WO2003031602A1 - Micro-organismes oncolytiques exprimant les proteines hsp, et leurs utilisations - Google Patents

Micro-organismes oncolytiques exprimant les proteines hsp, et leurs utilisations Download PDF

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Publication number
WO2003031602A1
WO2003031602A1 PCT/CN2001/001616 CN0101616W WO03031602A1 WO 2003031602 A1 WO2003031602 A1 WO 2003031602A1 CN 0101616 W CN0101616 W CN 0101616W WO 03031602 A1 WO03031602 A1 WO 03031602A1
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tumor
cells
antigen
oncolytic
microorganism
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PCT/CN2001/001616
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English (en)
French (fr)
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Siyi Chen
Fang Hu
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Hangzhou Conquer Biotech Co., Ltd.
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Priority to US10/472,798 priority Critical patent/US20040146488A1/en
Priority to EP01274525A priority patent/EP1435387A4/en
Priority to JP2003534572A priority patent/JP3952201B2/ja
Publication of WO2003031602A1 publication Critical patent/WO2003031602A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/761—Adenovirus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/763—Herpes virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16632—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the field of biomedical tumor immunotherapy. Specifically, the present invention relates to a new method for tumor immunotherapy, that is, a tumor patient's own tumor antigen is effectively used to simultaneously activate the patient's immune response, thereby suppressing tumor in situ while suppressing tumor metastasis and treating metastatic tumor method.
  • the invention also relates to an oncolytic microorganism, a composition containing the oncolytic microorganism, and the use of the oncolytic microorganism or composition for treating tumors or preparing a medicament for treating tumors.
  • viruses that can selectively infect tumor cells, such as herpes simplex virus iHerpesvir such as HSV-1 and G207, adenovirus such as ONYX-015 (also known as dll520), CN706, and CN787 et al., Newcastle disease virus (Newcarf / e ⁇ ⁇ «) as 73- ⁇ like, vesicular stomatitis virus (Vesicu vims, reovirus (eovz ⁇ ) as Reolysin 1 -?.
  • herpes simplex virus iHerpesvir
  • adenovirus such as ONYX-015 (also known as dll520), CN706, and CN787 et al.
  • Newcastle disease virus Newcarf / e ⁇ ⁇ «
  • vesicular stomatitis virus Vesicu vims
  • reovirus eovz ⁇
  • Cancer vaccination can stimulate the body's anti-tumor immune response, which will be a major advance in tumor treatment research.
  • tumor antigens are weak immunogens, that is, they cannot elicit a sufficient immune response against tumor antigens.
  • cancer vaccines are mainly used to treat tumors by: immunizing with isolated tumor-specific antigens; or treating tumor cells by fusing tumor cells with dendritic cells in vitro.
  • tumor-specific antigens because there are individual differences in tumor antigens among different patients; and the tumor development of the same patient is different
  • the specific antigens expressed by tumor cells are not the same; even in a certain stage of tumor development of the same patient, multiple tumor antigens exist in the tumor cells. Due to these limitations, there are many shortcomings in the current treatment of tumors with cancer vaccines: there is no antigen against multiple tumors, and corresponding tumor antigens must be designed for each patient.
  • cancer vaccine therapy is very expensive and not universally applicable. Therefore, until now, there is still a lack of a highly effective, low-toxicity, and convenient drug-administering tumor treatment substance, which can induce an effective anti-tumor immune response in the body after being administered to a tumor patient, thereby transforming the tumor, including metastatic The tumor is effectively killed. Summary of invention
  • the purpose of the present invention is to find a new method for tumor immunotherapy, in order to effectively use the tumor antigen of a tumor patient to simultaneously activate the immune response in the patient, thereby suppressing tumor in situ and suppressing tumor metastasis.
  • the present invention provides an oncolytic microorganism that can selectively grow in tumor cells and simultaneously express a protein that can adhere to tumor antigens and can be recognized by antigen-presenting cells (sometimes also hereinafter It is simply called "functional protein").
  • the present invention also provides an antigen presenting cell transformed with a vector containing a DNA sequence encoding a functional protein or a fragment thereof.
  • the present invention further provides a microbial composition, including:
  • an oncolytic microorganism that can selectively grow in tumor cells to lyse tumor cells; and b) a vector that expresses proteins and fragments thereof that adhere to tumor cell antigens and are recognized by antigen presenting cells;
  • the microorganism or vector described therein optionally further contains a DNA sequence encoding an immune enhancing molecule.
  • the present invention also provides a pharmaceutical composition, which mainly contains:
  • a oncolytic microorganism that is, a microorganism that can selectively grow in tumor cells
  • the present invention also provides an immunotherapeutic agent for treating tumors, which comprises the oncolytic microorganisms described above, or the antigen-presenting cells, or the microbial composition or the medicine. combination.
  • the present invention also provides a method for treating tumors, which comprises transforming a patient's antigen extract with a vector containing DNA encoding a protein or a fragment thereof that is capable of adhering to tumor cell antigens and recognized by antigen-presenting cells. Showing cells.
  • the present invention also provides a method for treating tumors, which comprises administering an immunologically effective amount of the oncolytic microorganisms, the antigen-presenting cells, the microbial composition, and The pharmaceutical composition or the immunotherapeutic agent.
  • an immunologically effective amount of an oncolytic microorganism carrying a functional protein gene is administered to a tumor patient.
  • the oncolytic microorganism carrying a functional protein gene is a recombinant oncolytic microorganism capable of expressing a protein or a fragment thereof capable of adhering to a tumor antigen and recognized by an antigen presenting cell; or an oncolytic microorganism and containing A plasmid encoding DNA of the functional protein or a fragment thereof; or an oncolytic microorganism and a replication-defective vector containing DNA encoding the functional protein or a fragment thereof.
  • the oncolytic microorganisms refer to viruses or bacteria that can selectively grow and lyse tumor cells in tumor cells.
  • Oncolytic microorganisms replicate in tumor cells in large numbers to lyse tumor cells, kill the tumor cells, release the patient's own tumor antigen, amplify danger signals, and synchronize the functional protein itself or its complex with the attached tumor antigen It stimulates and activates antigen-presenting cells, promotes the timely presentation of antigens, stimulates the patient's own immune system, and thereby kills local and metastatic tumors.
  • a patient's antigen-presenting cells are transformed with a vector containing DNA encoding a functional protein or a fragment thereof.
  • the protein is a protein that can adhere to tumor antigens and be recognized by antigen-presenting cells.
  • Antigen-presenting cells transformed by a vector containing a DNA encoding a functional protein or a fragment thereof are more sensitive to the released antigen.
  • the functional protein released by the presenting cells captures the antigen and submits it to the antigen-presenting cell, further enhancing the immune signal and activating body immunity. reaction.
  • the purpose is to induce specific anti-tumor immune response in tumor patients while killing tumor cells, to inhibit tumor metastasis, and to kill the metastatic distal tumor cells.
  • a tumor patient is administered a combination of an oncolytic microorganism capable of selectively growing in tumor cells and the above-mentioned functional protein or a variant thereof.
  • Oncolytic microorganisms replicate in tumor cells in large numbers to lyse the tumor cells, kill the tumor cells, and release the patient's own tumor antigens.
  • the functional protein itself and the antigen-presenting cells are administered to the patient simultaneously with the oncolytic microorganisms. of Affinity, its large appearance around the antigen presenting cells will also amplify the danger signal, activate the presenting cells more, stimulate the patient's own immune response, and thereby kill local and metastatic tumors.
  • the oncolytic microorganisms provided by the present invention are preferably viruses or bacteria that selectively grow in tumor cells.
  • viruses examples include adenovirus, herpes simplex virus, vesicular stomatitis virus, Newcastle disease virus, reovirus or other viruses that can selectively grow in tumor cells.
  • viruses are adenovirus, herpes simplex virus, vesicular stomatitis virus, Newcastle disease virus, reovirus or other viruses that can selectively grow in tumor cells.
  • An example of a bacterium is Salmonella typhi
  • the protein or functional protein capable of adhering a tumor antigen and being recognized by the antigen presenting cell is preferably a heat shock protein.
  • Heat shock proteins are derived from mammals and pathogenic microorganisms.
  • the mammals include, but are not limited to, humans, non-human primates, and rodents; the pathogenic microorganisms include, but are not limited to, M tuberculosis, M leprae, M.
  • the oncolytic microorganism containing the functional protein gene may further contain a DNA sequence encoding an immune-enhancing molecule, such as cells including IL-2, IL-12, TNF, IFN, G-CSF, GM-CSF, and chemokines. Factors, immune co-stimulatory molecules such as B7 and so on.
  • the oncolytic microorganism, antigen presenting cell, oncolytic microorganism composition, pharmaceutical composition or immunotherapeutic agent of the present invention can be used alone or in combination with other forms of immunotherapeutic agents, including cytokines, immune adjuvants and Traditional Chinese medicine.
  • the oncolytic microorganism, its composition or pharmaceutical composition in the present invention achieves the purpose of inhibiting or eliminating metastatic tumors through one or more of the following pathways: a) A lytic agent capable of selectively replicating in tumor cells Tumor microorganisms dissolve tumor cells, release tumor antigens, and synchronously express proteins that can adhere to tumor antigens and be recognized by antigen-presenting cells present the tumor antigens in real time, stimulating the antigen-presenting cells (mainly the dendritic state of the user) Cells), which further stimulates the activity of immune effector cells such as K cells, CTL cells, and Th cells, and activates the body's immune response; b) proteins that can adhere to tumor antigens and are recognized by antigen presenting cells have affinity with antigen presenting cells And its presence in large numbers around the antigen presenting cells will also amplify the danger signal and activate the presenting cells more; and / or c) contain a protein that encodes a tumor antigen and is recognized by the antigen presenting cells
  • the purpose is to induce specific anti-tumor immune response in tumor patients while killing tumor cells, to inhibit tumor metastasis, and to kill the metastatic distal tumor cells.
  • the present invention progresses in that the present invention overcomes the main drawbacks of using "cancer vaccine" for treatment in the prior art due to individual differences in tumor antigens: for example, a disease must be obtained Various defects of human autologous tumor cells that are cultured in vitro and a series of experimental operations.
  • the invention can present the tumor-specific antigen released after lysis of the patient's own tumor cells in real time, and can stimulate the body to be effective against its own specific tumor antigen regardless of the stage of development of the tumor cells and whether the types of tumor antigens are the same. Immune response.
  • the invention realizes the real-time and dynamic processing and presentation of its own specific tumor antigen in vivo in different individuals by using the individual's own antigen-presenting cells, thereby stimulating the specific immune response against the respective different tumor antigens.
  • Simple and effective treatment for example, in the present invention, tumor cells are lysed by an oncolytic microorganism, and at the same time, the released tumor antigen is adsorbed by a protein having affinity for antigen presenting cells expressed by the oncolytic microorganism, and then the antigen presenting cells such as Dendritic cells process and present tumor antigens to stimulate the body's own immune effector cells, including: CTL (cytotoxic T lymphocytes) that specifically kill tumors, and Th cells (helper T lymphocytes) that can regulate the body's immune response ), And NK cells (naturally kill T lymphocytes), to activate the body's immune response; not only can dissolve local tumors, but also further inhibit and kill metastatic tumors, compared with tumor treatment with oncolytic virus alone, it has broken
  • Figure 1 is a diagram of a recombinant HSV-1 virus plasmid expressing a heat shock protein constructed in the present invention.
  • the plasmid pHSV-HSP contains the HSP70 gene and the SV40 polyadenylation sequence (SV40poly (A)) under the control of the CMV promoter.
  • This plasmid also includes selectable markers, the origin of replication of E. coli (ColEl ori), HSV a (HSV cleavage and packaging signals), and HSV ori (HSV's DNA origin of replication).
  • Figure 2 is a graph showing the in vitro cytotoxic effect of the recombinant virus HSV-HSP. Among them, mouse tumor cell CT26 was cultured in a 24-well plate (1 ⁇ 10 5 per well) and infected by HSV-HSP or HSV with different multiplicity of infection (0.01-10).
  • Cytotoxicity (%) was measured 4 days after infection using a Promega kit. When the multiplicity of infection is 1, the cytotoxicity has reached more than 99%.
  • Figure 3 shows the good ability of recombinant HSV-HSP to inhibit distant tumors after intra-tumor injection. CT26 cells were inoculated symmetrically on both sides of the ribs of the mice.
  • HSV-HSP (1 X 10 6 ) (indicated by filled squares), HSV carrier (1 X 10 6 ) (in solid lines) Diamonds) and HSP70 protein (indicated by solid triangles) were used as controls (this day was set to day 0), and a second inoculation was performed after 7 days (6 animals in each group).
  • intratumorally injected tumors inoculated with HSV-HSP and tumors on the opposite side that were not inoculated with HSV-HSP had significantly slower tumor growth after injection (pO.001, 25 days after infection, non- Paired t-test).
  • HSV-HSP was significantly superior to HSV and HSP70 proteins alone in inhibiting tumor growth on both sides (pO.001, 25 days after infection, unpaired t test).
  • Figure 4 is a construction diagram of plasmid pLEP-HSP70-IRES-ElA.
  • Recombinant adenovirus was constructed using the dual plasmid method.
  • the HSP gene and the E1A gene necessary for adenovirus replication in tumor cells were ligated to the plasmid pLEP through the multiple cloning sites shown in the figure.
  • Figure 5 shows the construction of the recombinant virus Ad-HSP / ElA.
  • Fig. 6 is a map of a recombinant pcDNA-HSP plasmid expressing a heat shock protein constructed according to the present invention.
  • the HSP gene amplified by PCR was ligated to the eukaryotic expression plasmid pcDNA3.1 by blunt end.
  • the recombinant plasmid pcDNA-HSP contained the HSP70 gene under the control of the CMV promoter and the BGH polyadenylation sequence (BGHpolyA). It also includes a selection marker and a copy origin SV40ori.
  • Figure 7 shows the good ability of Salmonella expressing heat shock protein to inhibit distant tumors after intra-tumor injection. SMMC7721 cells were inoculated symmetrically on both sides of the ribs of mice.
  • Salmonella (3 ⁇ ⁇ ) (1 X 10 7 ) (indicated by solid squares) containing the plasmid pcDNA-HSP was injected into the tumor of the right tumor of the mouse and injected without plasmid.
  • Salmonella (aroA_) (1 X 10 7 ) (indicated by solid diamonds) and HSP70 protein (indicated by solid triangles) as controls (this day is set to day 0), and a second inoculation will be performed after 7 days (each group) 6 only).
  • oncolytic microorganism is defined herein as a naturally occurring or genetically modified type of microorganism that can enter tumor cells and cause the tumor cells to lyse and die by selectively replicating in large numbers within the tumor cells.
  • oncolytic microorganisms include oncolytic viruses and oncolytic bacteria as defined below.
  • oncolytic virus is defined herein as a tumor cell that is capable of selectively replicating and killing tumor cells regardless of whether the p53 or other proteins of the tumor cells are mutated.
  • oncolytic viruses include Herpes simplex virus (such as HSV-l), adenovirus (such as ONYX-015), Newcastle disease virus (NDV), vesicular stomatitis virus (VSV), etc.
  • oncolytic bacteria is defined herein as a genetically modified bacterium that is capable of replicating and killing tumor cells within tumor cells. It is known that some bacterial mutants can be used as tumor gene therapy vector 2Q .
  • a gene encoding purine or aromatic amino acid (aroA) in Salmonella cannot mutate these nutrients, which are essential to it. These substances do not exist in normal cells, and tumor cells can provide these nutrients due to changes in genetic characteristics. Therefore, these auxotrophic bacteria can selectively expand and lyse in tumor cells.
  • Salmonella typhimurium YS72 pur-
  • the ratio in is 9000: 1 2Q .
  • a gene expressing a heat shock protein is introduced into these bacterial mutant strains, and the characteristic that mutant bacteria can be enriched and lysed in tumor cells can be utilized as the oncolytic microorganisms of the present invention.
  • proteins that adhere to tumor antigens and are recognized by antigen-presenting cells is defined herein as a class of proteins that can be linked to tumor-specific antigen polypeptides by adsorption, adhesion, or binding to form proteins- Polypeptide complexes, and antigens are presented to antigen-presenting cells such as dendritic cells through antigen-peptide chaperone transfer.
  • antigens are processed and presented to stimulate the body's immune response.
  • chaperone proteins mainly composed of heat shock proteins.
  • replication-deficient vector is defined herein as a microorganism such as a virus that does not have the ability to replicate, but its ability to express foreign proteins is not affected.
  • heat shock protein is defined herein as a family of highly conserved molecules with ATPase activity, including heat shock protein variants. Heat shock proteins are found in all prokaryotes and most eukaryotic cells; whether or not there is external pressure, they play an important role in protein metabolism, including protein folding and transmembrane transport in the de novo synthesis pathway of proteins, and Degradation of misfolded proteins.
  • the heat shock proteins include hsp70, hsp90, and gp94 / gp96 obtained from tumor cells and bacterially infected cells, and they all can stimulate a cellular immune response.
  • plasmid expressing a heat shock protein is defined herein as a plasmid DNA capable of eukaryotic expression of a heat shock protein or a variant thereof in vivo, and a DNA sequence encoding a variant of the heat shock protein is appropriately located by the relevant Promoter, polyA, enhancer and other regulatory elements.
  • variant of heat shock protein is defined herein as a fragment or various modifications of a heat shock protein, including but not limited to the addition, deletion or substitution of one or more amino acids, and the fusion protein with an exogenous peptide, through This modification to the heat shock protein can enhance the ability of the heat shock protein to adhere to the antigen; or target targeting sequences such as signal peptides and nuclear localization sequences can be linked to the heat shock protein DNA so that the heat shock protein can change its intracellularity Distribution state, such as heat shock protein from non-secreted to secreted or The transformation from intracellular to extracellular, so that it can better bind to the antigen polypeptide.
  • a plasmid encoding DNA capable of adhering to tumor cell antigens and proteins or fragments thereof recognized by antigen presenting cells is defined herein as plasmid DNA capable of eukaryotic expression of said protein or variant thereof in vivo, encoding
  • the DNA sequence of the protein or a variant thereof is appropriately located in a sequence consisting of related regulatory elements such as a promoter, polyA, and an enhancer.
  • a replication-defective vector containing DNA encoding a protein or fragment thereof capable of adhering to tumor cell antigens and recognized by antigen-presenting cells is defined herein as a microorganism such as a virus that does not have the ability to replicate, but which expresses the protein or The ability of its variants is not affected.
  • an “adjuvant” is a substance that is used in conjunction with an antigen or can itself act as an antigen to enhance an immune response.
  • antigen is defined herein as a molecule that elicits an immune response.
  • the immune response includes the production of antibodies, the activation of cells with specific immune activity, or a combination of the two.
  • Antigens can be produced from organisms, proteins or subunits of antigens, whole cells or lysates that are killed or deactivated.
  • cancer is defined here as a malignant cellular tumor and is capable of invading other cells. Examples include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, uterine cancer, skin cancer, pancreatic cancer, colon cancer and lung cancer.
  • cancer vaccine is defined herein as a tumor-specific antigen molecule or an immune cell that has been exposed to tumor cells.
  • a protein expressed by a tumor cell that renders the tumor cell immunogenic and can be recognized by the immune system is a tumor-specific antigen, and the antigen protein or an antigenic epitope polypeptide having an immune effect is prepared in vitro; or The contact of tumor cells enables immune cells to recognize the antigen, and the above-mentioned protein or polypeptide or immune cells can be used as a therapeutic cancer vaccine.
  • kill tumor is defined herein as being capable of effectively inhibiting the expansion, invasion and metastasis of a tumor, as well as the regression or transformation of the tumor into benign.
  • expression is defined herein as the transcription and / or translation of a particular sequence initiated by its promoter.
  • MHC major histocompatibility complex
  • MHC I class I histocompatibility complex
  • MHC II class II histocompatibility complex
  • promoter is defined herein as a nucleic acid sequence that is capable of regulating the transcription of a particular nucleic acid sequence.
  • the term promoter includes enhancers, silencers, and other cis-regulatory elements.
  • T lymphocytes are defined herein as a type of cell of thymus origin that is capable of participating in a series of cell-mediated immune responses.
  • Antigen-presenting cells are defined herein as a class of cells that function to process and present antigens to T cells and B cells. These cells include dendritic cells, macrophages, and B cells.
  • Immuno-enhancing molecules are defined here as molecules that can enhance immune effects, including cytokines and immune co-influencers including IL-2, IL-12, TNF, IFN, G-CSF, GM-CSF, chemokines, etc.
  • Exciter molecules such as immune co-stimulatory molecule B 7 and so on.
  • immunoeffective amount is defined herein as a dose effective to stimulate the body to produce an immune response that suppresses or kills tumor cells.
  • the strategy we adopt is: without the need to prepare tumor antigens in vitro or even knowing in advance that patients have specific tumor antigens with individual differences, using the patient ’s own tumor antigens and antigens present in the body Cells, tumor immunotherapy in vivo.
  • oncolytic microorganisms carrying functional protein genes that can induce specific anti-tumor immune responses in tumor patients. After administering the microorganisms to tumor patients, they can play a role in lysing tumor cells.
  • one embodiment provided by the present invention is to construct a recombinant oncolytic microorganism capable of expressing a functional protein capable of adhering to tumor cell antigens and recognized by antigen presenting cells. It is used to infect a local tumor, and the microorganism grows in tumor cells and does not substantially grow in normal cells. While oncolytic microorganisms grow in tumor cells, they express proteins that adhere to tumor cell antigens and are recognized by antigen-presenting cells.
  • tumor cells Due to the large number of oncolytic microorganisms replicating in tumor cells, tumor cells are lysed, and various tumor cell antigens are released. These antigens bind to proteins expressed by oncolytic microorganisms that adhere to tumor cell antigens and are recognized by antigen presenting cells to form antigen-protein complexes. The complex is further recognized by antigen-presenting cells, and after processing by antigen-presenting cells, it is presented to cytotoxic T lymphocytes (CTLs) that can specifically kill tumor cells, and can stimulate Th cells (helper cells) that regulate the body's immune response. T lymphocytes), and NK cells (naturally killing T lymphocytes) and other immune effector cells to enhance the body's immune response.
  • CTLs cytotoxic T lymphocytes
  • Th cells helper cells
  • the combination of oncolytic microorganisms and a protein capable of adhering tumor cell antigens and recognized by antigen presenting cells or a vector expressing the protein can also achieve the purpose of the present invention.
  • the microorganisms can selectively grow in tumor cells to lyse the tumor cells and release tumor antigens.
  • the protein can adsorb antigen polypeptide to form a protein-polypeptide complex, and submit the antigen to antigen-presenting cells such as dendritic cells through antigen-peptide chaperone transfer.
  • an antigen-presenting cell of a tumor patient is transformed in vivo or in vitro with a vector containing a DNA encoding a protein or a fragment thereof that is capable of adhering to tumor cell antigens and recognized by antigen-presenting cells.
  • the transformed antigen-presenting cells can also be administered to a patient after transformation, which can also achieve the object of the present invention.
  • the transformed antigen presenting cells are more sensitive to the antigen, and the functional proteins expressed by the presenting cells capture the antigen and then return to the presenting cells, further enhancing the immune signal and activating the body's specific anti-tumor immune response.
  • the present invention in addition to the oncolytic microorganisms expressing the protein or fragments thereof capable of adhering to tumor cell antigens and recognized by antigen presenting cells, the present invention also includes but is not limited to the following combinations: Combinations, which include:
  • Another combination includes-a) microorganisms capable of selectively growing in tumor cells;
  • Another combination includes:
  • the microorganisms described above may further contain DNA sequences encoding immune-enhancing molecules, such as cytokines encoding IL-2, IL-6, IL-12, TNF, IFN, G-CSF, GM-CSF or chemokines, and And / or the sequence of an immune costimulatory molecule such as B7.
  • immune-enhancing molecules such as cytokines encoding IL-2, IL-6, IL-12, TNF, IFN, G-CSF, GM-CSF or chemokines
  • an immune costimulatory molecule such as B7.
  • the microorganism and the microbial composition of the present invention can be used alone or in combination with other forms of immunotherapeutic agents, for example, in combination with cytokines, immune adjuvants or traditional Chinese medicine.
  • the microbial composition may further contain a pharmaceutically acceptable carrier.
  • the "functional proteins” or “proteins that can adhere to tumor antigens and be recognized by antigen-presenting cells” in the present invention refer to a class of proteins that can bind tumor antigens and have affinity for antigen-presenting cells.
  • the proteinoid include chaperone proteins mainly composed of heat shock proteins. Heat shock proteins can bind to tumor-specific antigen polypeptides to form protein-peptide complexes, and submit antigens to antigen-presenting cells such as dendritic cells through antigen-peptide chaperone transfer.
  • the antigens are processed and presented to stimulate the body's NK cells and CTL
  • the activity of immune effector cells, such as cells and Th cells activates the body-specific immune response.
  • the heat shock protein itself has affinity for antigen-presenting cells and can also activate the presenting cells.
  • the heat shock protein expressed in the tumor in situ can bind to the tumor antigen and play an immunoregulatory role by means of cancer vaccine immunization, significantly triggering and enhancing the immune response to the tumor antigen, and inhibiting the tumor in situ while inhibiting Distal tumor.
  • Heat Shock Proteins are a family of highly conserved proteins with ATPase activity. They are found in all prokaryotes and most eukaryotic cells.
  • heat shock proteins play an important role in protein metabolism, including protein folding and transmembrane transport in the de novo synthesis pathway of proteins, and degradation after misfolding 5 .
  • Heat shock proteins including Hsp70 derived from tumor cells and bacteria infected cells, and of Hsp90 grp94 / gp96, can immunoblastic 6 _ 7 excitation reaction.
  • Alternative heat shock proteins can also be derived from pathogenic microorganisms, including M tuberculosis, leprae, Trypanoma cruzO, Plasmodium falciparum ⁇ iPla df m falciparum; and other species Mammals such as primates, mice, etc.
  • the immunogenicity of heat shock proteins is mainly produced by polypeptides that bind to their molecules. 8.
  • the specificity of the produced heat shock protein-antigen polypeptide is through the specificity of an endogenous antigen that is not expressed. After cells present antigens to T lymphocytes, they activate the immune system.
  • heat shock proteins as molecular chaperones can present antigenic polypeptides to antigen-presenting cells, mainly dendritic cells, and the possible mechanism is by making These peptides bind to MHC I and MHC II molecules and enter the MHC I and MHC II presenting antigen pathway; these antigen peptides will further activate the activity of immune effector cells such as CTL cells, NK cells, and Th cells, and stimulate the body-specific immune response.
  • Heat shock protein, fragment thereof, or various modifications (including but not limited to one or more amino acids Additions, deletions, and / or substitutions, etc.) variants can be used in the present invention, as long as the heat shock protein, fragment, or variant thereof retains the ability to bind to tumor cell antigen polypeptides.
  • the heat shock protein's ability to adhere to an antigen can be modified by adding, deleting or replacing one or more amino acids of the heat shock protein or by forming a fusion protein with a foreign peptide.
  • the target localization sequence such as Signal peptide), nuclear localization sequence, etc.
  • Protein linking enables heat shock proteins to change their distribution within the cell, such as heat shock proteins changing from non-secreted to secreted or from intracellular to extracellular, thereby making them better associated with antigens Polypeptide binding.
  • the oncolytic microorganisms of the present invention are viruses or bacteria that are natural or genetically modified to selectively replicate in tumor cells.
  • the viruses include herpes simplex viruses such as HSV-1 and G207, adenoviruses (AdV) such as ONYX-015 (also known as dll520), CN706 and CN787, Newcastle disease virus (DV) such as 73-T, vesicular stomatitis virus (VSV), reoviruses such as Reolysin 3 or other viruses that selectively grow in tumor cells.
  • AdV adenoviruses
  • ONYX-015 also known as dll520
  • CN706 and CN787 Newcastle disease virus (DV) such as 73-T
  • VSV vesicular stomatitis virus
  • reoviruses such as Reolysin 3 or other viruses that selectively grow in tumor cells.
  • the bacteria include Salmonella typhi, Bifidobacterium, Shigella, Listeria, Yersinia pestis, Clostridium 21, and other bacteria that are naturally occurring and can selectively grow
  • Oncolytic microorganisms also refer to various variants of the aforementioned microorganisms, as long as the microorganisms have the ability to selectively replicate and grow in tumor cells to lyse tumor cells. Therefore, one of ordinary skill in the art can recognize whether the gene encoding a protein, such as a heat shock protein, capable of adhering to tumor antigens and recognized by antigen-presenting cells is integrated into the genome of an oncolytic microorganism or is free from the microorganism If it exists in other expression vectors outside the genome, as long as the gene is effectively linked to regulatory sequences such as promoters and terminators, the oncolytic microorganisms such as HSV-1 or dll520 can be expressed with the Proteins that adhere to tumor antigens and are recognized by antigen-presenting cells, such as heat shock proteins, work together to achieve the object of the present invention.
  • a protein such as a heat shock protein
  • an oncolytic adenovirus Ad-HSP / ElA carrying the heat shock protein Hsp70 gene is disclosed.
  • the Hsp70 gene into dll520 (which can be reduced as a selective replication in tumor cells defective in p53 function venom virus 14 _ 9) construct a recombinant oncolytic adenovirus, see Example 2 specific process. Animal experiments were performed with the recombinant oncolytic adenovirus Ad-HSP / ElA.
  • This virus exhibits very strong antitumor activity and can effectively reduce or even eliminate tumor volume (see the table below).
  • the following table also lists the results of tests performed on different tumor model animals by combining heat shock protein Hsp70 or a vector expressing heat shock protein Hsp70 with oncolytic adenovirus Ad- ⁇ :
  • Group 1 Recombinant oncolytic adenovirus Ad-Hsp / El A capable of expressing heat shock protein Hsp70;
  • Group 2 Oncolytic adenovirus Ad- ⁇ and DNA plasmid expressing heat shock protein Hsp70;
  • Group 3 Oncolytic adenovirus Ad- ⁇ and replication defective virus expressing heat shock protein Hsp70;
  • Group 4 Oncolytic adenovirus Ad - ⁇ and heat shock protein Hsp70;
  • CT26 / Balb / c represents a CT26 tumor model of colon cancer established on Balb / c strain mice.
  • TRAMP-c2 / c57 represents a tumor model of prostate cancer TRAMP-c2 established on mice of the c57 strain.
  • B16 / C57 represents a melanoma B16 tumor model established on mice of the c57 strain. Were injected subcutaneously inoculated with the appropriate tumor cells in 2X 10 5 th ribs corresponding mouse symmetrical parts (left and right). These mice were used as models for animals with metastatic tumors.
  • asymmetric (right) tumors of experimental mice are inoculated with recombinant Ad-Hsp / El A at about 2 X 10 9 PFU; or Ad- ⁇ about 2 X 10 9 PFU and 50 micrograms of a DNA plasmid expressing heat shock protein Hsp70; or oncolytic adenovirus Ad- ⁇ about 2 X 10 9 PFU and a replication-deficient virus expressing heat shock protein Hsp70 about 2X 10 9 PFU ; Or oncolytic adenovirus Ad_- ⁇ E1B is about 2X 10 9 PFU and heat shock protein Hsp70 is about 10-25 micrograms.
  • the oncolytic virus used is herpes simplex virus. Have one kind of replication-competent herpes simplex virus mutants after a large number of different species of animal models we found that non-pathogenic and is in clinical trials for the treatment of primary brain tumors of 16 people.
  • This herpes simplex virus mutant in dividing cells can lead to cell death, its replication ability in non-dividing cells is very weak, and it can selectively replicate in tumors after inoculation to tumor-bearing mice, thereby inhibiting the original Tumor growth and prolong the survival of tumor-bearing mice by 15 ⁇ 17 .
  • the amplified plasmid encoding the heat shock protein HSP70 was introduced into this mutant strain of herpes simplex virus to construct a recombinant herpes simplex virus.
  • Two specific construction methods are as follows: One method: The cDNA of HSP70 was obtained by PCR amplification from a human cDNA library. DNA sequencing was performed.
  • Klenow enzyme was used to fill in both ends of the DNA fragment of the PCR product, and then the blunt end was inserted into the Spel site connected to the plasmid pHSV to obtain a plasmid pHSV-HSP.
  • the recombinant plasmid and helper virus are co-transfected into eukaryotic cells, and the helper virus packages the recombinant plasmid in the cell to obtain a recombinant herpes simplex virus capable of expressing heat shock protein.
  • the specific construction process is described in Example 1.
  • Another method First construct a vector containing HSP70 under the control of a promoter, and then insert HSP DNA into the genome of HSV according to the conventional methods in the art to obtain recombinant herpes simplex DNA with the HSP gene integrated into the viral genome.
  • the obtained recombinant herpes simplex virus can be directly administered to tumor patients without the need for co-transfection of HSV helper virus and plasmid DNA to prepare herpes simplex virus expressing heat shock protein in vitro.
  • the oncolytic virus used is an adenovirus.
  • the used adenovirus mutants can selectively replicate in tumor cells and cause the cells to lyse and die, and their replication capacity in normal cells The force is very weak 1Q .
  • This adenovirus mutant can selectively replicate in tumors after being inoculated into tumor-bearing mice, thereby inhibiting tumor growth in situ and prolonging the survival time of tumor-bearing mice.
  • 9 "" Recombinant construction with pLEP and pREP dual plasmid systems Adenovirus, the HSP gene and the E1A gene necessary for the adenovirus to have the ability to replicate in tumor cells were ligated to the plasmid pLEP, and the recombinant plasmid pLEP-HSP70-IRES-E1A was constructed to obtain the recombinant plasmid and plasmid pREP.
  • the resulting recombinant plasmid P Ad-Hsp70 / ElA was digested with I-Ceul and transfected into 293 cells to obtain a recombinant adenovirus capable of expressing Hsp70.
  • a Salmonella bacterium capable of specifically replicating in tumor cells is used as a vector for expressing heat shock protein, because the Salmonella used is aroA-deficient I9 , and its required nutrition can only be Provided by tumor cells, they cannot replicate and grow in normal cells, so this mutant can selectively replicate and pass through tumor cells.
  • Proliferation causes tumor cells to lyse and die.
  • the heat shock protein expressed by Salmonella can bind to the antigen released by tumor cell lysis and deliver it to antigen-presenting cells, thereby stimulating the CTL-mediated immune response that can specifically kill tumor cells. This inhibits tumor growth and prolongs the survival of tumor-bearing mice.
  • the specific process is described in Example 3.
  • the sequence encoding the heat shock protein Hsp70 is regulated by the transcription of the promoter.
  • Promoter means that it can be The DNA sequence recognized by the cell's own or introduced transcription complex is necessary for the initiation of transcription of a gene.
  • promoters consist of discrete functional regulatory elements, each about 7-20 bp DNA, and include one or more transcriptional activations Or repressor recognition sites.
  • Other types of promoters such as enhancers can regulate the frequency of transcription initiation.
  • the subsequences are located 30-110 bp upstream of the start site, although promoters containing regulatory elements downstream of the start site are now shown.
  • the distance between the promoter elements is usually variable, so when a These elements can still maintain the normal function of the promoter when inserted or removed.
  • the distance between the promoter elements can be increased up to 50bp under the premise of maintaining activity.
  • Each element can activate the transcription reaction in cooperation with each other or independently.
  • a promoter can be a gene or a sequence itself, that is, obtained from the coding region and / or the non-coding sequence 5 'upstream of the exon. Such a promoter Is an "endogenous" promoter. We can also get enhancers upstream or downstream of this gene or this sequence. Alternatively, a promoter that is not the gene or the sequence itself, that is, a recombinant or heterologous promoter, placed upstream of the coding sequence for transcriptional regulation would have special advantages.
  • Recombinant or heterologous promoters include enhancers that are not related to genes or sequences in the natural environment.
  • Such promoters and enhancers may include promoters and enhancers of other genes, as well as promoters and enhancers isolated from prokaryotes, viruses or eukaryotic cells, or promoters and enhancers that are not "naturally occurring", For example, the combination of different regulatory elements of different transcriptional regulatory regions, and / or the introduction of mutations that can affect function.
  • recombinant cloning and / or PCR Including nucleic acid amplification technology when synthesizing promoter or enhancer sequences, recombinant cloning and / or PCR Including nucleic acid amplification technology.
  • CMV cytomegalovirus
  • This promoter is a constitutively strong promoter capable of initiating ⁇ -level expression of its downstream sequences.
  • other constitutive promoters such as the pre-early promoter of simian virus SV40, the mouse mammary tumor virus (MMTV) promoter, the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, and the murine virus can also be used.
  • Promoters avian leukemia virus promoters, pre-EB virus promoters, Lloyd's sarcoma virus promoters, and human gene promoters such as (but not limited to): actin promoter, myosin promoter, hemoglobin promoter Promoter, creatine promoter.
  • the present invention is not limited to the use of a constitutive promoter, and an inducible promoter may also be used.
  • the use of an inducible promoter in the present invention provides a molecular switch that can open the expression of its connected downstream nucleic acid sequence when needed, and turn it off when its expression is not needed.
  • Inducible promoters include, but are not limited to: metallothionein promoter, glucocorticoid promoter, progesterone promoter, tetracycline promoter.
  • tissue-specific promoters include, but are not limited to, those known now such as: HER-2 promoter, PSA-related promoter sequences.
  • HER-2 promoter a typical polyadenylation signal is required to complete the correct polyadenylation tailing reaction. The nature of the polyadenylation is not critical to the successful implementation of the invention, and any type of stop sequence can be used.
  • Preferred embodiments include the SV40 polyadenylation signal, the LTR polyadenylation signal and / or the bovine growth hormone polyadenylation signal, which should be based on the principle of convenience and / or known to be able to be included in different targeted cells. The principle of proper function is to select polyadenylation signals. Transcription termination sites can also be used to stop transcription. These elements should be able to enhance the level of translation termination of transcription and / or avoid reading through to other sequences. The important point is that the correct expression of the heat shock protein expressing vector in the present invention does not require the vector to be integrated into the genome of the recipient cell. The vector may be present in the target cell in an episomal state.
  • the expression of a target protein by a vector in certain types of cells does not require the replication of the vector itself.
  • These cells such as muscle cells, usually cannot replicate normally, and expression vectors introduced into non-dividing cells can express the protein of interest without the vectors themselves replicating.
  • oncolytic microorganisms can be combined with a protein capable of adhering tumor cell antigens and recognized by antigen presenting cells or a vector expressing the protein. Combined administration or use of DNA containing the protein or a fragment thereof The vector transfects the patient's own antigen-presenting cells. This triggers the body's specific immune response against tumor antigens, killing local tumors and metastatic tumors.
  • the microorganism of the present invention or the composition containing the oncolytic microorganism can be used alone or in combination with other forms of immunotherapeutics, such as with cytokines or traditional Chinese medicine.
  • immune modulators such as adjuvants or so-called immunostimulants are used to enhance the immune response.
  • Numerous studies have shown that simultaneous administration of cytokines or expression Cytokine plasmids can enhance immune activity against tumors.
  • a person skilled in the art can easily recognize that the nucleic acid sequence of the cytokine and the nucleic acid sequence of the HSP in the embodiment of the present invention can be put into the same vector for common application, thus avoiding the use of two expression vectors.
  • the oncolytic microorganism, microbial composition, or pharmaceutical composition of the present invention can be made into an immunotherapeutic agent.
  • the oncolytic microorganism, oncolytic microorganism composition or pharmaceutical composition carrying the functional protein gene of the present invention can be processed into solid, semi-solid, liquid or aerosol forms according to the route of application. Known methods can be used to prevent the composition from being released and absorbed before reaching the target organ. Any acceptable pharmaceutical form can be used in the present invention to ensure the effectiveness of the components of the present invention.
  • the microorganisms in the present invention can be used alone or combined with other pharmaceutically active components in a proper ratio. It should be ensured that there is a sufficient amount of oncolytic microorganisms such as oncolytic viruses or oncolytic bacteria that carry DNA that can adhere to tumor antigens and be recognized by antigen presenting cells or fragments thereof, so that their gene products Can provide a pharmaceutically effective dose.
  • Oncolytic microorganisms can be used alone or in combination with different adjuvants.
  • the oncolytic microorganisms, microbial compositions, pharmaceutical compositions, or immunotherapeutics of the present invention described above can be used in immunologically effective amounts alone or in combination with other therapeutic agents by various conventional and acceptable means known in the art. Dosing.
  • the immunologically effective amount will vary greatly depending on the severity of the disease, the age and relative health of the individual, the effectiveness of the microorganisms used, and other factors. Generally, a person of ordinary skill in the art can determine the immunologically effective amount of the microorganism of the present invention for treating a given tumor based on personal knowledge and the content disclosed in this application.
  • Adjuvants that can be used with the microorganisms of the present invention include (but are not limited to) Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide, BCG, lipopolysaccharide (LPS), lipid A analogs , Muramyl dipeptide (MDP) and its analogs, endotoxin (LT) and cholera toxin (CT).
  • the medicament of the present invention can be administered by one of the following routes: intratumoral injection, oral, systemic (e.g., transdermal, intranasal or via suppository) or parenteral (e.g., intramuscular, intravenous) Internal or subcutaneous).
  • the pharmaceutical composition or immunotherapeutic agent may be in the form of a tablet, pill, capsule, semi-solid, powder, sustained-release preparation, solution, suspension, aerosol, or any other suitable composition, and is generally made by the present invention Oncolytic microorganisms and at least one pharmaceutically acceptable excipient.
  • a pharmaceutically acceptable excipient is a substance that is non-toxic, does not substantially impair the activity of the microorganism, facilitates administration, and does not adversely affect the immune or therapeutic effects of other active ingredients.
  • the excipient may be any solid, liquid, semi-solid, or in the case of an aerosol composition, it may be a gaseous excipient. These excipients are readily available to those skilled in the art.
  • Solid pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glyceryl monostearate, chlorinated Sodium, skimmed milk powder, etc.
  • Liquid and semi-solid excipients may be selected from water, ethanol, glycerol, propylene glycol, and various oils, including oils derived from petroleum, animal, vegetable, or synthetic sources (eg, peanut oil, soybean oil, mineral oil, sesame oil, etc.).
  • Preferred liquid carriers, especially those for injectable solutions include water, saline, aqueous dextrose and diethylene glycol.
  • the amount of the microorganism of the present invention in the pharmaceutical composition may vary greatly depending on the kind of the preparation, the size of the unit dose, the kind of the excipient, and other factors known to those skilled in the pharmaceutical field.
  • the actual dosage and treatment regimen will vary depending on other factors such as whether the components of the invention are combined with other therapeutic components, individual pharmacokinetics, drug distribution, and metabolism.
  • the amount of virus added to the cell also varies with the length, stability, and sequence of the therapeutic gene inserted into the vector. It is an empirically determined variable and is likely to be affected by factors other than the method of the invention change. A person skilled in the art can easily adjust according to the occurrence of specific conditions.
  • the present invention is described in detail below by way of examples. The following examples are exemplary, so these examples should not be considered as limiting the present invention.
  • HSP70 Human Heat Shock Protein
  • the PCR method was used to amplify the HSP70 gene from the cDNA of the human tumor cell line SKOV3 cells (ATCC HTB-77).
  • the upstream primer sequence is: GGT ATG GAA GAT CCC TCG AGA TC
  • the downstream primer sequence is: TA CTA ATC TAC CTC CTC AAT GGT GGG.
  • 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.
  • 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, the obtained product was purified using a PCR product purification kit from Qiagen. Construction of oncolytic virus-herpes simplex virus
  • HSV-1 herpes simplex virus
  • the Y 34.5 gene was deleted.
  • Replication HSV-1 mutant in the split-type cells lead to cell death; the ability to grow in a non-dividing cell type 16 is significantly reduced.
  • the herpes simplex virus mutant After the tumor-bearing mice replicated selectively in the tumor, thereby inhibiting the growth of orthotopic tumors and prolong survival of tumor-bearing mice 16.
  • HSV is used to deliver heat shock proteins in tumors.
  • the cDNA of human HSP70 was obtained from human cDNA library by PCR amplification and DNA sequencing was performed.
  • Klenow enzyme was used to fill in both ends of the DNA fragment of the PCR product, and then the blunt end was inserted into the Spel site connected to the plasmid pHSV (Gelle r , AI and Breakefield, X. 0. (1988), Science 241: 1667-1669).
  • plasmid pHSV-HSP (as shown in Figure 1).
  • the HSV-1 mutant strain in which the Y 34.5 gene was deleted was used as a helper virus to produce a recombinant virus HSV-HSP.
  • Herpes simplex virus host cells were co-transfected with the plasmid pHSV-HSP DNA and herpes simplex virus helper DNA.
  • pHSV-HSP is packaged in the shell of the herpes simplex virus, which produces a recombinant herpes simplex virus with the HSP70 gene.
  • the recombinant virus is amplified plasmid encoding the HSP 17 were obtained from packaged helper virus herpes simplex virus.
  • Each virus particle is capable of packaging 15 copies of the HSP70 gene and can efficiently transfect dividing and non-dividing cells.
  • Viral DNA is not integrated into the genome of infected cells, and the expression of HSP70 protein is regulated by the CMV promoter, which is transiently high expression.
  • the sequence of HSV-HSP was determined, and its sequence is listed in the sequence table. Titer determination of herpes simplex virus
  • the purified amplified plasmid DNA (pHSP70) and HSV virus were co-transfected into Vera cells with lipofect AMINE (product of Life Technologies), and then incubated at 34.5 ° C until the cells showed a complete cytopathic effect.
  • Recombinant virus was harvested from Vera cells and cultured in a 1: 5 dilution. Passages were generally passaged 5-6 until it was observed that helper virus replication was inhibited.
  • the HSV containing the pHSP70 plasmid was named HSV-HSP. After freeze-thaw and sonication and low-speed centrifugation (2000 X g, 4 ° C for 10 minutes), cell debris was removed, and the titer of the recombinant virus was measured.
  • Viral titers are expressed as plaque forming units (PFU) formed by the virus in Vero cells cultured at 34.5 ° C.
  • PFU plaque forming units
  • HSV-HSP determine the expression of Hsp70 and use the passage with the highest titer level to culture the virus.
  • HSV—HSP has a virus titer of 5 X 10 7 PFU / ml.
  • the titer of the helper virus HSV-1 was measured, and its production value was 6 X 10 7
  • HSV HSV oncolytic activity in vitro
  • HSV-1 is capable of replicating in many types of tumor cells. It has been found that murine colon cancer cell line CT26 is susceptible to HSV infection. This cell line is weakly immunogenic and fails to elicit detectable tumor-specific cytotoxic T lymphocytes (CTL). CT26 tumors are still prone to relapse after cytokine treatment.
  • the MHC-1 restricted dominant immune antigen of CT26 has been shown to be a nonapeptide derived from the membrane protein (gp70) of an endogenous tropic murine leukemia provirus. Studies have confirmed the antitumor effect of induced tumor-specific cytotoxic T lymphocytes (CTL) on CT26 tumors subcutaneously inoculated.
  • murine tumor cells CT26 were cultured in 24-well plates (1 ⁇ 10 5 per well) and infected by HSV-HSP or HSV with different multiplicity of infection (0.01-10).
  • CT26 cells were infected with HSV-HSP or HSV at a multiplicity of infection (MOI) of 0.1, which resulted in the death of 70% of tumor cells 4 days after infection.
  • MOI multiplicity of infection
  • CT26 cells were infected with HSV_HSP or HSV for 4 days. After the death of 99% of tumor cells (as shown in Figure 2).
  • the expression of HSP70 could be detected by radiolabeling and immunoprecipitation / SDS-PAGE.
  • HSV-HSP HSV-HSP on CT26 tumors was further evaluated on the same BALB / c mice.
  • BALB / c from Charles River (Wilmington, MA, USA). 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.
  • CT26 tumor cells (1 ⁇ 10 5 ) were injected subcutaneously into the symmetrical parts of the ribs of mice. These mice were used as models for animals with metastatic tumors.
  • asymmetric (right) tumors of each animal in the first group are inoculated with HSV-HSP (1 X 10 6 PFU), and the first inoculation is 7
  • a second inoculation was performed a few days later.
  • Asymmetric (right) intratumoral inoculation of HSV-1 (1 X 10 6 PFU) was performed for each animal in the second group, and the second inoculation was performed 7 days after the first inoculation.
  • HSV-1 was used as a control for HSV-HSP inoculation.
  • the asymmetrical (right) tumor of each group of mice was inoculated with heat shock protein HSP70, and a second inoculation was performed 7 days after the first inoculation to evaluate the heat shock protein itself.
  • the t-test was used for statistical analysis of variance, and the software was StaView4.5 (Abacus Concepts, Berkeley, CA).
  • HSV-HSP can cause very significant antitumor effects, whether it is the tumor on the injection side or the same animal body.
  • the growth of tumors at the symmetrical end of the injection was significantly inhibited (as shown in Figure 3).
  • the tumors in mice injected with HSV-HSP had regressed undetectably on both the injection and non-injection sides.
  • Injection of HSV-1 can only cause tumor growth inhibition after injection, and has only a certain effect on uninjected contralateral tumors (as shown in Figure 3).
  • the control group injected with HSP70 only showed that The tumors on the injection side had no effect.
  • the wild-type adenovirus Adl055 was used as a template, and the PCR-based method was used to amplify the type II adenovirus E1 region of bases 559 to 2262.
  • the resulting 1715 base pair fragment contains a Hindni restriction site at the 5 'end and a Xhol restriction site at the 3' end, and two mutation sites at bases 2253 (C ⁇ T) and 2262 (G ⁇ T).
  • pre-mature translation stop codons can be introduced at codons 79 and 82 of the reading frame of the Elb 55kD protein, respectively.
  • the PCR product fragment was digested with el / Xhol and cloned into the same digested pBS- (IRES) (Alexe V. Gordadze et al. J. of Virology 2001, vol 75: 5899-5912).
  • PBS- (IRES) -ElA was obtained and the (IRES) -EIA fragment was released by digestion with Spel / Xhol.
  • human heat shock protein Hsp70 DNA was obtained by PCR amplification in a similar manner as described in Example 1.
  • the amplified fragment includes a Hindlll site at the 5 'end and a Spel site at the 3' end.
  • the ligation product was packaged with Stratagen Y phage, and the packaged product was mixed with E. coli (DK1 or DH5 ct) and incubated, and the LB culture solution was added to a 37 ° C shaker and incubated for 30 minutes.
  • Escherichia coli infected with Y phage was coated with LB agar plates containing ampicillin and tetracycline antibiotics, and resistant colonies were selected and identified by enzyme digestion to obtain a recombinant plasmid pAd-Hsp70 / ElA.
  • the recombinant plasmid pAd-Hsp70 / ElA was digested with I-Ceul to release the recombinant adenovirus DNA from the plasmid.
  • the digested plasmid was added to HEBS buffer, and 2.5M CaCl 2 was added to form the DNA.
  • / Ca 3 (P0 4 ) 2 mixture was transfected into 293 cells. After cultured in a CO 2 incubator for 6-7 days, clear plaques (CPE) could be observed in the cultured cell layer, indicating that the recombinant virus particles began to form. .
  • Example 3 Construction of Salmonella expressing heat shock protein
  • the recombinant plasmid pcDNA-HSP was constructed by inserting the HSF70 sequence into the pcDNA3.1 plasmid (as shown in Figure 6).
  • the CMV promoter regulates the expression of HSP70 protein, which belongs to transient high expression. Antitumor activity
  • mice The therapeutic effect of Salmonella typhimurium SL3261 expressing heat shock protein on tumors was evaluated on BALB / c mice of the same line. 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.
  • SMMC7721 tumor cells (Shanghai Cell Preservation Center) (IX 10 "were injected subcutaneously at symmetrical locations on the shoulders of mice. These mice were used as models for animals with metastatic tumors.
  • the asymmetric (right) tumor of each animal in the first group was inoculated with SL3261 (1 X 10 7 PFU) carrying the expression plasmid pcDNA-HSP, and the second one was performed 7 days after the first inoculation ..
  • each animal a second set of non-symmetric (right) intratumoral inoculation SL3261 (1 X 10 6 PFU) , and at 7 days after the first vaccination for a second vaccination, the bacteria themselves as a control to evaluate the factors
  • Tumor rejection antigen gp96 / grp94 is an ATPase: implications for protein folding and antigen presentation. EMBO Journal, VI,> ⁇ ⁇ >.

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Description

表达热休克蛋白的溶瘤微生物及其应用 技术领域
本发明涉及生物医学的肿瘤免疫治疗领域。 具体地讲， 本发明涉及一种肿瘤免 疫治疗新方法， 即一种有效利用肿瘤患者自身肿瘤抗原， 同步激活患者免疫应 答， 从而在杀灭原位肿瘤的同时抑制肿瘤转移并治疗转移性肿瘤的方法。 本发 明还涉及一种溶瘤微生物、 含有该溶瘤微生物的组合物以及所述的溶瘤微生物 或组合物用于治疗肿瘤或制备用于治疗肿瘤的药物的应用。 技术背景
目前， 治疗肿瘤最常用的还是传统的肿瘤手术切除和放化疗疗法， 它给患者带 来很大的痛苦和负担。 而且， 对转移性肿瘤患者而言， 一方面很难通过手术将 肿瘤彻底切除或通过放化疗疗法将其彻底杀灭； 另一方面， 切除原位肿块， 往 往有引发肿瘤在身体其它部位生长的风险， 结果反而加速了肿瘤的扩散。 通过改变遗传特性， 人们创建了一些能够选择性地感染肿瘤细胞的病毒， 例如 单纯疱疹病毒 iHerpesvir 如 HSV-1 和 G207等， 腺病毒 Adenovirus) 如 ONYX-015 (也称为 dll520)、 CN706和 CN787等，新城疫病毒 (Newcarf/e β η«)如 73-Τ 等， 水泡性口炎病毒 ( Vesicu vims , 呼肠孤病毒 ( ?eovz∞)如 Reolysin 1- 3。 虽然这其中的某些病毒具备很强的抗肿瘤活性， 在临床试验中能够 使肿瘤体积变小甚至完全消除 3， 但存在的问题是： 对于转移性肿瘤的抑制作用 不强。 原因在于： 虽然这种病毒通过裂解肿瘤细胞来使肿瘤消退， 但实体瘤的 病人体内缺乏肿瘤特异性的细胞毒性 T淋巴细胞介导的免疫反应 4。 缺乏免疫 反应的原因可能是大部分肿瘤抗原是在肿瘤细胞内， 而并不是释放出来， 发生 坏死而释放肿瘤抗原的肿瘤细胞数量也很有限， 抗原提呈细胞吸纳肿瘤抗原的 几率极小。 即使在病毒介导的肿瘤细胞裂解后通过增强肿瘤抗原的释放， 呈递
"危险信号"， 来激发机体肿瘤特异性免疫反应， 但由于缺乏有效呈递， 只有很 少的一部分肿瘤抗原能够被提呈， 激发的免疫反应也非常有限， 仍然需要特殊 的机制来进一步增强抗肿瘤的免疫反应。 用癌症疫苗进行肿瘤免疫治疗的策略主要是将被杀死的肿瘤细胞或其裂解产物 同时与佐剂或细胞因子共同使用。 通过接种癌症疫苗能激发机体的抗肿瘤免疫 反应， 这在肿瘤治疗研究中将是一个重大的进展。 但是， 在很多情况下， 肿瘤 抗原是弱免疫原， 也即不能够引起机体足够的针对肿瘤抗原的免疫应答反应。 现阶段利用癌症疫苗治疗肿瘤的方式主要为： 用分离的肿瘤特异性抗原进行免 疫； 或通过体外将肿瘤细胞与树突状细胞融合后对病人进行细胞治疗。 但是， 因为不同病人存在着肿瘤抗原的个体差异性； 而且同一病人在肿瘤发展的不同 阶段， 肿瘤细胞所表达的特异性抗原也不相同； 即使在同一病人的肿瘤的某一 发展时期， 其肿瘤细胞内也存在着多种肿瘤抗原。 由于这种种限制， 导致现阶 段用癌症疫苗治疗肿瘤存在着许多缺点： 不存在一种针对多种肿瘤的抗原， 必 须针对每个病人设计相应的肿瘤抗原， 技术操作细节各不相同， 操作环节多， 每个环节的技术要求高， 操作中容易造成污染； 而且由于病人体内多种抗原的 动态发展， 使得这种体外操作技术不一定有效。 在临床治疗实践中， 必须针对 每个病人制备独特的癌症疫苗， 难度大， 费用高， 可行性差。 综上所述， 传统的手术治疗和放化疗对机体本身造成伤害而且难以消除转移性 肿瘤。 现阶段用病毒等作为载体对肿瘤进行基因治疗也只能针对局部肿瘤， 对 转移性肿瘤也无能为力。 用白介素， 干扰素等细胞因子对肿瘤进行免疫治疗只 能激发免疫调节反应和很少的 CTL细胞介导的对肿瘤细胞的特异性杀伤效应。 最近发展的 "癌症疫苗"疗法非常昂贵且没有普遍适用性。 因此， 到目前为止， 还缺乏一种高效低毒、 给药方便的肿瘤治疗物质， 该物质在被施用给肿瘤患者 后能在体内诱导出有效的抗肿瘤免疫反应， 从而将肿瘤， 包括转移性肿瘤有效 地杀灭。 发明概述
本发明的目的在于找到一种肿瘤免疫治疗新方法， 以便有效地利用肿瘤患者自 身的肿瘤抗原， 同步激活患者体内的免疫应答， 从而在杀灭原位肿瘤的同时抑 制肿瘤转移。 为实现上述目的， 本发明提供了一种溶瘤微生物， 该微生物能选择性地在肿瘤 细胞中生长并同时表达能粘附肿瘤抗原并能被抗原提呈细胞所识别的蛋白 （在 下文中有时也将之简称为"功能蛋白"）。 为实现上述目的， 本发明还提供了一种抗原提呈细胞， 其是被含有编码功能蛋 白或其片段的 DNA序列的载体转化的。 为实现上述目的， 本发明还提供了一种微生物组合物， 包括：
a) 溶瘤微生物， 该微生物能选择性地在肿瘤细胞中生长以裂解肿瘤细胞； 和 b)表达能粘附肿瘤细胞抗原的并被抗原提呈细胞所识别的蛋白及其片段的载 体；
其中所述的微生物或载体任选地进一步含有编码免疫增强分子的 DNA序列。 为实现上述目的， 本发明还提供了一种药物组合物， 其主要含有：
a. 溶瘤微生物， 即能选择性地在肿瘤细胞中生长的微生物；
b. 能粘附肿瘤细胞抗原的并被抗原提呈细胞所识别的蛋白或其片段； 和 c. 任选地含有的免疫增强因子、 免疫佐剂和可药用载体， 其中免疫增强因子可 以由溶瘤微生物产生。 为实现上述目的， 本发明还提供了一种治疗肿瘤的免疫治疗剂， 其包含以上所 述的溶瘤微生物， 或者所述的抗原提呈细胞、 或者所述的微生物组合物或者所 述的药物组合物。 为实现上述目的， 本发明还提供了一种治疗肿瘤的方法， 包括用含有编码能粘 附肿瘤细胞抗原的并被抗原提呈细胞所识别的蛋白或其片断的 DNA 的载体转 化患者的抗原提呈细胞。 为实现上述目的， 本发明还提供了治疗肿瘤的方法， 包括给肿瘤患者施用一种 免疫有效量的以上所述的溶瘤微生物、 所述的抗原提呈细胞、 所述的微生物组 合物、 所述的药物组合物或所述的免疫治疗剂。 在本发明的一个实施方案中， 给肿瘤患者体内施用免疫有效量的携有功能蛋白 基因的溶瘤微生物。 具体地说， 所述的携有功能蛋白基因的溶瘤微生物为能表 达能粘附肿瘤抗原并被抗原提呈细胞所识别的蛋白或其片段的重组溶瘤微生 物； 或者是溶瘤微生物和含有编码上述功能蛋白或其片段的 DNA 的质粒； 或 者是溶瘤微生物和包含编码上述功能蛋白或其片断的 DNA的复制缺陷性载体。 所述溶瘤微生物是指能选择性地在肿瘤细胞中生长裂解肿瘤细胞的病毒或细 菌。 通过溶瘤微生物在肿瘤细胞中大量复制而裂解肿瘤细胞， 将肿瘤细胞杀死， 释放患者自身的肿瘤抗原， 放大危险信号， 同步表达的功能蛋白自身或其与所 粘附的肿瘤抗原形成的复合体， 剌激并激活抗原提呈细胞， 促进抗原及时提呈， 激发患者自身免疫系统， 从而杀死局部的及转移的肿瘤。 在本发明的另一实施方案中， 用含有编码功能蛋白或其片段的 DNA的载体转 化患者的抗原提呈细胞。 所述蛋白是能粘附肿瘤抗原并能被抗原提呈细胞所识 别的蛋白。 含有编码功能蛋白或其片段 DNA 的载体转化的抗原提呈细胞， 对 释放的抗原更加敏感， 该提呈细胞释放的功能蛋白捕捉抗原后提交给抗原提呈 细胞， 进一步增强免疫信号， 激活机体免疫反应。 达到在杀灭肿瘤细胞的同时 诱导肿瘤患者体内特异性抗肿瘤免疫反应， 抑制肿瘤转移， 并杀灭已转移的远 端肿瘤细胞的目的。 在本发明的再一实施方案中， 给肿瘤患者施用能选择性地在肿瘤细胞中生长的 溶瘤微生物和上述功能蛋白或及其变体的组合物。 溶瘤微生物在肿瘤细胞中大 量复制而裂解肿瘤细胞， 将肿瘤细胞杀死， 释放患者自身的肿瘤抗原； 另外， 由于和溶瘤微生物同时施用给患者的所述功能蛋白本身具有和抗原提呈细胞的 亲和性， 其在抗原提呈细胞周围的大量出现也会放大危险信号， 更多地激活提 呈细胞， 激发患者自身的免疫应答， 从而杀死局部的及转移的肿瘤。 本发明提供的溶瘤微生物优选为选择性地在肿瘤细胞中生长的病毒或细菌。 病 毒实例是腺病毒、 单纯疱疹病毒、 水泡性口炎病毒、 新城疫病毒、 呼肠孤病毒 或其他能选择性地在肿瘤细胞中生长的病毒。 细菌的实例是伤寒沙门氏菌
{Salmonella)^ f (Bifidobacterium) 志贺氏菌 (>S 2 ge¾ 、李斯特菌 (ϋ·? 'β)、
Figure imgf000005_0001
梭菌 (c¾wtn'i¾ ) 21或其它能选择性地在肿瘤细胞中生长的 细菌。 所述的能粘附肿瘤抗原并能被抗原提呈细胞所识别的蛋白或功能蛋白优选热休 克蛋白。 热休克蛋白来源于哺乳动物和病原微生物。 所述哺乳动物包括但不限 于人、 非人灵长类动物和啮齿动物； 所述病原微生物包括但不限于结核分支杆 菌 (M tuberculosis) 麻风分支杆菌 (M leprae) 克氏椎虫 (2 ya".oiww crazz')禾口恶
Figure imgf000005_0002
fakipamm~)。 所述含有功能蛋白基因的溶瘤微生物可进一 步含有编码免疫增强分子的 DNA序列， 如 IL-2、 IL-12、 TNF、 IFN、 G-CSF、 GM-CSF、 趋化因子等在内的细胞因子， 免疫共刺激分子如 B7等。 本发明的溶瘤微生物、 抗原提呈细胞、 溶瘤微生物的组合物、 药物组合物或免 疫治疗剂可以单独使用， 也可以和其它形式的免疫治疗剂联合使用， 包括细胞 因子、 免疫佐剂和传统中药。 据推测， 本发明中的溶瘤微生物、 其组合物或药物组合物是通过如下一种或多 种途径达到抑制或消除转移性肿瘤的目的的： a) 能选择性在肿瘤细胞中复制的 溶瘤微生物溶解肿瘤细胞， 释放肿瘤抗原， 同步表达的能粘附肿瘤抗原并被抗 原提呈细胞识别的蛋白将肿瘤抗原实时提呈， 刺激抗原提呈细胞 （主要是被施 用者自身的树突状细胞）， 进一步激发 K细胞、 CTL细胞、 Th细胞等免疫效 应细胞的活性， 激活机体免疫反应； b ) 能粘附肿瘤抗原并被抗原提呈细胞识别 的蛋白本身具有和抗原提呈细胞的亲和性， 其在抗原提呈细胞周围的大量出现 也会放大危险信号， 更多地激活提呈细胞； 和 /或 c) 含有编码能粘附肿瘤抗原 并被抗原提呈细胞识别的蛋白或其片段 DNA的载体转化的抗原提呈细胞， 对 释放的抗原更加敏感， 该提呈细胞释放的功能蛋白捕捉抗原后提交给抗原提呈 细胞， 进一步增强免疫信号， 激活机体免疫反应。 达到在杀灭肿瘤细胞的同时 诱导肿瘤患者体内特异性抗肿瘤免疫反应， 抑制肿瘤转移， 并杀灭已转移的远 端肿瘤细胞的目的。 综上所述， 可以看出本发明的进步在于， 本发明克服了现有技术中的由于肿瘤 抗原的个体差异性， 用 "癌症疫苗"进行治疗的主要缺陷： 例如， 必须得到病 人自体的肿瘤细胞， 并在体外进行培养及一系列的实验操作的种种缺陷。 本发 明能够将患者自身肿瘤细胞裂解后释放的肿瘤特异性抗原实时提呈， 并且不论 肿瘤细胞所处的发展阶段以及肿瘤抗原的种类是否相,同， 都能激发机体针对自 身特定的肿瘤抗原有效的免疫反应。 本发明实现了在不同个体内利用个体自身 的抗原提呈细胞在体内实时动态地将其自身特异性的肿瘤抗原进行加工和提 呈， 从而激发针对各自不同肿瘤抗原的特异性免疫反应， 达到了既简单又有效 的治疗效果。 举例来说， 本发明通过溶瘤微生物将肿瘤细胞裂解， 同时由溶瘤 微生物所表达的对抗原提呈细胞具有亲和性的蛋白对释放的肿瘤抗原进行吸 附， 并随后由抗原提呈细胞如树突状细胞将肿瘤抗原加工提呈， 来激发机体本 身的免疫效应细胞， 包括： 特异性杀伤肿瘤的 CTL (细胞毒性 T淋巴细胞)， 能够调节机体免疫反应的 Th细胞 （辅助性 T淋巴细胞）， 和 NK细胞（自然杀 伤 T淋巴细胞）， 激活机体的免疫反应； 不但能够溶解局部肿瘤， 还可以进一步 抑制并杀灭转移肿瘤， 与单独用溶瘤病毒进行肿瘤治疗相比， 突破了其只能治 疗局部肿瘤的局限性。
附图简述 图 1 是本发明所构建的表达热休克蛋白的重组 HSV-1病毒质粒的图。
质粒 pHSV-HSP含有 CMV启动子控制下的 HSP70基因和 SV40多聚腺苷酸序 列 （SV40poly(A))。 该质粒还包括选择标记、 E. coli的复制起点 （ColEl ori)、 HSV a (HSV切割和包装信号)和 HSV ori (HSV的 DNA复制起点）。 图 2是显示重组病毒 HSV-HSP 的体外细胞毒性效果的图。 其中将鼠肿瘤细胞 CT26培养于 24孔板 （每孔 1 X 105) 并由 HSV-HSP或 HSV以不同的感染复数 (0.01-10)感染。 感染 4天后用 Promega试剂盒测定细胞毒性 ( % )。 图 2中 当感染复数为 1时， 细胞毒性已达 99%以上。 图 3 表示重组的 HSV-HSP在原位肿瘤内注射后能抑制远端肿瘤的良好能力。 在小鼠的肋部双侧对称接种 CT26细胞。 当肿瘤分别长到最大直径为 0.5cm时， 仅在鼠的右侧肿瘤瘤内注射接种 HSV-HSP ( 1 X 106) (以实心方块表示)、 HSV 载体 （1 X 106) (以实心菱形表示)和 HSP70蛋白 (以实心三角表示)作为对照 （该 日设为第 0日）， 过 7天再进行第二次接种 （每组 6只）。 与对照组相比， 经瘤 内注射接种 HSV-HSP的肿瘤和其对侧未被接种的肿瘤在注入 HSV-HSP后都会 有显著的肿瘤生长减慢（pO.001,感染后 25天， 非配对的 t检验）。 HSV-HSP 在抑制两侧的肿瘤生长显著优于 HSV以及 HSP70蛋白分别单独使用 （pO.001, 感染后 25天， 非配对 t检验）。 图 4是质粒 pLEP-HSP70-IRES-ElA的构建图。
利用双质粒法构建重组腺病毒， 首先将 HSP基因和对腺病毒在肿瘤细胞内具备 复制能力必须的 E1A基因通过图示的多克隆位点连接到质粒 pLEP上， 构建得 到重组质粒 pLEP-HSP70-IRES-ElA。 图 5表示重组病毒 Ad-HSP/ElA的构建过程。
将重组质粒 pLEP-HSP70-I ES-ElA和质粒 pREP通过酶切连接， 并将连接产物 用 Y噬菌体包装并感染大肠杆菌， 得到的重组质粒 pAd-Hsp70/ElA经 I-Ceul酶 切和转染 293细胞后得到能够表达 Hsp70的重组腺病毒。 图 6是本发明所构建的表达热休克蛋白的重组 pcDNA-HSP质粒图。
将 PCR扩增得到的 HSP基因通过平端连接连到真核表达质粒 pcDNA3.1上， 重 组质粒 pcDNA-HSP含有 CMV启动子控制下的 HSP70基因和 BGH多聚腺苷酸 序列 （BGHpolyA)，该质粒还包括选择标记以及复制起点 SV40ori.。 图 7表示表达热休克蛋白的沙门氏菌在原位肿瘤内注射后能抑制远端肿瘤的良 好能力。 在小鼠的肋部双侧对称接种 SMMC7721细胞。当肿瘤分别长到最大直径为 0.5cm 时， 仅在鼠的右侧肿瘤瘤内注射接种含有质粒 pcDNA-HSP 的沙门氏菌 (3τοΑ·) ( 1 X 107) (以实心方块表示)、 不含质粒的沙门氏菌 (aroA_) ( 1 X 107) (以实心菱 形表示)和 HSP70蛋白 (以实心三角表示)作为对照 （该日设为第 0 日）， 过 7天 再进行第二次接种 （每组 6 只）。 与对照组相比， 经瘤内注射接种含有质粒 pcDNA-HSP 的沙门氏菌 (aroA_)的肿瘤和其对侧未被接种的肿瘤都会有显著的肿 瘤生长减慢（pO.001,感染后 25天， 非配对的 t检验）。 HSV-HSP在抑制两侧 的肿瘤生长显著优于 HSV和 HSP70蛋白分别单独使用 （pO.001,感染后 25天， 非配对 t检验）。 发明详述
在本专利申请的上下文中， 采用的术语具有本领域中所熟知的一般含义。 为清 楚起见， 本专利申请中的下列术语具有如下所说明的含义-
"溶瘤微生物 "这一术语在此定义为天然存在的或经过遗传改造的一类能够进入 肿瘤细胞， 并通过选择性地在肿瘤细胞内大量复制而导致肿瘤细胞裂解死亡的 微生物。 溶瘤微生物的实例包括如下定义的溶瘤病毒和溶瘤细菌。
"溶瘤病毒"这一术语在此定义为一种能够选择性地在肿瘤细胞内复制并杀死 肿瘤细胞而不论肿瘤细胞的 p53 或其它蛋白是否突变。 溶瘤病毒的实例包括单 纯疱疹病毒 （如 HSV-l ), 腺病毒 （如 ONYX-015), 新城疫病毒（NDV)， 水 泡性口炎病毒 （VSV)等。
"溶瘤细菌" 这一术语在此定义为一种经过遗传改造的细菌， 该细菌能够在肿 瘤细胞内复制并杀死肿瘤细胞。 已知一些细菌的突变株可以作为肿瘤的基因治 疗载体 2Q。 例如， 沙门氏菌中编码嘌昤 （pur) 或芳香族氨基酸（aroA) 的基因 突变后， 不能自己合成这些对其必不可少的营养物质。 这些物质在正常细胞中 并不存在， 而肿瘤细胞由于遗传特性的改变可以提供这些营养物质。 所以这些 营养缺陷型细菌能够选择性地在肿瘤细胞内扩增而使其裂解， 如 Salmonella typhimurium YS72(pur-)在荷瘤鼠腹膜接种 2天后， 可检测到在肿瘤细胞中与在 正常肝细胞中的比例为 9000:12Q。 将表达热休克蛋白的基因引入这些细菌突变 株， 再利用突变型细菌能够在肿瘤细胞中富集并使其裂解的这种特性， 这些细 菌完全可以作为本发明的溶瘤微生物使用。
"粘附肿瘤抗原的并被抗原提呈细胞所识别的蛋白"这一术语在此定义为一类蛋 白， 该蛋白能够通过吸附、 粘附或结合等作用与肿瘤特异抗原多肽连接而形成 蛋白-多肽复合物， 并通过抗原肽伴侣转移作用将抗原提交给树突状细胞等抗原 提呈细胞， 抗原被加工提呈， 激发机体免疫反应。 这类蛋白的实例包括以热休 克蛋白为主的分子伴侣蛋白。
"复制缺陷型载体"这一术语在此定义为不具备复制能力的病毒等微生物， 但 其表达外源蛋白的能力不受影响。
"热休克蛋白" 这一术语在此定义为一族高度保守具有 ATP酶活性的分子， 包括热休克蛋白变体。 热休克蛋白在所有的原核生物以及大多数真核细胞中都 存在； 无论是否存在外界压力， 其在蛋白质代谢中都起着重要作用， 包括在蛋 白质从头合成途径中蛋白质的折叠和跨膜转运以及错误折叠后的蛋白的降解。 所述热休克蛋白， 包括从肿瘤细胞和细菌感染的细胞中得到的 hsp70、 hsp90和 gp94/gp96, 他们都能够激发细胞性的免疫反应。
"表达热休克蛋白的质粒 " 这一术语在此定义为能够在体内进行真核表达热休 克蛋白或其变体的质粒 DNA， 编码热休克蛋白其变体的 DNA序列以适当的方 式位于由相关的启动子、 polyA、 增强子等调控元件组成的序列之中。
"热休克蛋白的变体" 这一术语在此定义为热休克蛋白的片段或各种修饰， 包 括但不限于一个或多个氨基酸的添加、 缺失或替换以及和外源肽组成融合蛋白， 通过对热休克蛋白的这种修饰可以增强热休克蛋白对抗原的粘附能力； 或者可 以将靶定位序列如信号肽， 核定位序列等与热休克蛋白 DNA连接使热休克蛋 白能够改变其在细胞内的分布状态， 如热休克蛋白由非分泌型转变为分泌型或 由定位于胞内转变为定位于胞外， 从而使其更好的与抗原多肽结合。
"编码能粘附肿瘤细胞抗原并被抗原提呈细胞所识别的蛋白或其片段的 DNA 的质粒" 在此定义为能够在体内进行真核表达所述蛋白或其变体的质粒 DNA, 编码所述蛋白或其变体的 DNA序列以恰当的方式位于由相关的启动子、 polyA、 增强子等调控元件组成的序列之中。
"包含编码能粘附肿瘤细胞抗原并被抗原提呈细胞所识别的蛋白或其片段的 DNA的复制缺陷型载体"在此定义为不具备复制能力的病毒等微生物， 但其表 达所述蛋白或其变体的能力不受影响。
"佐剂" 是一类和抗原共同使用或其本身就能够充当抗原来能够增强免疫反应 的物质。
"抗原" 这一术语在此定义为能激发免疫应答的一种分子。 免疫反应包括产生 抗体， 具备特异的免疫活性细胞的活化， 或两者的组合。 抗原可以产生于生物 体、 蛋白或抗原的亚单位、 被杀死或被去活化的全细胞或裂解物。
"癌症" 这一术语在此定义为一种恶性的细胞性肿瘤并能够侵袭其它细胞。 其 实例包括但并不限于乳腺癌， 前列腺癌， 卵巢癌， 子宫癌， 皮肤癌， 胰腺癌， 结肠癌和肺癌。
"癌症疫苗"这一术语在此定义为肿瘤特异性抗原分子或接触过肿瘤细胞的免疫 细胞。 肿瘤细胞所表达的， 使肿瘤细胞具有免疫原性从而能被免疫系统识别的 蛋白质为肿瘤特异性抗原， 通过体外制备该抗原蛋白质或其具有免疫效应的抗 原表位多肽； 或通过体外免疫细胞与肿瘤细胞接触使免疫细胞能够识别该种抗 原， 上述蛋白或多肽或免疫细胞即可作为治疗性的癌症疫苗。
"杀灭肿瘤"这一术语在此定义为能够有效地抑制肿瘤的扩增、 浸润和转移以 及使肿瘤消退或转化成良性。
"表达" 这一术语在此定义为由其启动子启动的一段特定序列的转录和 /或翻 译。
"主要组织相容性复合物"或 "MHC" 这一术语在此定义为一簇特殊的基因， 其中大部分编码在细胞表面进行抗原提呈的蛋白， 其中对决定组织相容性最重 要的包括 I类组织相容性复合物， 或 MHC I,其功能主要是将抗原提呈给 CD8 T 淋巴细胞； II类组织相容性复合物，或 MHC II,其功能主要是将抗原提呈给 CD4 T淋巴细胞。
"启动子"这一术语在此定义为一段核酸序列， 能够调控一段特殊的核酸序列 的转录。 启动子这一术语包括增强子、 沉默子和其它顺式调控原件。
"T淋巴细胞" 在此定义为胸腺来源的能够参与一系列的细胞介导的免疫反应 的一类细胞。
"抗原提呈细胞" 在此定义为其功能为加工提呈抗原给 T细胞和 B细胞的一 类细胞， 该类细胞包括树突状细胞， 巨噬细胞和 B细胞。
"免疫增强分子"在此定义为能增强免疫作用的分子， 包括 IL-2、 IL-12、 TNF、 IFN、 G-CSF、 GM-CSF、 趋化因子等在内的细胞因子和免疫共剌激分子如免疫 共刺激分子 B 7等。
"免疫有效量 "这一术语在此定义为能够有效地激发机体产生抑制或杀死肿瘤细 胞的免疫反应的剂量。 在本发明中， 我们采用的策略是： 在不需要体外制备肿瘤抗原甚至预先不知道 患者体内具有个体差异性的特异性肿瘤抗原的情况下， 利用患者自身的肿瘤抗 原和自体中存在的抗原提呈细胞， 在体内实现肿瘤免疫治疗。 为实现本发明的目的， 我们提供了能在肿瘤患者体内诱导特异性抗肿瘤免疫反 应的携带有功能蛋白基因的溶瘤微生物， 将该微生物对肿瘤患者施用后， 能够 起到在溶解肿瘤细胞的同时刺激抗原提呈细胞， 激发机体同步免疫反应， 达到 抑制肿瘤转移， 杀灭远端肿瘤细胞的目的。 具体说， 本发明提供的一个实施方案是构建一种能表达能粘附肿瘤细胞抗原并 被抗原提呈细胞所识别的功能蛋白的重组溶瘤微生物。 用其感染局部肿瘤， 该 微生物在肿瘤细胞中生长而在正常细胞中基本上不生长。 溶瘤微生物在肿瘤细 胞中生长的同时， 表达能粘附肿瘤细胞抗原的并被抗原提呈细胞所识别的蛋白。 由于溶瘤微生物在肿瘤细胞中的大量复制， 导致肿瘤细胞裂解， 释放出各种肿 瘤细胞的抗原。 这些抗原与溶瘤微生物所表达的能粘附肿瘤细胞抗原的并被抗 原提呈细胞所识别的蛋白结合， 形成抗原一蛋白复合物。 该复合物进一步被抗 原提呈细胞识别， 经抗原提呈细胞加工后提呈给能够特异性杀伤肿瘤细胞的细 胞毒性 T淋巴细胞 （CTL)， 并能够激发调节机体免疫反应的 Th细胞 （辅助性 T淋巴细胞）， 和 NK细胞 （自然杀伤 T淋巴细胞）等免疫效应细胞来增强机体 的免疫反应。 在本发明的另一实施方案中， 将溶瘤微生物和能粘附肿瘤细胞抗原并被抗原提 呈细胞所识别的蛋白或表达该蛋白的载体联合施用， 也能实现本发明的目的。 其中所述微生物能选择性的在肿瘤细胞中生长以溶解肿瘤细胞， 使肿瘤抗原释 放出来。 所述蛋白能够吸附抗原多肽而形成蛋白-多肽复合物， 并通过抗原肽伴 侣转移作用将抗原提交给树突状细胞等抗原提呈细胞， 抗原一蛋白复合物经抗 原提呈细胞加工， 进一步激发 K细胞、 CTL细胞、 Th细胞等免疫效应细胞的 活性， 激活机体特异性免疫反应； 同时， 由于所述蛋白本身具有和抗原提呈细 胞的亲和性， 起到放大危险信号并激活提呈细胞的作用， 最后激活机体的特异 性抗肿瘤的免疫反应。 在本发明的另一实施方案中， 用含有编码能粘附肿瘤细胞抗原并被抗原提呈细 胞所识别的蛋白或其片段 DNA的载体在体内或体外转化肿瘤患者的抗原提呈 细胞。 如果在体外转化抗原提呈细胞， 则在转化后将所述被转化的抗原提呈细 胞施用给患者， 同样也能实现本发明的目的。 转化的抗原提呈细胞对抗原更加 敏感， 提呈细胞表达的功能蛋白捕捉抗原后又返回提呈细胞， 进一步增强免疫 信号， 激活机体的特异性抗肿瘤免疫反应。 综上所述， 除本发明的表达能粘附肿瘤细胞抗原并被抗原提呈细胞所识别的蛋 白或其片段的溶瘤微生物外， 本发明还包括但不限于以下几种组合形式- 一种组合， 其包括：
a) 能选择性地在肿瘤细胞中生长的微生物； 和
b) 包含编码能粘附肿瘤细胞抗原并被抗原提呈细胞所识别的蛋白或其片段 的 DNA的质粒。
另一种组合， 其包括- a) 能选择性地在肿瘤细胞中生长的微生物； 和
b) 复制缺陷性载体， 该载体包含编码能粘附肿瘤细胞抗原并被抗原提呈细 胞所识别的蛋白或其片段的 DNA。
再一种组合， 其包括：
a) 能选择性地在肿瘤细 1¾中生长的微生物； 和
b) 能粘附肿瘤细胞抗原并被抗原提呈细胞所识别的蛋白。 以上所述微生物还可进一步含有编码免疫增强分子的 DNA序列， 如编码 IL-2、 IL-6、 IL-12、 TNF、 IFN、 G-CSF、 GM-CSF或趋化因子等的细胞因子和 /或免 疫共刺激分子如 B7等的序列。 本发明的微生物、 微生物组合物可以单独使用， 也可以和其它形式的免疫治疗剂联合使用， 例如， 和细胞因子、 免疫佐剂或传 统中药联合使用。 所述的微生物组合物可进一步含有可药用载体。 本发明中所述的 "功能蛋白"或 "能粘附肿瘤抗原并被抗原提呈细胞所识别的 蛋白"是指能够结合肿瘤抗原并对抗原提呈细胞有亲和性的一类蛋白， 这类蛋 白的实例包括以热休克蛋白为主的分子伴侣蛋白。 热休克蛋白能够结合肿瘤特 异抗原多肽而形成蛋白-多肽复合物， 并通过抗原肽伴侣转移作用将抗原提交给 树突状细胞等抗原提呈细胞， 抗原被加工提呈， 激发机体 NK细胞、 CTL细胞、 Th细胞等免疫效应细胞的活性， 激活机体特异性免疫反应。 热休克蛋白本身具 有和抗原提呈细胞的亲和性， 也能够激活提呈细胞。 在一个具体实施例中， 我们采用表达热休克蛋白的溶瘤微生物感染局部肿瘤。 试验中， 在原位肿瘤表达的热休克蛋白能够结合肿瘤抗原并通过癌症疫苗免疫 的方式起到免疫调节的作用， 显著引发、 增强对肿瘤抗原的免疫反应， 在杀灭 原位肿瘤的同时抑制远端肿瘤。 热休克蛋白 (Heat Shock Proteins, HSP)是一类高度保守并具有 ATP酶活性的蛋白 家族， 在所有的原核生物以及大多数真核细胞中都存在。 无论是否存在外界压 力， 热休克蛋白在蛋白质代谢中都起着重要作用， 包括在蛋白质从头合成途径 中蛋白质的折叠和跨膜转运以及错误折叠后的降解 5。 热休克蛋白， 包括从肿瘤 细胞和细菌感染的细胞中得到的 Hsp70、 Hsp90和 grp94/gp96， 都能够激发细胞 性的免疫反应 6_7。 可以替代的热休克蛋白也可以来源于病原微生物， 包括结核 分支杆菌 (M tuberculosis) ,麻风分支杆菌 ( leprae) ,克氏锥虫 ( Trypanoma cruzO ,恶性疟原虫 <iPla画 df m falciparum ; 以及其它物种如灵长类、 鼠等 哺乳动物。 热休克蛋白的免疫原性主要是由结合到其分子上的多肽所产生 8。 产生的热休克 蛋白一抗原多肽通过一种不表达内生性抗原的特异性细胞将抗原呈递给 T淋巴 细胞后， 从而激活免疫系统。 这些结果表明热休克蛋白作为分子伴侣能够将抗 原性的多肽呈递给抗原提呈细胞 主要为树突状细胞， 其可能的机制是通过使 这些多肽与 MHC I和 MHC II分子结合而进入 MHC I和 MHC II呈递抗原途径； 这些抗原多肽会进一步激活 CTL细胞、 NK细胞、 Th细胞等免疫效应细胞的活 性， 激发机体特异性免疫反应。 各种热休克蛋白、 其片段或各种修饰 （包括但不限于一个或多个氨基酸的添加、 缺失和 /或替换等）变体均可用于本发明， 只要该热休克蛋白、 片段或其变体保 持与肿瘤细胞抗原多肽结合的能力即可。 在本发明的具体应用中， 为增强热休克蛋白对抗原的粘附能力， 可通过对热休 克蛋白一个或多个氨基酸的添加、 缺失或替换或者与外源肽组成融合蛋白等手 段进行修饰。 也可以根据需要将靶定位序列 （如信号肽)、 核定位序列等与热休 克蛋白连接使热休克蛋白能够改变其在细胞内的分布状态， 如热休克蛋白由非 分泌型转变为分泌型或由定位于胞内转变为定位于胞外， 从而使其更好地与抗 原多肽结合。 本发明的溶瘤微生物是天然的或经遗传改造后能选择性地在肿瘤细胞中复制的 病毒或细菌。 所述病毒包括单纯疱疹病毒如 HSV-1 和 G207, 腺病毒 （AdV) 如 ONYX-015 (也称为 dll520)、 CN706和 CN787, 新城疫病毒 ( DV)如 73-T， 水泡性口炎病毒 （VSV)， 呼肠孤病毒如 Reolysin 3或其他能选择性地在肿瘤细 胞中生长的病毒。 所述细菌包括伤寒沙门氏菌、 双岐杆菌、 志贺氏菌、 李斯特 菌、 鼠疫杆菌、 梭菌 21以及其他天然存在和经诱变筛选后能选择性地在肿瘤细 胞中生长的细菌。 溶瘤微生物也指上述微生物的各种变体， 只要该微生物具有 选择性地在肿瘤细胞中复制和生长从而将肿瘤细胞裂解的能力即可。 因此， 本领域的普通技术人员可以认识到， 无论编码能粘附肿瘤抗原的并被抗 原提呈细胞所识别的蛋白如热休克蛋白的基因是整合到溶瘤微生物的基因组还 是游离于所述微生物的基因组之外如存在于其它表达载体中， 只要保证所述的 基因与调控序列如启动子和终止子有效地连接， 所述的溶瘤微生物如 HSV-1或 dll520就可以同所表达的能粘附肿瘤抗原的并被抗原提呈细胞所识别的蛋白如 热休克蛋白共同作用， 达到本发明的目的。 在不背离本发明的范围和精神情况 下， 可对本发明申请中各种实例进行不同的改变或修改， 这一点对于本领域的 技术人员是很显而易见的。 在本发明的一个具体的实施例中， 公开了携带热休克蛋白 Hsp70基因的溶瘤腺 病毒 Ad-HSP/ElA。 将 Hsp70基因导入 dll520 (其为一种能够选择性的在 p53 功能缺陷的肿瘤细胞内复制的减毒腺病毒 9_14) 中构建重组溶瘤腺病毒， 具体过 程参见实施例 2。 采用重组溶瘤腺病毒 Ad-HSP/ElA进行了动物试验， 这种病 毒表现非常强的抗肿瘤活性， 能够有效地使肿瘤体积减小甚至消除（见下表）。 下表还列出了热休克蛋白 Hsp70或表达热休克蛋白 Hsp70的载体与溶瘤腺病毒 Ad-ΔΕΙΒ组成联合体， 在不同的肿瘤模型动物上进行的试验结果：
Figure imgf000013_0001
注：
组 1： 能表达热休克蛋白 Hsp70的重组溶瘤腺病毒 Ad-Hsp/El A; 组 2: 溶瘤腺病毒 Ad-ΔΕΙΒ和表达热休克蛋白 Hsp70的 DNA质粒； 组 3: 溶瘤腺病毒 Ad-ΔΕΙΒ和表达热休克蛋白 Hsp70的复制缺陷型病毒； 组 4: 溶瘤腺病毒 Ad-ΔΕΙΒ和热休克蛋白 Hsp70;
CT26/Balb/c表示在 Balb/c品系的小鼠上建立的结肠癌 CT26肿瘤模型。
TRAMP-c2/c57表示在 c57品系的小鼠上建立的前列腺癌 TRAMP-c2肿瘤模 型。
B16/C57表示在 c57品系的小鼠上建立的黑素瘤 B16肿瘤模型。 在相应的小鼠肋部对称部位（左侧和右侧） 分别进行皮下注射接种相应的肿瘤 细胞 2X 105个。 将这些小鼠作为患有转移性肿瘤的动物的模型。 当皮下注射的 肿瘤迅速生长 （在接种后大约 8-10天） 时， 将实验小鼠的非对称 （右面） 的瘤 内接种重组 Ad-Hsp/El A约 2 X 109 PFU; 或 Ad-ΔΕΙΒ约 2 X 109 PFU和表达热 休克蛋白 Hsp70的 DNA质粒 50微克； 或溶瘤腺病毒 Ad-ΔΕΙΒ约 2 X 109 PFU 和表达热休克蛋白 Hsp70的复制缺陷型病毒约 2X 109 PFU; 或溶瘤腺病毒 Ad_- ΔΕ1Β约 2X 109 PFU和热休克蛋白 Hsp70约 10-25微克。 瘤内注射治疗后每隔 3-4天测量肿瘤的体积。 表中列出了初步的实验结果， 其中 " + "表示已有初步 的实验结果表明不但被瘤内注射治疗侧的肿瘤的体积明显缩小， 对侧的未被注 射的肿瘤的体积也明显缩小。 "一"表示实验数据尚在进一步的整理之中。 在另一个具体的实施例中， 采用的溶瘤病毒是单纯疱疹病毒。 一种具备复制能 力的单纯疱疹病毒的突变株经过大量的不同种的动物模型试验发现为非病原性 的并被用于人的原发性脑部肿瘤治疗的临床试验 16。 这种单纯疱疹病毒突变株 在分裂细胞内的复制能够导致细胞死亡， 其在非分裂细胞内的复制能力则非常 弱， 在接种到荷瘤鼠后能够选择性的在肿瘤内复制， 从而抑制原位肿瘤的生长 并延长荷瘤鼠的生存期 15·17。 将编码热休克蛋白 HSP70 的扩增质粒导入这种单 纯疱疹病毒突变株，构建重组单纯疱疹病毒， 两种具体构建方法如下： 一种方法：从人的 cDNA文库中通过 PCR扩增得到 HSP70的 cDNA，进行了 DNA 序列测定。 用 Klenow酶补平上述 PCR产物的 DNA片段两端， 然后平末端插 入连接于质粒 pHSV中的 Spel位点， 得到质粒 pHSV— HSP。 通过重组质粒和 辅助病毒共转染真核细胞， 在细胞内辅助病毒对重组质粒进行包装， 从而得到 能够表达热休克蛋白的重组单纯疱疹病毒。 具体构建过程见实施例 1。 另一种方法： 先构建含有在启动子控制下 HSP70的载体， 然后按照本领域的常 规方法将 HSP DNA插入到 HSV的基因组中， 以得到 HSP基因整合到病毒基因 组中的重组单纯疱疹 DNA。 所得到的这种重组单纯疱疹病毒可以直接给肿瘤患 者施用， 而不需要体外通过 HSV辅助病毒和质粒 DNA的共转染来制备表达热 休克蛋白的单纯疱疹病毒。 在另一个具体的实施例中， 采用的溶瘤病毒是腺病毒。 所釆用的腺病毒突变株 能够选择性的在肿瘤细胞中复制而使细胞裂解死亡， 其在正常细胞内的复制能 力则非常弱 1Q。 这种腺病毒突变株在接种到荷瘤鼠后能够选择性的在肿瘤内复 制， 从而抑制原位肿瘤的生长并延长荷瘤鼠的生存期 9— "。 通过 pLEP和 pREP 双质粒系统构建重组腺病毒， 将 HSP基因和腺病毒在肿瘤细胞内具备复制能力 所必须的 E1A基因连接到质粒 pLEP 上， 构建得到重组质粒 pLEP- HSP70- IRES-E1A。 将所得的重组质粒和质粒 pREP通过酶切连接， 并将连接产物用 Y 噬菌体包装并感染大肠杆菌， 得到的重组质粒 PAd-Hsp70/ElA经 I-Ceul酶切和 转染 293细胞后得到能够表达 Hsp70的重组腺病毒。 具体过程见实施例 2。 在另一个具体实施例中， 采用能够特异性的在肿瘤细胞内复制的沙门氏细菌作 为表达热休克蛋白的载体， 因为所用的沙门氏菌为 aroA缺陷型 I9， 其所需要的 营养只能由肿瘤细胞提供， 在正常细胞中不能够复制生长。 所以该突变株能够 选择性的在肿瘤细胞内复制并通过其增殖使肿瘤细胞裂解死亡。 沙门氏菌表达 的热休克蛋白能够结合肿瘤细胞裂解所释放的抗原并将其传送给抗原提呈细 胞， 从而激发了 CTL介导的能够特异性杀死肿瘤细胞的免疫反应， 从而抑制了 肿瘤的生长， 延长了荷瘤鼠的生存期。 具体过程见实施例 3。 在本发明中， 编码热休克蛋白 Hsp70的序列受启动子的转录调控。 "启动子" 是指能被细胞自体的或引入的转录复合物所识别的 DNA序列， 其对于一个基 因地转录起始是必须的。 对于启动子是如何组织的， 人们通过分析多种不同病 毒如 CMV (巨细胞病毒）， HSV胸苷激酶 （tk)和 SV40的启动子来进行研究。 近来的研究表明， 启动子由不连续的功能调控元件组成， 每个约 7— 20bp DNA， 并且包括一个或更多的由转录活化或抑制蛋白的识别位点。 其它类型的启动子 例如增强子能够调控转录起始的频率。 典型的启动子序列都是位于起始位点上 游 30— llObp 处， 尽管现在表明有包含在起始位点下游的调控元件的启动子。 启动子各元件之间的距离通常是可改变的， 所以当某些元件被插入或移去时仍 能保持启动子的正常功能。 在 tk启动子中， 在保持活性的前提下， 启动子各元 件之间的距离最多可以增加到 50bp。 根据各启动子类型不同， 各元件可互相协 同或独立地活化转录反应。 一个启动子可以是一个基因或一段序列自身的， 即从编码区和 /或外显子的上游 5'端非编码序列得到。 这样的启动子为 "内源性"启动子。 我们同样可以得到 这个基因或这段序列上游或下游的增强子。 可替代的， 将并非基因或序列自身 的启动子也即重组或异源启动子置于编码序列的上游进行转录调控会有特殊优 势。 重组或异源的启动子包括并非在自然环境中与基因或序列相关的增强子。 这种启动子和增强子可包括其它基因的启动子和增强子， 以及从原核生物， 病 毒或真核细胞中分离的启动子和增强子， 或者并非 "天然存在"的启动子和增 强子， 例如不同转录调控区的不同调控元件的组合， 和 /或引入能够影响功能的 突变。 而且在合成启动子或增强子序列时， 还可以使用重组克隆和 /或包括 PCR 在内的核酸扩增技术。 而且， 也可以使用其它细胞器如线粒体， 叶绿体的能够 调控转录和 /或表达的序列。 在实施例中所釆用的为巨细胞病毒 （CMV) 的前早期启动子序列。 这个启动子 属于组成型的强启动子能够启动其下游序列的髙水平表达。 同时， 也可使用其 它组成型的启动子如猴病毒 SV40前早期启动子， 小鼠乳腺瘤病毒（MMTV) 启动子， 人免疫缺陷病毒 （HIV)长末端重复序列 （LTR) 启动子， 鼠病毒启动 子， 鸟白血病病毒启动子， EB病毒前早期启动子， 劳氏肉瘤病毒启动子， 以及 人类基因启动子， 例如 （但不限于）： 肌动蛋白启动子， 肌球蛋白启动子， 血红 蛋白启动子， 肌酸启动子。 而且本发明并不仅限于用组成型启动子， 也可以选 用诱导型启动子。 本发明中诱导型启动子的使用提供了在需要时能打开其相连 的下游核酸序列表达的分子开关， 并在不需要其表达时将其关闭。 诱导型启动 子包括但并不局限于： 金属硫蛋白启动子， 糖皮质激素启动子， 孕酮启动子， 四环素启动子。 而且， 本发明还包括使用组织特异性启动子， 其转录活化只能 在特定组织内进行。 组织特异性启动子包括但不限于现在已知的如： HER— 2 启动子， PSA相关启动子序列。 对于热休克蛋白的表达， 需要一个典型的多聚腺苷酸信号来完成转录的正确多 聚腺苷酸加尾反应。 多聚腺苷酸的性质对于成功的实施本发明并不是至关重要 的， 任何类型的中止序列都可以使用。 优选的实施例包括 SV40 多聚腺苷酸信 号， LTR多聚腺苷酸信号和 /或牛生长激素多聚腺苷酸信号， 对于不同的靶向细 胞应以方便原则和 /或已知能在其中正确行施功能的原则选择多聚腺苷酸信号。 也可以选用转录中止位点来中止转录。 这些元件应能够增强转录的翻译中止水 平和 /或避免通读到其它序列。 重要的一点是， 本发明中表达热休克蛋白的载体正确进行蛋白表达并不需要载 体整合到受体细胞的基因组中。 载体可以以游离体状态存在于靶细胞中。 例如， 在某些类型的细胞中载体表达目标蛋白并不需要载体本身的复制。 这些细胞如 肌肉细胞通常是不能正常复制的， 被导入非分裂细胞的表达载体在载体本身不 复制的情况下能够表达目的蛋白。 本发明是还可以将溶瘤微生物与能粘附肿瘤细胞抗原并被抗原提呈细胞所识别 的蛋白或表达该蛋白的载体进.行联合施用， 或用含有编码所述蛋白或其片段的 DNA的载体转染患者自身的抗原提呈细胞。 从而引发机体针对肿瘤抗原的特异 性免疫反应， 杀死局部肿瘤和转移的肿瘤。 本发明的微生物或含有所述溶瘤微 生物的组合物可以单独使用， 也可以和其它形式的免疫治疗剂联合使用， 例如 和细胞因子或传统中药一起使用。 在许多实际应用中， 免疫调节剂如佐剂或称 为免疫刺激剂被用来增强免疫反应。 大量研究表明， 同时给予细胞因子或表达 细胞因子的质粒能够增强抗肿瘤的免疫活性。 一个本领域的技术人员很容易认 识到可以将细胞因子的核酸序列和本发明实施方案中的 HSP的核酸序列放入同 一载体来共同应用， 这样就避免使用两个表达载体。 另外， 一个本领域的技术 人员很容易认识到， 为了提高热休克蛋白在肿瘤细胞中的表达量可以将多个拷 贝的 HSP基因引入同一载体中。 在适当的情况下， 可以将本发明的溶瘤微生物、 微生物组合物或药物组合物制 成免疫治疗剂。 本发明的携有功能蛋白基因的溶瘤微生物、 溶瘤微生物的组合 物或药物组合物可根据其被施用的途径不同被加工成固体， 半固体， 液体或气 雾剂形式。 可以利用已知的方法来阻止组合物在到达靶器官前被释放和吸收。 本发明中可釆用任何一个可以接受的药剂形式保证本发明组分的有效性。 在制 剂时， 本发明中的微生物可以单独使用， 也可和其它的药学上活性组分以适当 的比例结合 /配合使用。 应保证在给药时有足够多量的携有能粘附肿瘤抗原的并 被抗原提呈细胞所识别的蛋白或其片段的 DNA的溶瘤微生物如溶瘤病毒或溶瘤 细菌， 从而其基因产物能提供药学上有效的剂量。 溶瘤微生物可以单独使用也 可以和不同的佐剂配合使用。 通常， 可将上述本发明的溶瘤微生物、 微生物组合物、 药物组合物或免疫治疗 剂以免疫有效量通过本领域已知的各种常规的和可接受的方式单独或与其它治 疗剂一起联合给药。 免疫有效量将根据疾病的严重程度、 个体的年龄和相对健 康状况、 所用微生物的效力以及其它因素有很大变化。 通常， 本领域普通技术 人员可以根据个人知识以及本申请所公开的内容确定出用于治疗给定肿瘤时的 本发明的微生物的免疫有效量。 可以和本发明的微生物共同使用的佐剂包括（但不限于） 弗氏完全佐剂、 弗氏 不完全佐剂、 氢氧化铝、 卡介苗（BCG)、 脂多糖 (LPS)、 脂质 A类似物、 胞壁 酰二肽 （MDP) 及其类似物、 内毒素（LT)和霍乱毒素 （CT)。 本发明的药物可通过下列途径之一给药： 瘤内注射、 口服、 全身性给药 (例如， 经皮、 鼻内或通过栓剂给药)或胃肠外给药 (例如， 肌肉内、 静脉内或皮下)。 药 物组合物或免疫治疗剂可以是片剂、 丸剂、 胶囊、 半固体、 散剂、 缓释制剂、 溶液剂、 混悬剂、 气雾剂的形式或是任何其它适宜的组合物， 并且通常由本发 明溶瘤微生物以及至少一种可药用赋形剂组成。 可药用赋形剂是无毒的、 对所 述微生物的活性没有实质性损害的、 有助于给药的并且不会对其他活性成分的 免疫或治疗效果产生不利影响的物质。 所述赋形剂可以是任何固体、 液体、 半 固体， 或者在气雾剂组合物的情况下， 可以是气体赋形剂。 这些赋形剂是本领 域技术人员易于得到的。 固体药物赋形剂包括淀粉、 纤维素、 滑石、 葡萄糖、 乳糖、 蔗糖、 明胶、 麦芽、 米、 面粉、 白垩、 硅胶、 硬脂酸镁、 硬脂酸钠、 甘油单硬脂酸酯、 氯化钠、 脱 脂奶粉等。 液体和半固体赋形剂可以选自水、 乙醇、 甘油、 丙二醇和各种油， 包括来源于石油、 动物、 植物或合成的油 (例如， 花生油、 大豆油、 矿物油、 芝 麻油等)。 优选的液体载体， 特别是用于可注射溶液的液体载体包括水、 盐水、 葡萄糖水溶液和二甘醇。 药物组合物中本发明微生物的量可以根据制剂的种类、 单位剂量的大小、 赋形 剂的种类以及药学领域技术人员已知的其它因素有很大变化。 而且， 实际的剂 量和治疗方案会根据其它因素如该发明的组分是否和其它治疗组分结合， 个体 的药物动力学， 药物分布和代谢等各不相同而有所变化。 而且， 加入细胞的病 毒量也随着插入载体的治疗基因的长度、 稳定性， 和序列的性质而不同， 是一 个由经验决定的变量， 很可能被非本发明中方法之外的其它因素所改变。 一个 本领域的技术人员可以根据具体情况的出现很容易的进行调整。 下面以实施例的方式具体描述本发明。 以下的实施例是举例性的， 因此不能将 这些实施例看成是对本发明的限制。
实验实施例 实施例 1 人热休克蛋白 （HSP70)基因的扩增及克隆- 利用 PCR方法从人肿瘤细胞系 SKOV3细胞 （ATCC HTB-77) 的 cDNA中扩增 HSP70基因。 上游引物序列为： GGT ATG GAA GAT CCC TCG AGA TC， 下游 引物序列为： TA CTA ATC TAC CTC CTC AAT GGT GGG。使用 PE公司的 PCR 仪， 反应条件是： 每个反应的引物终浓度是 30pM, dNTP为 lOOmM, 模板 DNA 为 100ng， Taq DNA聚合酶为 2.5个单位。 其他反应条件依照 GeneAmp DNA扩 增试剂盒中的使用说明进行， 每个反应总体积为 100微升。 每份反应混合物上 覆盖以 75微升矿物油。 扩增共进行 30个循环， 每一循环包括变性步骤 92 °C 1 分钟， 退火步骤 50°C 1分钟， 延长步骤 72°C 2分钟。反应结束后， 使用 Qiagen 公司的 PCR产物纯化试剂盒纯化所得产物。 溶瘤病毒一单纯疱疹病毒的构建
我们采用的是具备复制能力的、 突变型 I型单纯疱疹病毒 （HSV-1 ) 15 18。 其中 Y 34.5基因被缺失。 HSV-1 突变株在分裂型细胞中的复制会导致细胞死亡； 其 在非分裂型细胞中的生长能力显著减弱 16。 这种单纯疱疹病毒突变株在接种到 荷瘤鼠后能够选择性的在肿瘤内复制， 从而抑制原位肿瘤的生长并延长荷瘤鼠 的生存期 16。 HSV被用来在肿瘤内投递热休克蛋白。 人 HSP70的 cDNA是从人 的 cDNA文库中通过 PCR扩增得到并进行了 DNA序列测定。 用 Klenow酶补 平上述 PCR产物的 DNA片段两端 ，然后平末端插入连接于质粒 pHSV (Geller, A. I. and Breakefield, X. 0.(1988), Science 241 :1667-1669) 中的 Spel位点， 得到 质粒 pHSV-HSP(如图 1所示)。 Y 34.5基因被缺失的 HSV— 1突变株， 作为辅助 病毒来生产重组病毒 HSV— HSP。 使用质粒 pHSV-HSP DNA和单纯疱疹病毒 辅助病毒 DNA共转染疱疹病毒宿主细胞。 在 HSV辅助病毒存在的条件下， pHSV-HSP被包装于单纯疱疹病毒的外壳中， 这样就产生了带有 HSP70基因的 重组单纯疱疹病毒。 重组病毒是由单纯疱疹病毒辅助病毒对编码 HSP的扩增质 粒进行包装得到的 17。 每个病毒颗粒能够包装 15个 HSP70基因的拷贝， 并且 能够高效的转染分裂和非分裂的细胞。 病毒 DNA并不整合到被感染的细胞的 基因组中， 由 CMV启动子调控 HSP70蛋白的表达， 属于瞬时高表达。 对 HSV 一 HSP进行了序列测定， 其序列列于序列表中。 单纯疱疹病毒的滴度测定
用脂质体 lipofectAMINE(Life Technologies 产品）将纯化的扩增质粒 DNA (pHSP70 ) 和 HSV病毒共转染 Vera细胞， 然后在 34.5Ό温育培养， 直到细胞 表现出完全的细胞病变效应。 从 Vera细胞中收获重组病毒并按 1 :5比例稀释培 养， 一般传代 5-6代， 直到观察到辅助病毒的复制被抑制。 含有 pHSP70质粒 的 HSV被命名为 HSV— HSP。 经过冻融和超声以及低速离心 （2000 X g，4°C 10 分钟） 去除细胞碎片， 测定重组病毒的滴度。 病毒滴度以病毒在 34.5 Ό培养的 Vero细胞中形成的溶斑形成单位 （PFU) 来表示。 对于 HSV— HSP来说， 确定 Hsp70 的表达， 并采用滴度水平最高的传代用于培养病毒。 HSV— HSP 的病毒 滴度为 5 X 107 PFU/ml。 同时， 测定辅助病毒 HSV-1 的滴度， 其制值为 6 X 107
HSV— HSP的体外溶瘤活性
HSV- 1 能够在很多类型的肿瘤细胞中都能够复制。 人们发现鼠的结肠癌细胞 系 CT26易受 HSV感染的影响。 这种细胞系免疫原性很弱并且不能激发可检测 到的肿瘤特异性细胞毒性 T淋巴细胞（CTL)。 CT26型肿瘤经细胞因子治疗后 仍然容易复发。 现已证明 CT26的 MHC— I限制性优势免疫抗原是一个九肽， 来源于内生性亲嗜性鼠白血病前病毒的膜蛋白 （gp70)。 研究已证实了诱导产生 的肿瘤特异性细胞毒性 T淋巴细胞 （CTL)对皮下接种的 CT26肿瘤的抗瘤效 果。 其中将鼠肿瘤细胞 CT26培养于 24孔板（每孔 1 X 105) 并由 HSV-HSP或 HSV 以不同的感染复数 （0.01-10) 感染。 在感染复数 （MOI) 0.1 时， 用 HSV 一 HSP或 HSV对 CT26细胞感染， 感染 4天后导致 70%的肿瘤细胞的死亡。 在 感染复数 （MOI) 为 1时， 用 HSV_HSP或 HSV对 CT26细胞感染， 感染 4天 后导致 99%的肿瘤细胞的死亡（如图 2所示）。 感染的肿瘤细胞经培养后， 用 放射性标记及免疫沉淀 /SDS— PAGE可以检测到 HSP70的表达。 这些数据表明 插入的 HSP70并没有降低 HSV的复制能力和细胞毒性。 HSV-HSP抑制远端肿瘤的生长
同系的 BALB/c小鼠上进一步评价 HSV— HSP对 CT26肿瘤的治疗效果。 BALB/c 来自于 Charles River (Wilmington, MA, USA)。 所有动物实验都经动物关心和使 用委员会（Animal Care and Use Committee)批准。 将动物分成 3组， 第一和第 二组作为实验组， 第三组作为对照组。 将小鼠用 CT26肿瘤细胞 （1 X 105) 在小 鼠肋部对称部位分别进行皮下注射。 将这些小鼠作为患有转移性肿瘤的动物的 模型。 当皮下注射的肿瘤迅速生长 （最大直径达到 5 毫米） 时， 将第一组每只 动物非对称 （右面） 的瘤内接种 HSV-HSP ( 1 X 106 PFU) , 并于第一次接种 7 天后进行第二次接种。 将第二组每只动物非对称 （右面） 的瘤内接种 HSV— 1 ( 1 X 106 PFU, 并于第一次接种 7天后进行第二次接种。 HSV-1作为接种 HSV -HSP 的对照以评价病毒因素的影响。 将第三组小鼠每只动物非对称 （右面） 的瘤内接种热休克蛋白 HSP70, 并于第一次接种 7天后进行第二次接种， 来评 价热休克蛋白本身的作用。 肿瘤体积用卡尺测量， 体积的计算公式 (V=hXwX d)。 如果小鼠垂死或其肿瘤直径超过 18毫米， 将被处死， 并记录下日期来研究 其存活率。用非配对法 t检验进行方差统计，软件为 StaView4.5(Abacus Concepts, Berkeley, CA)。 用 HSV— HSP接种能引起非常显著的抗肿瘤效果， 无论是被注 射一侧的肿瘤， 还是同只动物身体上未经注射的对称端的肿瘤的生长都明显受 到抑制 （如图 3所示）。 经注射 HSV— HSP的小鼠身上的肿瘤无论在注射侧还 是在非注射侧都已经消退得无法检测到。 而注射 HSV— 1 只能引起经过注射的 肿瘤生长的抑制， 对于未被注射的对侧肿瘤仅有一定的影响 （如图 3所示）。 仅 注射 HSP70的对照组显示出在对注射侧和非注射侧的肿瘤都没有影响。 这些结 果表明重组的 HSV通过表达 HSP70能够激发强的免疫应答从而抑制了远端肿 瘤的生长。 实施例 2 重组腺病毒的构建
通过 pLEP和 pREP双质粒系统来构建重组腺病毒
首先用腺病毒 Adl055野生型为模板， 通过 PCR方法扩增得到碱基 559到 2262 的 Π型腺病毒 E1区。 得到的 1715碱基对的片段包含 5'末端的 Hindni限制位 点和 3'末端的 Xhol限制位点， 以及在碱基 2253处（C→T)和 2262处（G→T) 两个突变位点， 能够在 Elb 55kD蛋白的读码框的 79和 82密码子处分别引入成 熟前翻译终止密码子。 PCR产物片段用 el/Xhol酶切后克隆到经同样酶切处 理的 pBS-(IRES) (Alexe V. Gordadze et al. J. of Virology 2001, vol 75:5899-5912)中 得到 pBS-(IRES)-ElA,并通过用 Spel/Xhol酶切释放 (IRES)-EIA片段。以 SKOV3 细胞的 cDNA为模板， 通过实施例 1中所述的类似方法 PCR扩增得到人的热休 克蛋白 Hsp70 DNA。扩增得到的片段包括 5'末端的 Hindlll位点和 3'末端的 Spel 位点。 利用 pLEP上的多克隆位点， 将 Hindlll/Spel酶切的 HSP片段， Spel/Xhol 酶切的 (IRES)-EIA片段和 Hindlll/Xhol酶切的 pLEP载体片段， 连接后得到重 组质粒 pLEP-HSP-El A。 （过程如图 4所示） 上述质粒 pLEP-HSP-ElA用 Pi-Pspl酶切， 同时将 pREP质粒用 Pi-Pspl酶切， 并将两者于 16°C过夜进行连接反应。 连接产物用 Strategen公司的 Y噬菌体进行 包装， 将包装产物与大肠杆菌 （DK1或 DH5 ct )混合孵育， 并加入 LB培养液 37°C摇床孵育 30分钟。 将被 Y噬菌体感染的大肠杆菌涂含有氨苄青霉素和四环 素两种抗生素的 LB琼脂平板， 筛选具有抗性的菌落， 并酶切鉴定， 得到重组 质粒 pAd-Hsp70/ElA。 将重组质粒 pAd-Hsp70/ElA用 I-Ceul酶切将重组的腺病 毒 DNA从质粒中释放出来， 并将酶切后的质粒加入 HEBS缓冲液， 在其中加 入 2.5M的 CaCl2，形成的 DNA/ Ca3(P04)2混合物转染 293细胞， 在 C02孵箱中 培养 6-7天后， 能观察到培养的细胞层中出现清亮的空斑（CPE), 这表明重组 病毒颗粒开始形成。 当整个平板都出现 CPE时， 1500转离心 10分钟收获细胞， 冻融三次裂解细胞收获重组病毒 Ad-Hsp/E1A，重组病毒的滴度约为 107-108。 （过 程如图 5所示） 体外溶瘤活性和肿瘤抑制能力
釆用与实施例 1 相似的步骤， 进行体外溶瘤试验和在动物模型上的抑瘤试验， 取得了相似的试验结果。 重组腺病毒通过表达 HSP70能够激发潜在的免疫应答 从而抑制了远端肿瘤的生长。 实施例 3 表达热休克蛋白的沙门氏菌的构建
我们釆用鼠伤寒沙门氏菌 (Salmonella typhimurium) SL3261[19], 其中芳香族氨基 酸 aroA突变， 其所需要的营养物质只有肿瘤细胞才能够提供， 所以突变株能够 选择性的侵袭肿瘤细胞， 在肿瘤细胞中复制并导致肿瘤细胞裂解死亡； 而在正 常细胞中 SL3261则不能够生存。 通过将 HSF70序列插入到 pcDNA3.1质粒中 构建得到重组质粒 pcDNA-HSP质粒， （如图 6所示）。由 CMV启动子调控 HSP70 蛋白的表达， 属于瞬时高表达。 抑瘤活性
在同系的 BALB/c小鼠上评价表达热休克蛋白的鼠伤寒沙门氏菌 SL3261对肿瘤 的治疗效果。 将动物分成 3组， 第一和第二组作为实验组， 第三组作为对照组。 用 SMMC7721 肿瘤细胞（上海细胞保藏中心） ( I X 10" 在小鼠肩部对称部位 分别进行皮下注射。 将这些小鼠作为患有转移性肿瘤的动物的模型。 当皮下注 射的肿瘤迅速生长（最大直径达到 5 毫米） 时， 将第一组每只动物非对称 （右 面） 的瘤内接种携带表达质粒 pcDNA-HSP的 SL3261 ( 1 X 107PFU)， 并于第一 次接种 7天后进行第二次接种。.将第二组每只动物非对称 （右面） 的瘤内接种 SL3261 ( 1 X 106 PFU) , 并于第一次接种 7天后进行第二次接种， 作为对照以评 价细菌本身因素的影响。 将第三组小鼠每只动物非对称 （右面） 的瘤内接种热 休克蛋白 HSP70, 并于第一次接种 7天后进行第二次接种， 来评价热休克蛋白 本身的作用。 肿瘤体积用卡尺测量， 体积的计算公式 (V=h X w X d)。 如果小鼠 垂死或其肿瘤直径超过 18毫米， 将被处死， 并记录下日期来研究其存活率。 用 非配对法 t检验进行方差统计， 软件为 StaView4.5(Abacus Concepts, Berkeley, CA)。 用表达热休克蛋白的鼠伤寒沙门氏菌 SL3261接种能弓 I起非常显著的抗肿 瘤效果， 无论是被注射的一侧肿瘤， 还是同只动物身体上未经注射的对称端的 肿瘤的生长都明显收到抑制 （如图 7所示）。 而注射鼠伤寒沙门氏菌 SL3261只 能引起经过注射的肿瘤生长的抑制， 对于未被注射的对侧肿瘤的影响甚微 （如 图 7所示）。 这些结果表明伤寒沙门氏菌 SL3261也能够通过表达 HSP70能够激 发潜在的免疫应答从而抑制了远端肿瘤的生长。 本专利申请将下列文献以其全文引入作为参考。
参考文献
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Claims

权利要求书
1.一种溶瘤微生物， 其中该微生物表达能粘附肿瘤细胞抗原的并被抗原提呈细 胞所识别的蛋白， 并且该微生物能选择性地在肿瘤细胞中生长以裂解肿瘤细 胞。
2.如权利要求 1 所述的微生物， 其中所述的能粘附肿瘤细胞抗原的蛋白是热休 克蛋白或其变体。
3.如权利要求 2所述的溶瘤微生物， 其中所述的热休克蛋白来源于哺乳动物或 病原微生物。
4.如权利要求 3所述的微生物， 其中所述的热休克蛋白来源于人， 包括热休克 蛋白 Hsp70、 Hsp90、 Hsp94、 Hsp96和其他的热休克蛋白。
5.如权利要求 3所述的溶瘤微生物， 其中所述的热休克蛋白来源于病原微生物， 所述的病原微生物包括结核分支杆菌 (M tuberculosis) , 麻风分支杆菌 (M leprae) ^ 克 ^^^.{Tryanosoma cruzi)^H '\i^ ^(Pl smodium falciparum)。
6.如权利要求 1所述的溶瘤微生物， 其中所述的微生物是病毒。
7.如权利要求 2所述的溶瘤微生物， 其中所述的微生物是病毒。
8.如权利要求 6 或 7 所述的溶瘤微生物， 其中所述的病毒包括腺病毒 (Adenovirus 、单纯疱疫病毒 (Herpesvirus)^ 7_K泡性口炎病毒 ( Vesiculovirus )、 新城疫病毒 (Newowt/e Disease Virus), 呼肠孤病毒 (^0^ ?«)以及其他能选择 性地在肿瘤细胞中生长的病毒。
9.如权利要求 8所述的溶瘤微生物， 其中所述的病毒为腺病毒。
10. 如权利要求 8所述的溶瘤微生物， 其中所述的病毒为单纯疱疹病毒。
11.如权利要求 1或 2所述的溶瘤微生物， 其中所述的微生物是细菌。
12. 如权利要求 11 所述的溶瘤微生物， 其中所述的细菌包括伤寒沙门氏菌 (Salmonella) , 双歧杆菌
Figure imgf000024_0001
、 志贺氏菌 、 李斯特菌 (Listeria) 、 鼠疫杆菌 (Je ' 'a)和梭菌 (C/ostn ¾'M )以及其他能选择性地在肿 瘤细胞中生长的细菌。
13.一种抗原提呈细胞， 其是被含有编码能粘附肿瘤细胞抗原的并被抗原提呈 细胞所识别的蛋白或其片段的 DNA序列的载体转化的， 所述载体可任选地 进一步含有编码免疫增强分子的 DNA序列。
14.如权利要求 13所述的抗原提呈细胞， 其中所述能粘附肿瘤细胞抗原的蛋白 为热休克蛋白或其变体。
15. 一种微生物组合物， 包括：
a)溶瘤微生物， 该微生物能选择性地在肿瘤细胞中生长以裂解肿瘤细胞； 和 b) 表达能粘附肿瘤细胞抗原的并被抗原提呈细胞所识别的蛋白及其片段的载 体；
其中所述的微生物或载体任选地进一步含有编码免疫增强分子的 DNA序列。
16.如权利要求 15所述的微生物组合物， 其中所述能粘附肿瘤细胞抗原的蛋白 为热休克蛋白或其变体。
17. 如权利要求 15或 16所述的微生物组合物， 其中所述的溶瘤微生物是溶瘤 病毒或溶瘤细菌。
18. 如权利要求 17所述的微生物组合物， 其中所述的溶瘤病毒包括腺病毒、 单 纯疱疹病毒、 水泡性口炎病毒、 新城疫病毒、 呼肠孤病毒以及其他能选择性 地在肿瘤细胞中生长的病毒。
19. 如权利要求 17所述的微生物组合物， 其中所述的溶瘤细菌包括伤寒沙门氏 菌、 双歧杆菌、 志贺氏菌、 李斯特菌、 鼠疫杆菌、 梭菌以及其他能选择性地 在肿瘤细胞中生长的细菌。
20.如权利要求 15所述的微生物组合物， 其中所述的载体为包含编码能粘附肿 瘤细胞抗原的并被抗原提呈细胞所识别的蛋白或其片段的 DNA的质粒。
21.如权利要求 15所述的微生物组合物， 其中所述的载体为复制缺陷型载体。
22.如权利要求 20或 21所述的微生物组合物， 其中所述的粘附肿瘤细胞抗原 并被抗原提呈细胞所识别的蛋白是热休克蛋白或其变体。
23.一种药物组合物， 其主要含有：
a)溶瘤微生物， 即能选择性地在肿瘤细胞中生长的微生物； b)能粘附肿瘤细胞抗原的并被抗原提呈细胞所识别的蛋白或其片段； 和
c)任选地含有的免疫增强因子、 免疫佐剂和可药用载体， 其中免疫增强因子可 以由溶瘤微生物产生。
24.权利要求 23所述的药物组合物， 其中所述的微生物是溶瘤病毒， 包括腺病 毒、 单纯疱疹病毒、 水泡性口炎病毒、 新城疫病毒、 呼肠孤病毒和其他能选 择性地在肿瘤细胞中生长的病毒。
25.权利要求 23所述的药物组合物， 其中所述的微生物是溶瘤细菌， 包括伤寒 沙门氏菌、 双歧杆菌、 志贺氏菌、 李斯特菌、 鼠疫杆菌、 梭菌和其他能选择 性地在肿瘤细胞中生长的细菌。
26.权利要求 23— 25 中任一项所述的药物组合物， 其中所述的能粘附肿瘤细胞 抗原的并被抗原提呈细胞所识别的蛋白是热休克蛋白或其变体。
27.一种治疗肿瘤的免疫治疗剂， 其包含权利要求 1-12 中任一项所述的溶瘤微 生物， 或者权利要求 13或 14所述的抗原提呈细胞、 或者权利要求 15-22中 任一项所述的微生物组合物或权利要求 23-26中任一项所述的药物组合物。
28.权利要求 27所述的免疫治疗剂， 其进一步含有免疫增强因子、 免疫佐剂或 可药用载体。
29.一种治疗肿瘤的方法， 用含有编码能粘附肿瘤细胞抗原的并被抗原提呈细 胞所识别的蛋白或其片断的 DNA的载体转化患者的抗原提呈细胞。
30.权利要求 29所述的治疗肿瘤的方法， 其中所述的能粘附肿瘤细胞抗原的并 被抗原提呈细胞所识别的蛋白是热休克蛋白或其变体。
31.一种治疗肿瘤的方法， 包括给肿瘤患者施用一种免疫有效量的权利要求 1一 12中任一项所述的溶瘤微生物、 权利要求 13或 14所述的抗原提呈细胞、 权 利要求 15-22中任一项所述的微生物组合物、 权利要求 23— 26中任一项所述 的药物组合物或权利要求 27或 28所述的免疫治疗剂。
32.权利要求 29— 31 中所述的治疗肿瘤的方法， 其中所述的肿瘤包括良性肿瘤 和恶性肿瘤。
33.权利要求 32所述的治疗肿瘤的方法， 其中所述的恶性肿瘤包括黑素瘤、 乳 腺癌、 前列腺癌、 肝癌、 肺癌、 鼻咽癌、 结肠癌、 卵巢癌、 宫颈癌和白血病。
34.权利要求 32所述的治疗肿瘤的方法， 其中所述的施用是通过肿瘤内注射、 肌肉内注射、 口服、 粘膜内给药、 腹膜内给药、 静脉内给药或直肠内给药方 式完成的。
35. 权利要求 1-12中任一项所述的溶瘤微生物、 权利要求 13或 14所述的抗原 提呈细胞、 权利要求 15-22 中任一项所述的微生物组合物、 权利要求 23-26 中任一项所述的药物组合物， 用于制备抗肿瘤药物的应用。
PCT/CN2001/001616 2001-10-09 2001-12-12 Micro-organismes oncolytiques exprimant les proteines hsp, et leurs utilisations WO2003031602A1 (fr)

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JP2003534572A JP3952201B2 (ja) 2001-10-09 2001-12-12 Ｈｓｐを発現するオンコリティック微生物およびその使用

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