WO2004024181A1 - Nouveau vaccin a base d'antigene tumoral, procede de production dudit vaccin et composition de vaccin associee - Google Patents

Nouveau vaccin a base d'antigene tumoral, procede de production dudit vaccin et composition de vaccin associee Download PDF

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WO2004024181A1
WO2004024181A1 PCT/CN2003/000776 CN0300776W WO2004024181A1 WO 2004024181 A1 WO2004024181 A1 WO 2004024181A1 CN 0300776 W CN0300776 W CN 0300776W WO 2004024181 A1 WO2004024181 A1 WO 2004024181A1
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tumor antigen
antigen vaccine
vaccine
cells
toxin
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PCT/CN2003/000776
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Chinese (zh)
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Jin Li
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Jin Li
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to the fields of biological engineering and medicine. More specifically, the present invention relates to a novel tumor antigen vaccine, a preparation method thereof, and a vaccine composition. Background of the invention
  • Tumors are still one of the leading causes of death in humans. Although the level of diagnosis and treatment of tumors has been continuously improved and improved in recent years, and the chemotherapy and radiotherapy programs have also been continuously improved, most patients still cannot escape the doom of death. In recent years, advances in molecular biology and a better understanding of immune system functions have led to the rapid development of research and development of biotherapeutic methods. The development of tumor vaccines is one of the main directions of tumor biotherapy.
  • T-cell antigen receptor recognizes antigens in a way that involves only peptide fragments located on the surface of target cells and embedded in major histocompatibility complex (MHC) class I and II molecules [3].
  • MHC major histocompatibility complex
  • Class I MHC molecules present peptides produced by endogenous proteolysis. During the synthesis of tumor antigens by tumor cells, the degraded peptides are presented by MHC class I molecules to cause CD8 + T-cells to be activated on the surface of tumor cells.
  • Class II molecules are recognized by CD4 + T-cells and are mainly located on the surface of special antigen-presenting cells (APCs), including dendritic cells, B-cells, and macrophages. Exogenous proteins secreted by tumor cells or released by tumor lysis are captured by APCs. In APC, the antigen is processed into peptide fragments and presented to CD4 + cells by MHC class II. CD4 + T-cells are closely related to the immune response of humoral cells. The direct interaction with B-cells stimulates the production of antibodies and stimulates the expansion of CD8 + T-cell responses through secreted cytokines. Activated antigen-specific CD8 + cells eventually become cytotoxic T cells and lyse tumor cells [4]. APCs can also process peptides and present them to CD8 + T- cells via MHC class I molecules.
  • APCs can also process peptides and present them to CD8 + T- cells via MHC class I molecules.
  • the ideal tumor-specific antigen should be immunogenic and expressed by tumor cells but not in normal cells. Unfortunately, most tumor antigens are not sufficiently immunogenic to induce an effective immune response, and many tumor antigens are expressed to some extent in normal tissues. Therefore, these antigens are only tumor-related and not real Tumor-specific antigen.
  • the designed tumor vaccine must overcome the body's immune tolerance disorders.
  • Cancer vaccines are mainly divided into whole cell vaccines, protein molecular vaccines, peptide vaccines, recombinant molecular vaccines and Dendritic cell vaccine.
  • DNA vaccines and RNA vaccines are still molecular vaccines, but they use different expression systems.
  • Dendritic cell vaccine For an effective T-cell-mediated immune response, T-cells require antigen presentation and sensitization of the original T-cells, and the sensitized T-lymphocytes are restimulated. To initiate effective T-cell-mediated tumor immunity, tumor antigen polypeptides derived from any part of the body must be recognized by circulating T-cells. The lack of MHC molecules and costimulatory molecules on the surface of tumor cells does not activate T-cell immunity. Therefore, the presentation of the antigen is a key step in obtaining an effective immune response. The immune response stimulated by the vaccine mainly depends on the effective APC for the initial processing and further presentation of the antigen. Dendritic cells (DC) are the most effective APCs [5].
  • DCs can enable the immune system to overcome this obstacle.
  • DCs are currently available in large numbers from the isolation of CD34 + hematopoietic stem cells or peripheral blood mononuclear lymphocytes.
  • DCs exist in immature state in most tissues and cannot directly stimulate T-cells but have special ability to capture and process antigens. These captured antigens are effectively presented to the cell surface in DCs cells via class I and class II MHC molecules. The capture of the antigen serves as a stimulus to promote cell maturation and migration to local lymph nodes.
  • the cell surface of these mature DCs also highly express co-stimulatory molecules and adhesion molecules, which has a strong function of activating T-lymphocytes [6-8].
  • the DCs-based vaccine is the most ideal vaccine of all programs.
  • the in vitro isolation and culture of DC requires high technical requirements and costs. If the tumor antigen can be effectively delivered to DC and activated in vivo, the treatment cost can be greatly reduced.
  • Delivering antigens to DCs through antigen-antibody complexes is a viable option because binding of the Fc segment of immunoglobulin to Fc receptors on the surface of DC cells can promote DC phagocytosis of antigen-antibody complexes [12, 13].
  • the use of recombinant DNA vaccines has also confirmed that the Fc segment of immunoglobulins can promote the immune response to hepatitis B virus, can increase the level of interferon production of immunologically active cells, and increase the activity of CD8 + to a certain extent [13], It has not been reported whether this DNA vaccine can obtain satisfactory therapeutic effect on hepatitis in animal experiments.
  • Another object of the present invention is to provide a nucleotide sequence encoding the tumor antigen vaccine.
  • Another object of the present invention is to provide a vaccine composition containing the tumor antigen vaccine.
  • Another object of the present invention is to provide a method for preparing the tumor antigen vaccine.
  • the first aspect of the present invention relates to an isolated tumor antigen vaccine, the tumor antigen vaccine comprising a sequence of 7 or more amino acids from a tumor antigen and an amino acid sequence containing a CH3 portion of an immunoglobulin These two sequences are connected to each other.
  • a second aspect of the present invention relates to a DNA molecule containing a nucleotide sequence encoding the aforementioned tumor antigen vaccine.
  • a third aspect of the invention relates to a vaccine composition
  • a vaccine composition comprising a tumor antigen vaccine and a pharmaceutically acceptable carrier.
  • the fourth aspect of the present invention relates to a method for preparing the above-mentioned tumor antigen vaccine, the method comprising: a) providing an expression vector, the expression vector comprising a nucleotide sequence encoding the above-mentioned tumor antigen vaccine and operable with the nucleotide sequence Linked expression control sequences;
  • step b) transforming the host cell with the expression vector in step a);
  • step b) culturing the host cell obtained in step b) under conditions suitable for expressing the tumor antigen vaccine; and d) isolating and obtaining the expressed tumor antigen vaccine.
  • the tumor antigen vaccine of the present invention is obtained by recombinantly expressing a tumor antigen or a polypeptide thereof and an immunoglobulin CH3 portion at the DNA level.
  • the tumor antigen vaccine can bind to the Fc receptor on the surface of DC through its CH3 part, thereby promoting the endogenization of tumor antigen carried by the fusion protein and stimulating DC maturity, stimulating the presentation of DC to the antigen and activating T lymphocytes.
  • the polypeptide presented by CH3 mediated antigen endogenization mainly binds to class I MHC molecules and activates CD8 + cytotoxic T cells, it can generate a powerful immune attack against tumor cells expressing the antigen and kill such Tumor cells.
  • the method of the present invention is simple, and the very complicated step of preparing special antibodies is omitted.
  • the antigen and CH3 are recombinant proteins, and the binding is firm.
  • the antigen-antibody complex is easy to detach.
  • the molecular weight of the vaccine of the present invention is many times smaller than that of the antigen-antibody complex, and it is easily phagocytosed by dendritic cells and produces a strong immune response. Especially after additional toxins are added, the antigenicity of tumor antigens can be significantly improved, and the T cell activation effect can be further increased by a factor of two.
  • the invention provides an isolated tumor antigen vaccine.
  • the tumor antigen vaccine comprises a sequence of 7 or more amino acids from a tumor antigen and an amino acid sequence containing a CH3 portion of an immunoglobulin, and the two sequences are connected to each other.
  • the term "isolated" when applied to a protein means that the protein is substantially free of other cellular components associated in its natural state, and is preferably in a homogeneous state, but may also be dry or aqueous. Purity and homogeneity can usually be determined by analytical chemistry methods such as polyacrylamide gel electrophoresis or high performance liquid chromatography.
  • the tumor antigen vaccine of the present invention contains two amino acid sequences.
  • the first amino acid sequence is a polypeptide sequence of 7 or more amino acids from a tumor antigen.
  • polypeptide and protein as used herein are interchangeable and include 7 or more amino acid chains of any length, including full-length proteins (ie, the tumor antigen itself), in which amino acid residues are passed through covalent peptides Key to connect.
  • tumor antigen is well known to those skilled in the art.
  • the tumor antigen is preferably selected from any tumor-associated antigen that can be recognized by T cells.
  • the tumor antigen is preferably selected from: 707-AP, AFP, ART-4, BAGE B, p-catenin / m, bcr-abK CAMEL, CAP-CASP-8, CDC27m, CDK4 / m, CEA, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, ETS, G250, GAGE, GnT-V, GP100, HAGE, HER-2 NEU, HLA-A * 0201-R170I, HPV-E7, HSP70-2M, HST-2, hTERT, iCE, KIAA0205, LAGE, LDLR / FUT, GDP-Lf icose, MAGE.
  • tumor antigens need only 7 or more amino acid polypeptide sequences to be presented by antigen presenting cells (CAP-1 is the amino acid sequence derived from CEA is YLSGANLNL [16], VISNDVCAQV Is the amino acid sequence derived from PSA [17], and KIFGSLAFL is the amino acid sequence derived from HER2 / neu [18]).
  • CAP-1 is the amino acid sequence derived from CEA is YLSGANLNL [16]
  • VISNDVCAQV Is the amino acid sequence derived from PSA [17]
  • KIFGSLAFL is the amino acid sequence derived from HER2 / neu [18]
  • tumor antigen polypeptide sequence There may be one or more amino acid deletions, substitutions or additions in the tumor antigen polypeptide sequence, and the variants thus produced are also included in the term "tumor antigen" of the present invention, as long as the variant retains the tumor antigen polypeptide sequence Of antigenicity.
  • the second amino acid sequence contained in the tumor antigen vaccine of the present invention is an amino acid sequence containing a CH3 portion of an immunoglobulin.
  • the inventors have discovered that the CH3 portion of the immunoglobulin Fc fragment is a key sequence that causes the immunoglobulin Fc fragment to bind to the Fc receptor on the surface of DC cells. Therefore, any amino acid sequence containing the CH3 portion of the immunoglobulin is expected to bind to the Fc receptor on the surface of DC cells.
  • the amino acid sequence and DNA sequence of CH3 are shown in the sequence listing SEQ ID NO: 1 and SEQ ID NO: 2, respectively. however,
  • CH3 variants resulting from deletion, substitution, or addition of one or more amino acids in the CH3 amino acid sequence are also included in the term "CH3 part" of the present invention, as long as the variant retains the ability to bind to the Fc receptor on the surface of DC cells .
  • the "CH3 variant” is preferably more than about 80% identical to the CH3 sequence, and more preferably more than about 95%. Common substitutions are conservative amino acid substitutions, such as the aliphatic amino acids Ala,
  • amino acid sequence containing a CH3 portion of an immunoglobulin may be a CH3 amino acid sequence alone. In another embodiment, it may be an immunoglobulin Fc fragment containing the CH3 amino acid sequence.
  • the above two amino acid sequences need only be linked without any sequence.
  • the linkage may be directly linked (i.e., no amino acids involved therein) or may be linked via a linker sequence that does not significantly affect the antigenicity of the tumor antigen polypeptide sequence.
  • the tumor antigen vaccine of the present invention further comprises a toxin.
  • the toxin may be any bacterium or virus and other biological toxins, such as diphtheria toxin, pertussis toxin, pseudomonas toxin, anthrax toxin, tetanus toxin and the like.
  • the toxin may be tandemly linked to a tumor antigen vaccine with any toxin fragment of 30 amino acids or more, wherein the toxin fragment may be ligated at any position before, after, and between the tumor antigen polypeptide fragment and the CH3 fragment.
  • the present invention also provides a DNA molecule containing a nucleotide sequence encoding a tumor antigen vaccine of the present invention.
  • nucleotide sequences encoding various tumor antigen polypeptide sequences are known to those skilled in the art and can be retrieved from a gene bank.
  • the nucleotide sequence encoding the CH3 portion is shown, for example, in SEQ ID NO: 2.
  • those skilled in the art can also use the degeneracy of the genetic code known in the art to obtain all other nucleic acid sequences encoding the amino acid sequences described above.
  • the tumor antigen vaccine of the present invention can be obtained by the following method.
  • the nucleotide sequence encoding the sequence of seven or more amino acids of the tumor antigen and the coding sequence are obtained by conventional means known to those skilled in the art, such as artificial synthesis or PCR amplification. Contains the nucleotide sequence of the CH3 portion of the immunoglobulin. Then, various methods well known in the art, such as genetic engineering methods, can be used to ligate the tumor antigen polypeptide coding sequence and CH3 coding sequence into an appropriate expression vector using an optional linker sequence, and operably interact with the expression control sequence. Connected.
  • the coding sequence of a toxin can also be ligated into the expression vector by genetic engineering methods.
  • expression control sequence generally refers to a sequence involved in controlling the expression of a nucleotide sequence.
  • the expression control sequence includes a promoter and a termination signal operably linked to the target nucleotide sequence. They also typically include sequences required for proper translation of the nucleotide sequence. "Operationally linked” means that certain parts of a linear DNA sequence can affect the activity of other parts of the same linear DNA sequence. For example, if a promoter or enhancer increases transcription of a coding sequence, it is operably linked to the coding sequence.
  • various commercially available expression vectors known to those skilled in the art can be used.
  • the term "host cell” includes prokaryotic cells and eukaryotic cells.
  • prokaryotic host cells include E. coli, Bacillus subtilis, and the like.
  • Commonly used eukaryotic host cells include yeast cells, insect cells, and mammalian cells.
  • a mammalian cell line is preferably used as a host cell, and a commercially available immortalized cell line such as a Chinese hamster ovary (CHO) cell, a Vero cell, and a Hella cell is more preferable. , Baby hamster kidney (BHK) cells, monkey kidney cells (COS), etc.
  • transformation refers to the direct introduction of an expression vector containing a nucleic acid of interest into a host cell using methods well known to those skilled in the art. Transformation methods vary by host cell type and typically include: electroporation; transfection with calcium chloride, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; infection and other methods (see Sambrook et al., Guide to Molecular Cloning Experiments, 2nd edition, 1989).
  • the transformed host cells are cultured under conditions suitable for expression of the tumor antigen vaccine of the present invention.
  • the cells are then lysed with a cell lysis buffer, and then purified by conventional separation and purification means well known to those skilled in the art, such as ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, and affinity chromatography to obtain the tumor antigen vaccine of the present invention.
  • the invention also provides a vaccine composition containing a pharmaceutically effective amount of the tumor antigen vaccine of the invention and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means that when the molecular body and composition are properly administered to an animal or human, they do not cause adverse, allergic or other adverse reactions.
  • a "pharmaceutically acceptable carrier” should be compatible with the tumor antigen vaccine of the present invention, that is, it can be blended with it without significantly reducing the effect of the vaccine composition under normal circumstances.
  • Suitable carriers are usually large, slow-metabolizing macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, amino acid polymers, amino acid copolymers, lipid agglomerates (such as oil droplets or liposomes), and inactivity Virus particles. These vectors are well known to those of ordinary skill in the art.
  • the vaccine composition of the present invention can be prepared into various dosage forms according to needs, and can be administered by a physician according to the patient's type, age, weight, general disease status, administration mode and other factors. Traditional methods are given by injection from the parenteral (subcutaneous, intramuscular, or transdermal / transdermal) route.
  • the therapeutic dose may be a single dose schedule or a multiple dose schedule.
  • the vaccine can be administered in combination with other immunomodulators or immune adjuvants.
  • MUC1 The full sequence of MUC1 can be obtained from the gene bank (NM ⁇ 002456).
  • CDNA was synthesized from X-108 gastric cancer cell line (gastric cancer cell line derived from surgical specimens) by mRNA reverse transcription method using the Invitrogene reverse transcription kit according to the manufacturer's instructions, and the DNA of MUC1 was obtained.
  • MUC1 (5 'PCR primer sequence AACCCGGTACCACAGGTTCTGGTCATGCAAGC (SEQ ID NO: 3), 3 * PCR primer sequence AACCCCTCGAGGGGGGCGGTGGAGCCCGGGGCC (SEQ ID NO: 4)) was synthesized using the DNA of MUC1 obtained as a template PCR method. MUC1 was cloned into the multiple cloning site in the pcDNA3.1 vector (purchased from Invitrogene) using the restriction enzymes Kpn I / Xho I.
  • the CH3 fragment of immunoglobulin Fc was synthesized by PCR (5 'PCR primer sequence AACCCCTCGAGGGCAGCCCCGAGAACCAC (SEQ ID NO: 5), 3' PCR primer sequence AACCCTCTAGATCATTTACCCGGGGACAG (SEQ ID NO: 6) ).
  • the restriction enzymes Xho I / Xba I were used to clone the CH3 fragment into the corresponding site in the pcDNA3.1 vector to make it compatible with MUC1. Connected in series.
  • pcDNA3.1 was amplified in DH-5ot (purchased from Invitragene) and plasmid DNA was purified using Miniprep kit. Take 5-10ug of digested DNA, transfect it into CHO cells (purchased from ATCC, USA) using Superfectine kit (Qiagene), select by G418, and monoclonalize.
  • cell lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 10 mM DTT, 1 mM benzylsulfonyl fluoride PMSF, 10 ⁇ g / ml aprotinin, 10 ⁇ g / ml leupeptin 5 ⁇ g / ml gastrostatin
  • the expression of the fusion protein was detected by Western blotting.
  • the primary antibody was a mouse anti-human MUC1 monoclonal antibody
  • the secondary antibody was a rabbit anti-mouse IgG
  • the kit was a product of American Vector Company. The results suggest that fusion proteins can be detected at approximately 23,000 molecular weights.
  • mice were immunized with 2 mg of purified fusion protein three times a week, and mouse splenocytes 1X10 7 were irradiated with MC38 cells (derived from an 8000 cobalt source) at a ratio of 20: 1. (NIH) mixed culture for 5 days, and then mixed with wild-type MC38 at different concentrations of target-effect ratio to determine the killing activity of spleen cells on MC38. It was found that 80% of MC38 cells could be killed in four hours with a 1:20 target ratio. The mice immunized with the above method can get 100% immune protection and reject the attack of MC38 tumor cells up to 1 ⁇ 10 6 . Its therapeutic effect is 5-10 times higher than that of ordinary antigen-antibody complexes.
  • Example 2 CEA Tumor Antigen Vaccine (CAP-1)
  • the DNA coding sequence of CAP-1 is known as TACCTTTCGGGAGCGAACCTCAACCTCTCC (SEQ ID NO: 8).
  • the Fc segment cDNA obtained as a template the CAP-1-Fc recombinant protein DNA (5 'PCR primer sequence AACCCGGTACCATGTACCTTT) was synthesized by PCR using the PCR method.
  • pcDNA3.1 was amplified in DH-5a (purchased from Invitragene) and purified using Miniprep kit Plasmid DNA. Take 5-10ug of digested DNA, transfect it into CHO cells (purchased from ATCC, USA) using Superfectine kit (Qiagene), select by G418, and monoclonalize.
  • cell lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 10 mM DTT, 1 mM benzylsulfonyl fluoride PMSF, 10 ⁇ g / ml aprotinin, 10 ⁇ g / ml leupeptin 5 ⁇ g / ml gastrostatin
  • the primary antibody was a mouse anti-human CH3 monoclonal antibody
  • the secondary antibody was a rabbit anti-mouse IgG
  • the kit was a product of the American vector company. The results suggest that a fusion protein monomer can be detected at a molecular weight of approximately 30,000.
  • the fusion protein was isolated and purified, lyophilized, and packed.
  • Example 3 P53 tumor antigen vaccine
  • the full sequence of human P53 can be obtained from the gene bank (M14695).
  • a plasmid containing the P53 gene can be purchased from ATCC in the United States.
  • the full amino acid sequence is shown in SEQ ID NO: 11.
  • reverse transcription from human B lymphocyte mRNA to synthesize cDNA to obtain CH3 DNA.
  • P53 (5 'PCR primer sequence AACCCGGTACCATGGAGGAGCCGCAGTCAGAT (SEQ ID NO: 12) was synthesized using the DNA of P53 obtained as a template by the PCR method, and P53 was cloned into the pcDNA3.1 vector by the 3' PCR primer endonuclease Kpn I / Xho I ( Purchased from the Invitrogene company).
  • the CH3 DNA obtained above was used as a template to synthesize a CH3 fragment of immunoglobulin Fc (5 'PC bow I sequence AACCCCTCGAGGGCAGCCCCGAGAACCAC (SEQ ID NO: 5) ), 3 'PCR primer sequence AACCCTCTAGATCATTTACCCGGGGACAG (SEQ ID NO: 6)).
  • restriction enzymes Xho I / Xba I the CH3 fragment was cloned into the corresponding site in the pcDNA3.1 vector and tandemly linked to P53 .
  • Partial DNA sequence of diphtheria toxin was synthesized by PCR (its full-length sequence can be retrieved from the gene bank A04646) (SEQ ID NO: 14) (5 'PCR primer sequence is AACCCGGTACCAACTTTTCTTCGTACCACG (SEQ ID NO: 15), 3' PCR primer sequence Is AACCCGGTACCACTATAAAACCCTTTCCAA (SEQ ID NO: 16)).
  • the restriction enzyme Kpn I was used to connect the toxin sequence in series to the front end of P53 in the order of toxin-P53-CH3.
  • pcDNA3.1 was amplified in DH-5a (purchased from Invitragene) and purified using Miniprep kit Plasmid DNA. Take 5-10ug of digested DNA, transfect it into CHO cells (purchased from ATCC, USA) using Superfectine kit (Qiagene), select by G418, and monoclonalize.
  • cell lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 10 mM DTT, 1 mM benzylsulfonyl fluoride PMSF, 10 ⁇ g / ml aprotinin, 10 ⁇ g / ml leupeptin 5 ⁇ g / ml gastrostatin
  • the primary antibody was a mouse anti-human P53 monoclonal antibody
  • the secondary antibody was a rabbit anti-mouse IgG
  • the kit was a product of the American company Vector. The results suggest that a fusion protein can be detected at a molecular weight of approximately 60,000.
  • mice were immunized with 2 mg of purified fusion protein, three times a week, and mouse splenocytes 1X10 7 were irradiated with L002 cells (human P53 transgene) at a ratio of 20: 1 to irradiated (8000 cobalt source) Cells) were mixed and cultured for 5 days, and then mixed with wild-type L002 at different concentrations of target-effect ratio to determine the killing activity of splenocytes on L002. It was found that a 1:20 target ratio could kill 95% of L002 cells in four hours, which was about 20% higher than the P53-CH3 vaccine without toxin.
  • Example 4 Her2 / neu tumor antigen vaccine
  • the full Her2 / neu sequence can be retrieved from the gene bank (M11730).
  • a plasmid containing this gene is available from ATCC in the United States.
  • a part of Her2 / neu DNA was synthesized by PCR (5 'PCR primer sequence AACCCGGTACCAGCACCCAAGTGTGCACC (SEQ ID NO: 17), 3' PCR primer sequence AACCCCTCGAGTTGGTTGTGCAGGGGGCA (SEQ ID NO: 18)).
  • a part of Her2 / neu DNA was cloned into the multiple cloning site in pcDNA3.1 vector (purchased from Invitrogene) using restriction enzymes Kpn l / Xho l.
  • an Invitrogene reverse transcription kit was used according to the manufacturer's instructions to synthesize cDNA from human B lymphocyte mRNA by reverse transcription to obtain Fc DNA (SEQ ID NO: 7).
  • a fragment of immunoglobulin Fc was synthesized by the PCR method (5 'PCR bow I sequence AACCCCTCGAGGCAGAGCCCAAATCTTGTGA (SEQ ID NO: 8), 3' primer sequence AACCCTCTAGATCATTTACCCGGAGACAG (SEQ ID NO: 9) ).
  • the restriction enzymes Xho I / Xba I were used to clone the Fc fragment into the corresponding site in the pcDNA3.1 vector and ligate it with Her2 / neu in tandem.
  • the endonuclease Kpn l connects the toxin sequence in series to the front end of P53 in the order of toxin-Her2 / n eU -F C.
  • pcDNA3.1 was amplified in DH-5a (purchased from Invitragene) and plasmid DNA was purified using Miniprep kit. Take 5-10ug of digested DNA, transfect it into CHO cells (purchased from ATCC, USA) with Superfectine kit (Qiagene), select by G418, and monoclonalize.
  • lysing cell buffer (20mM Tris pH7.5, 150mM NaCl, 10mM DTT, 1mM benzylsulfonyl fluoride PMSF, 10 ⁇ g / ml aprotinin, 10 ⁇ g / ml leupeptin 5 ⁇ g / ml gastrostatin) to harvest lysed CHO cells.
  • the primary antibody was a mouse anti-human Her2 / neu monoclonal antibody
  • the secondary antibody was a rabbit anti-mouse IgG
  • the kit was a product of American Vector Company. The results suggest that the fusion protein can be detected at approximately 66,000 molecular weights.

Abstract

Nouveau vaccin à base d'antigène tumoral qui contient une séquence d'antigène tumoral ayant sept acides aminés ou plus et une séquence d'acides aminés de la partie CH3 de l'immunoglobuline, ces deux séquences étant jointes l'une à l'autre. La présente invention concerne également la séquence d'ADN codant le vaccin à base d'antigène tumoral, le procédé de production de ce vaccin et la composition de vaccin associée. Le poids moléculaire du vaccin selon la présente invention est de nombreuses fois plus petit que le composé antigène-anticorps. Ce vaccin peut être avalé facilement par les cellules dendritiques, engendrant ainsi une immunoréaction très élevée.
PCT/CN2003/000776 2002-09-13 2003-09-15 Nouveau vaccin a base d'antigene tumoral, procede de production dudit vaccin et composition de vaccin associee WO2004024181A1 (fr)

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CN104800838B (zh) * 2015-04-14 2018-01-09 深圳市中联生物科技开发有限公司 MUC1‑Fc多肽疫苗及其制备方法和应用
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