperitoneally.

In addition, the actual dosage and regimen may vary depending on whether the composition is administered in combination with other pharmaceutical compositions, or on individual differences in pharmacokinetics, drug configuration, and metabolism. In addition, the amount of vector added to each cell will vary with the length and stability of the therapeutic gene inserted into the vector and the nature of the sequence. Specifically, it is a parameter that needs to be determined experimentally, and can vary with non-factors that are not related to the nature of the method of the present invention. Those skilled in the art can easily make necessary adjustments according to the urgency of the specific situation.

Generally, the amount of Hsp protein administered is 1-200 ug / mm ³ tumor, preferably 5- 100 ug / mm ³ tumor. Expression constructs administered in an amount of Hsp 10 ³ - ¹⁰¹¹ Hsp coding sequence copies / mm ³ of tumor, preferably ¹ × ¹⁰ ⁴ -1 × ^{10 9} Hsp coding sequence copy / mm ³ tumor, more preferably 1 × ¹⁰ 5-1x10 ¹¹ Hsp coding sequence copy / mm ³ tumor. In addition, the administration amount can also be expressed as "pfu / tumor". Adenovirus expressing Hsp example, the administration of an amount of lxlO ⁷ - 5xl0 ¹³ pfu / tumor, preferably 5xl0 ⁷ - lxl0 ¹² pfu / tumor.

As for the application amount of the cytotoxic agent, they can be used in their usual amounts. It is usually 0.01-200 mg / g tumor, or 0.001--50 mg / kg body weight. For example, the amount of cisplatin is 0.2-2 mg / g tumor, preferably 0.5-1.5 mg / g tumor. The amount of GCV used is 5-50 mg / kg body weight, preferably 20-50 mg / kg body weight.

Although these substances may be administered alone, they are preferably administered in the form of a pharmaceutical preparation. The formulations of the invention include at least one active ingredient as described above, and one or more pharmaceutically acceptable carriers, and optionally other therapeutic ingredients. The carrier must be "pharmaceutically acceptable", that is, it must be compatible with other ingredients in the formulation and not harmful to the recipient, such as liposomes. Suitable liposomes include, for example, lipids containing positively charged lipid N-[1-(2,3-dioleoyloxy) propyl] -N, N, N-triethylammonium (DOTMA) Body, liposomes containing dioleosylethanolamine (DOPE), and 3β- [N-(N ', N'-dimethylaminoethane)-carbamoyl] cholesterol (DC- Choi) Of liposomes.

The formulations include formulations suitable for oral, rectal, nasal, topical (including oral and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) administration. The formulations may conveniently be presented in unit dosage forms and may be prepared by any method well known in the pharmaceutical arts. These methods include the steps of: mixing the active ingredient with a carrier as one or more accessory ingredients. Generally, formulations are prepared by intimately mixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The preparations suitable for oral administration of the present invention may exist as independent units such as hard capsules, capsules or tablets, each containing a predetermined amount of active ingredient; or as a powder or granule; or as a solution in an aqueous liquid or a non-aqueous liquid Or suspension; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be in the form of a pill, sugar, or paste.

The medicament of the present invention can be administered to mammals including humans by any route suitable for the condition to be treated. Suitable routes include oral, rectal, nasal, topical (including intratumor, oral and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and epidural) administration. Intratumoral administration is preferred. It should be understood that the preferred route will vary depending on, for example, the condition of the recipient and the type to be treated. Therefore, existing tumors can be treated systemically, or a route to deliver vectors or cells directly to the lesion can be chosen. The purpose of preventative treatment is to stimulate protective immunity of tissues that may be affected by the target tumor.

In a preferred example, the immunomodulatory effect of the local HSP expression caused by the Hsp-expressing construct is combined with the tumoricidal effect of the cytotoxic agent, so that not only tumor antigens are effectively presented to each of them by forming a complex with Hsp This immune cell also provides a local danger signal to the organism. such, Inducing local anti-tumor immune response and systemic anti-tumor activity against metastatic tumors in patients, thereby achieving the effect of effective treatment of tumors. The present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples are generally based on conventional conditions, such as Sambrook et al., Molecular Cloning: The conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions Conditions recommended by the manufacturer. Example 1

Preparation of a replication-deficient adenovirus construct expressing Hsp70

See Figure 1 and Figure 3. The E1 region of the replication-deficient adenovirus vector expressing Hsp70 was replaced by the Hsp70 expression cassette, which contains the complete coding sequence of human heat shock protein 70 (HSP70). The coding sequence is driven by the CMV-IE promoter and the transcription direction is parallel. For adenovirus El 0RF transcription direction.

Ad_HSP70 was produced by cloning human HSP70 in the form of a HI- Seal fragment obtained from plasmid pH2.3 (ATCC 57494, American Type Culture Collection (ATCC)) into the adenovirus shuttle plasmid pAVC3 [Lozier, JN, Yankaskas, JR, Ramsey, WJ, Chen, L., Ber Schneider, H., and Morgan, RA Gut epithelial cells as targets for gene therapy of hemophilia, Human Gene Therapy. 8: 1481-90, 1997.] point. In the subsequent steps, pAVC3. HSP70 was obtained as described previously [Prevec, L. and Graham, FL Methods for construction of adenovirus vectors, Molecular Biotechnology. 3: 207-20, 1995]. Ad-HSP70 was rescued by homologous recombination of pJM17 (Quantum Biotechnologies, J2C.) And pAVC3. HSP70 in 293 cells. Ad. HSP70 was propagated in 293 cells, purified by 2 rounds of CsCl density centrifugation, and 1500 mL of PBS containing 1 mmol / L MgCl tl 10% glycerol at 4 was used. C. Dialysis was performed 4 times (1 hour each) in a Slide-A-Lyzer box (Pierce), and then stored at -80 ° C. The concentration of the virus was determined by measuring the absorbance at 260 nm, and the titer was estimated by plaque analysis on 293 cells. The virus titer is 2-8xl0 ^1Q plaque-forming units (pfu) / ml, and the particle: plaque ratio is about 30-80: 1. The presence of replicable adenovirus in the plaque-purified Ad. HSP70 preparation was evaluated by infection of A549 cells. No lysis of the indicator cells occurred 21 days after infection, excluding the possibility of existence. Example 2

Preparation of a replication-deficient adenovirus construct expressing TK See Figure 1. A replication-deficient adenovirus construct expressing TK was prepared in the same manner as in Example 1. The difference is that the Hsp70 expression cassette is replaced with a TK expression cassette.

The E1 region of the replication-deficient adenovirus vector expressing TK was replaced by the TK expression cassette, which contained the complete coding sequence of HSV-TK, which was driven by the CMV-IE promoter and had a transcription direction parallel to the adenovirus El 0RF Transcription direction.

The HSV-TK gene was obtained by PCR amplification using Ad-TK (Chen S. et al. PNAS 91: 3054, 1994) containing the TK gene as a template. Primer sequence: upstream primer: ACT GCG GCC GCA GCT TCG TAC CCC TGC (SEQ ID NO: 1), downstream primer: AGT TCT AGA CTC GAG TCA GTT AGC CTC CCC CAT (SEQ ID NO: 2). A PCR instrument from PE company was used. The reaction conditions were: the final primer concentration of each reaction was 30 pM, dNTP was 100 mM, template DNA was 100 ng, and Taq DNA polymerase was 2.5 units. Other reaction conditions were performed according to the instructions in the GeneAmp DNA Amplification Kit. The total volume of each reaction was 100 microliters. Each reaction mixture was covered with 75 microliters of mineral oil. Amplification was performed for a total of 30 cycles, each cycle including a denaturation step of 92 ° C for 1 minute, an annealing step of 50 ° C for 1 minute, and an extension step of 72 ° C for 2 minutes. After the reaction was completed, the obtained product was purified using Qiagen's PCR product purification kit. The HSV-TK gene amplified by PCR was 1132 bp and contained Nhel and Xhol loci. The gene was double digested with Nhel and Xhol, and then cloned into pRc / CMV vector (InVitrogen, Inc.) and Ad- Easy vector (Quantum Biotechnologies, Inc.). Example 3

Construction of replication-deficient adenoviruses that co-express HSP and TK

See Figure 2 and Figure 4. An easy (AdEasy) adenovirus production method is used for the construction and production of HSP and TK adenoviruses. The human inducible HSP70 cDNA was obtained by PCR amplification from the plasmid pH2.3 (ATCC 57494) in Example 1 as a template. The upstream primer sequence is: GGT ATG GAA GAT CCC TCG AGA TC (SEQ ID NO: 3), and the downstream primer sequence is: TA CTA ATC TAC CTC CTC AAT GGT GGG (SEQ ID NO: 4). The reaction conditions were the same as those in Example 2 using a PCR instrument from PE. After the reaction was completed, the obtained product was purified using Qiagen's PCR product purification kit. The DNA fragments of the TK gene and the HSP70 gene prepared as in Example 2 were cloned into the Nhel and Xhol sites in the multicloning site in the Ad- Easy vector (Quantum Biotechnologies, Inc.), respectively. Recombinant Ad-HSP-TK virus was generated by Ad- Easy kit (Quantum Biotechnologies, Inc.).

The connection diagram of the CMV promoter, Hsp coding sequence, IRES and TK in the virus Ad_HSP-TK is shown in Figure 5. See SEQ ID NO: 5 for the specific sequence. Example 4

Replication-Defective Ad-Hsp and Ad-Hsp-TK Expression Hsp

Human tumor cells Hep3B were seeded on 6-well plates and infected with Ad-HSP, Ad-HSP-TK or Ad-negative control with a multiplicity of infection (MOI) of 10. HSP70 virus was detected by radiolabeling and immunoprecipitation / SDS-PAGE 48 hours after virus infection.

As shown in Fig. 6, there were high levels of HSP70 and TK expression in cells infected with Ad-HSP-TK (lanes B and 0, and high levels of HSP70 expression in cells infected with Ad-HSP70 (not shown). However, only low levels of HSP70 expression was detected in Ad-negative control infected cells (lane A). Example 5

Ad-HSP-TK and cytotoxic agents (GCV) inhibit distant tumor growth

The effect of Ad-HSP-TK on CT26 tumors was further evaluated on the same BALB / c mice. BALB / c is from Charles River (Wilmington, MA, USA). ₀ All animal experiments were approved by the Animal Care and Use Committee. The animals were divided into 3 groups, the first and second groups were used as the experimental group, and the third group was used as the control group. Each group of 6 to 8 mice was injected subcutaneously with CT26 tumor cells (2 × ^{10 6} ) in the ribs of the mice.

When the subcutaneous tumor growth to a maximum of 5 mm in diameter, the first set of each animal (right) tumor inoculation ^{Ad- HSP- TK (5xl0 8 PFU)} , once a day for three consecutive days. And GCV (10mg / g mouse body weight) was injected intraperitoneally twice a day for 5 days. The second set of intratumoral inoculation Ad- TK right of each animal (5xl0 ⁸ PFU), once a day for three consecutive days, and the same treatment with GCV. The third group of mice was inoculated with PBS intratumorally on the right side of each animal.

Tumor volume was measured with a caliper. If the mouse was dying or its tumor diameter was more than 12 mm, the tumor was surgically removed. After 2 weeks, the mice were seeded with 1 × 10 ⁶ CT26 cells on the left side. Then observe the growth of the inoculated tumor.

Ad-HSP-TK intratumoral injection caused a very significant antitumor effect, and tumor growth on the non-injection side of the symmetrical end was significantly suppressed. However, injection of Ad-TK or PBS had no effect on tumors on the non-injection side (Figure 7). These results indicate that recombinant Ad-HSP-TK can stimulate a strong immune response by expressing HSP70 / TK, thereby inhibiting the growth of distant tumors. Example 6

Inhibitory effect of Ad-HSP and cytotoxic agent (cisplatin) on distant tumor growth in a mouse model

The murine colorectal cancer cell line CT26 has been found to be sensitive to adenovirus infection. The cell is low in immunogenicity and It does not induce detectable tumor-specific CTL.

When the maximum diameter of the subcutaneous tumors on both sides reached about 0.5 cm, on day 0, mice were inoculated unilaterally with Ad-Hsp or Ad-negative control vector (lxl0 ⁶ PFU) on the right tumor. , Or PBS buffer (blank control), and then a second inoculation (6 animals per group) and a dose of 2 mg / kg cisplatin were used on day 7. Isogenic BALB / c mice containing CT26 tumors on both sides were vaccinated on one side of the tumor, while tumors on the other side (contralateral) were not vaccinated. In the immunization with Ad-Hsp, compared with the blank control, both the vaccinated tumor and the non-vaccinated contralateral tumor showed a significant decrease in tumor growth. Ad-Hsp immunization can inhibit tumor growth on both sides more effectively than Ad-negative control vector inoculation. Example 7

Inhibitory effect of HSP protein and cytotoxic agent (cisplatin) on distant tumor growth in a mouse model

The experiment of Example 6 was repeated, except that the purified Hsp70 protein (20 ug / mm ³ tumor) was used instead of Ad-Hsp.

Compared with the blank control (PBS buffer), when immunized with Hsp protein, both the inoculated tumor and the uninoculated contralateral tumor showed a significant decrease in tumor growth.

Claims

Rights request

1. A pill box, characterized in that it contains:

(a) a first composition comprising a pharmaceutically acceptable carrier and a Hsp protein and / or a construct expressing Hsp, said construct containing a coding for Hsp operably linked to an expression control sequence

5 sequence;

(b) a second composition comprising a cytotoxic agent and a pharmaceutically acceptable carrier.

The kit according to claim 1, wherein the second composition is administered before, simultaneously with, or after the first composition is administered.

3. The kit of claim 1, wherein the construct is selected from the group consisting of a replication-deficient 10 virus, an expression vector, an RNA molecule, a DNA molecule, and a combination thereof.

4. The kit according to claim 3, wherein the replication-deficient virus is selected from the group consisting of replication-deficient adenovirus, herpes simplex virus (HSV), vesicular stomatitis virus (VSV), and Newcastle disease virus (NDV) and combinations thereof.

The kit according to claim 1, wherein the construct further comprises a suicide gene operably linked to 15 sequences of expression control sequences.

6. The kit according to claim 1, wherein the HSP is an Hsp selected from the group of organisms: human, Tuberculosis, Mycobacterium leprae (Leprae), Tryanosoma cruzi, H malaria, A Plasinoc I falciparum), non-human primates, mice, and combinations thereof.

7. The kit of claim 1, wherein the HSP is selected from the group consisting of: HSP70, HSP96, HSP90, 20 HSP20, and combinations thereof.

8. The kit of claim 1, wherein the cytotoxic agent is selected from the group consisting of: i). Chemotherapeutic agent: cisplatin, an antimetabolite, fluorouracil, methotrexate, aminopterin Ii) Suicide gene toxin agents: GCV, cyclophosphamide, cytarabine, 5-fluorocytosine; ii i). Cytotoxins from plants, fungi and bacteria: Pseudomonas exotoxin, soap Oxytocin, ricin 25 protein, lutein, diphtheria toxin, ribonuclease, or expression vector expressing the above cytotoxin; iv) radioactive isotopes;

V). Inhibitors of hormones / steroids: hydroxyflutamide, letrozole;

vi) a combination of (i)-(V) above.

9. The kit of claim 5, wherein the suicide gene is selected from the group consisting of: HSV chest 30 adenine kinase (TK), cytochrome P450, human deoxycytidine kinase, and cytosine deamination Enzyme (CD) gene.

10. A composition, characterized in that it contains:

(i) a Hsp protein and / or a Hsp-expressing construct expressing a Hsp construct, said construct containing a coding sequence for Hsp operably linked to an 'expression control sequence;

(ii) a cytotoxic agent; and

35 (iii) a pharmaceutically acceptable carrier.