WO2021110120A1 - Molécule vaccinale antitumorale, son procédé de préparation et son utilisation - Google Patents

Molécule vaccinale antitumorale, son procédé de préparation et son utilisation Download PDF

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WO2021110120A1
WO2021110120A1 PCT/CN2020/133798 CN2020133798W WO2021110120A1 WO 2021110120 A1 WO2021110120 A1 WO 2021110120A1 CN 2020133798 W CN2020133798 W CN 2020133798W WO 2021110120 A1 WO2021110120 A1 WO 2021110120A1
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antigen
tumor
protein
tumor vaccine
agonist
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Chinese (zh)
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郭军
杨光富
杜晶晶
王昌伟
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华中师范大学
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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
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001152—Transcription factors, e.g. SOX or c-MYC
    • A61K39/001153—Wilms tumor 1 [WT1]
    • 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
    • A61K39/001169—Tumor associated carbohydrates
    • A61K39/00117—Mucins, e.g. MUC-1
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001184—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K39/001186—MAGE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K39/0011—Cancer antigens
    • A61K39/001184—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K39/001188—NY-ESO
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/00119—Melanoma antigens
    • A61K39/001192—Glycoprotein 100 [Gp100]
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4727—Mucins, e.g. human intestinal mucin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6081—Albumin; Keyhole limpet haemocyanin [KLH]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the invention relates to the field of anti-tumor vaccines, in particular to an anti-tumor vaccine molecule and its preparation method and application.
  • TACAs tumor-associated antigens
  • tumor-associated antigens due to the commonly used tumor-associated antigens as self-antigens, they have poor antigenicity and poor immune regulation tolerance, resulting in failure to elicit an effective immune response. Therefore, improving the immunogenicity of tumor-associated antigens has become an urgent problem to be solved.
  • the vaccine is equipped with optimized uncovalently linked MUC1 or nicotine antigen and ⁇ GalCer, which significantly improves the immunogenicity of the antigen.
  • semi-synthetic anti-cancer vaccines usually combine tumor-associated antigens with different carrier proteins, and then mix them with adjuvants to form a vaccine.
  • the carrier proteins include bovine serum albumin (BSA), CRM197 (non-toxic diphtheria toxin mutant), and Cold toxoid (TTOX) and keyhole limpet hemocyanin (KLH).
  • BSA bovine serum albumin
  • CRM197 non-toxic diphtheria toxin mutant
  • TTOX Cold toxoid
  • KLH keyhole limpet hemocyanin
  • TLR Toll-like receptor
  • the purpose of the present invention is to overcome the problem of poor immunogenicity of tumor-associated antigens in the prior art.
  • the first aspect of the present invention provides an anti-tumor vaccine molecule having a structure represented by formula (I).
  • A is an adjuvant
  • B is an antigen
  • m The A is covalently connected to the protein through a covalent linking arm, and the n Bs are covalently connected to the protein through a covalent linking arm, and the number of amino acid molecules in the protein is greater than or equal to 100;
  • m is an integer greater than or equal to 1
  • n is an integer greater than or equal to 1;
  • the second aspect of the present invention provides a method for preparing the anti-tumor vaccine molecule having the structure represented by formula (I) as described in the first aspect, the method comprising:
  • the adjuvant is coupled to the protein for the third time, and the obtained second intermediate is coupled to the antigen for the fourth time.
  • the third aspect of the present invention provides the application of the anti-tumor vaccine molecule having the structure represented by formula (I) as described in the aforementioned first aspect in an anti-tumor vaccine.
  • the number of amino acids in the peptides is small ( ⁇ 100, generally used peptides ⁇ 50 amino acids), and the molecular weight is small; proteins are composed of amino acids. Large numbers (>100), large molecular weight, large volume;
  • Polypeptides are generally synthesized chemically, with few modification sites, and are easy to modify during chemical synthesis or after synthesis. They can be purified by HPLC; proteins are generally synthesized through biosynthesis.
  • the peptide is small in size, easy to diffuse, and not easy to be enriched in lymphoid tissues. Generally, it contains less than 3 Th and Tc epitopes.
  • the immunogenicity is weak; the protein is large in size, not easy to spread, and is easy to be enriched in lymphoid tissues. It contains many Th and Tc epitopes, and the immunogenicity is strong.
  • the design of the built-in adjuvant protein conjugate strategy provided by the present invention is an effective strategy for designing high-efficiency immunotherapy anticancer vaccines.
  • the anti-tumor vaccine of the present invention is a new anti-tumor molecule, has good immune performance, can produce higher titer IgG antibodies and stronger cellular immunity, has good thermal stability, is easy to store and transport.
  • Fig. 1 is an anti-tumor vaccine molecule according to a preferred embodiment of the present invention.
  • Fig. 1A shows the anti-tumor vaccine molecule according to a preferred embodiment of the present invention.
  • the immune agonist is an adjuvant, LK1 and LK2 both represent covalently linked arms;
  • Figure 1B shows an anti-tumor vaccine molecule according to another more preferred embodiment of the present invention.
  • the TLR agonist is an adjuvant, and both LK1 and LK2 represent Covalently connected arms.
  • Figure 2 shows the cytokine IFN- ⁇ and IL-6 test for the serum taken 2h after the first immunization as described in Example 9.
  • Figure 3 shows the test results of the IgG antibody titer against MUC1 of the three immunity described in Example 10.
  • Figure 4 shows the comparison of the IgG antibody titer test results against MUC1 for the first, second, and third immunity described in Example 10.
  • Figure 5 shows the comparison of IgM antibody titer test results against MUC1 for the first, second, and third immunity described in Example 10.
  • Fig. 6 shows the comparison of the test results of the IgG subtype antibody titers against MUC1 of the three immunity described in Example 10.
  • Fig. 7 shows the comparison of the IgG antibody titer test results of the three immunity against BSA described in Example 11.
  • Fig. 8 shows the MTT method described in Example 12 to measure the survival rate of MCF-7 cells.
  • Figure 9 shows the measurement of the binding of vaccine-induced antiserum to MCF-7 cells by flow cytometry as described in Example 13.
  • Figure 10 shows the lysis rate of MCF-7 cells described in Example 15.
  • the first aspect of the present invention provides an anti-tumor vaccine molecule having a structure represented by formula (I).
  • A is an adjuvant
  • B is an antigen
  • m The A is covalently connected to the protein through at least one covalent linking arm
  • n Bs are covalently connected to the protein through at least one covalent linking arm.
  • the number of amino acid molecules in the protein is greater than or equal to 100;
  • m is an integer greater than or equal to 1
  • n is an integer greater than or equal to 1;
  • n in the formula (I) is 1, and n is an integer greater than or equal to 1; for example, m is 1, and n is 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.
  • n in the formula (I) is 1, and n is an integer greater than or equal to 2; for example, m is 1, and n is 2, 3, 4, 5, 6, 7, 8. , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.
  • n in the formula (I) is an integer greater than or equal to 2
  • n is an integer greater than or equal to 1, for example, m is 2, 3, 4, 5, 6, 7, 8. , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, etc.
  • n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, etc.
  • m in the formula (I) is an integer greater than or equal to 2
  • n is an integer greater than or equal to 2; for example, m is 2, 3, 4, 5, 6, 7, 8. , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, etc., n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 etc.
  • the adjuvant and the protein can be covalently linked to each other through any covalently linked sites, and the antigen and the protein can also be covalently linked to each other through any covalently linked sites. ⁇ Price connection.
  • the antigen includes at least one of a tumor-associated antigen, a tumor-specific antigen, a pathogen antigen, a biotoxin, and a biomolecular antigen.
  • the tumor-associated antigens of the present invention refer to antigen molecules present on tumor cells or normal cells, including, for example, embryonic proteins, glycoprotein antigens, squamous cell antigens, etc., and are commonly used in clinical tumor diagnosis. Tumor-associated antigens are not unique to tumor cells. Normal cells can be synthesized in small amounts and are highly expressed when tumor cells proliferate, so they are called "related antigens.”
  • the tumor-specific antigen of the present invention refers to a new antigen that is only expressed on the surface of a certain tumor cell but does not exist on normal cells, so it is also called a unique tumor antigen.
  • the tumor-associated antigen is selected from at least one of a tumor-associated polypeptide antigen, a tumor-associated glycopeptide antigen, and a tumor-associated carbohydrate antigen.
  • the antigen contains at least one polypeptide or glycopeptide selected from MUC1, MUC16, NY-ESO-1, MAGE-A1/3/4, WT1, STAT3, HER2 and GP100.
  • the MUC1 of the present invention is a glycosylated, high molecular weight (Mr>200 ⁇ 103) mucin 1, which is a transmembrane molecule whose transmembrane sequence is inserted into the cell membrane like a rod, and has a structure consisting of 69 amino acid residues.
  • the composed tail extends into the cytoplasm.
  • the MUC16 of the present invention also known as CA125, is a high-molecular-weight glycoprotein expressed on the surface of various types of epithelial cells, and mainly plays the role of protecting and repairing the epithelium.
  • the NY-ESO-1 of the present invention represents New York esophageal squamous cell carcinoma 1 (NY-ESO-1), which is a cancer-testis antigen (CTA) and is re-expressed in many tumors.
  • NY-ESO-1 New York esophageal squamous cell carcinoma 1
  • CTA cancer-testis antigen
  • the MAGE-A1/3/4 of the present invention means the detection of tumor-testis antigen (CTA) melanin antigen A (MAGE-A).
  • the WT1 of the present invention represents the Wilms tumor protein, which is expressed by the human WT1 gene.
  • the STAT3 of the present invention represents signal transduction and activation transcription factor 3.
  • the HER2 of the present invention represents human epidermal growth factor receptor-2 (HER2).
  • the GP100 of the present invention represents a melanin-related antigen.
  • the antigen contains MUC1, and more preferably, the MUC1 antigen is selected from at least one of the following structures:
  • each of R 1 , R 2 , R 3 , R 4 and R 5 is connected to an amino acid residue modification group, and each of R 1 , R 2 , R 3 , R 4 and R 5 are each independently selected from hydrogen and the sugar structure shown below:
  • the tumor-associated carbohydrate antigen is selected from at least one of the following structures:
  • the protein is preferably selected from bovine serum albumin (BSA), chicken ovalbumin (OVA), keyhole hemocyanin (KLH), tetanus toxoid (TT), Diphtheria toxoid (DT), Haemophilus influenzae D protein, group B meningococcal outer membrane protein complex (OMP), pertussis toxoid, typhoid flagella, pneumolysin (PLY) and non-toxic mutations of diphtheria toxin At least one of the body (CRM197).
  • BSA bovine serum albumin
  • OVA ovalbumin
  • KLH keyhole hemocyanin
  • TT tetanus toxoid
  • DT Diphtheria toxoid
  • Haemophilus influenzae D protein group B meningococcal outer membrane protein complex
  • PLY pneumolysin
  • non-toxic mutations of diphtheria toxin At least one of the body (CRM197).
  • the adjuvant is a pattern recognition receptor agonist.
  • the pattern recognition receptor agonist is selected from at least one of Toll-like receptor agonists and NKT agonists; more preferably, the Toll-like receptor agonist is selected from TLR7 agonists, TLR8 agonists , At least one of TLR9 agonist, TLR3 agonist, TLR2 agonist and TLR4 agonist.
  • the representative structure of a TLR7 agonist is as follows:
  • R 1 is selected from any one of the following structures:
  • R 2 is selected from any one of the following structures:
  • the representative structure of the TLR7 agonist is as follows:
  • TLR8 agonist the representative structure of a TLR8 agonist is as follows:
  • the TLR9 agonist is CpG-ODN, and the representative structure is as follows:
  • the TLR3 agonists are poly(I:C) and poly-ICLC, and the representative structures are as follows:
  • Poly(I:C) is polyinosic acid, which is polycreatic acid-polycytidysic acid, which is an analog of double-stranded RNA, one chain is Poly(I) and the other chain is Poly(C).
  • Poly-ICLC is a synthetic double-stranded polyriboinosine-polyribocytidylic acid Poly(I:C) stabilized with polylysine and carboxymethylcellulose (LC).
  • the representative structure of a TLR2 agonist is as follows:
  • the representative structure of a TLR4 agonist is as follows:
  • R 3 represents a hydrogen atom or a phosphate group
  • R 4 , R 5 , R 6 , and R 7 each independently represent a fatty acyl group with a carbon number of 1-20;
  • R 8 represents a hydrogen atom or a phosphate group
  • R 9 represents a hydroxyl group, an amino group or a carboxyl group
  • a, b, c, and d represent an alkyl group having 1-20 carbon atoms.
  • the representative structure of a TLR4 agonist is as follows:
  • R 8 represents a hydrogen atom or a phosphate group
  • R 9 represents a hydroxyl group, an amino group, a carboxyl group, a phosphoric acid group
  • R 10 represents a hydrogen atom or a methyl group, including R/S configuration
  • R 11 and R 12 each independently represent a hydroxyl group, an amino group, or a carboxyl group
  • R 13 , R 14 and R 15 each independently represent a fatty acyl group with a carbon number of 1-20;
  • X represents oxygen or sulfur or selenium atom or amino group
  • Z represents oxygen or amino
  • e, f and g represent alkyl groups with 1-20 carbon numbers
  • h, i, j, and k each independently represent an integer of 0-6.
  • the representative structure of the NKT cell agonist is as follows:
  • each covalent linking arm is independently selected from the following structures:
  • Each x is independently selected from an integer of 1-60;
  • Each Y is independently selected from at least one of -NH-, -O-, -S- and -S-S-;
  • Each p is independently selected from an integer of 1-60.
  • the present invention does not specifically limit the method for preparing the aforementioned anti-tumor vaccine molecule.
  • Those skilled in the art can combine the structural characteristics of the anti-tumor vaccine molecule in the art and the conventional synthetic methods in the field to determine a suitable method to prepare the anti-tumor vaccine molecule.
  • the example part of the present invention exemplarily lists the specific preparation methods of some anti-tumor vaccine molecules, and those skilled in the art can also combine the exemplified preparation methods of the present invention to determine the specific preparation of all anti-tumor vaccine molecules of the present invention.
  • these skilled in the art should not understand that this is a limitation of the present invention.
  • the adjuvants, antigens and proteins in the anti-tumor vaccine molecules of the present invention can be synthesized by using existing methods, or can be obtained commercially, and the present invention is not limited to this.
  • the second aspect of the present invention provides a method for preparing the anti-tumor vaccine molecule according to the first aspect of the present invention, the method comprising:
  • the adjuvant is coupled to the protein for the third time, and the obtained second intermediate is coupled to the antigen for the fourth time.
  • the first one is: the first coupling of the antigen and the protein, and the second coupling of the obtained first intermediate and the adjuvant.
  • the second method is to perform a third coupling between the adjuvant and the protein, and perform a fourth coupling between the obtained second intermediate and the antigen.
  • the present invention has no particular restrictions on the specific conditions of each coupling, and those skilled in the art can determine suitable conditions in combination with the context of the present invention and the routine in the art.
  • the third aspect of the present invention provides the application of the anti-tumor vaccine molecule described in the first aspect of the present invention in an anti-tumor vaccine.
  • the present invention provides an anti-tumor vaccine molecule with the structure shown in Figure 1A.
  • the antigen molecule and the adjuvant molecule are shared with the same protein (that is, the carrier protein). After valency connection, an embedded adjuvant three-in-one protein conjugate is obtained, which can produce high-titer IgG antibody immune response when used as an anti-tumor vaccine molecule.
  • both LK1 and LK2 represent covalent link arms.
  • the adjuvant, antigen, protein, and covalent linking arm are all as described above.
  • TLR7 agonists when used as adjuvants, they can significantly better enhance immune stimulating activity and reduce side effects. Therefore, in another more preferred embodiment of the present invention, a TLR7 agonist is used as an adjuvant, and BSA is used as a protein, and the three components of the tumor-associated glycopeptide antigen MUC1 are covalently combined to form an adjuvant-protein-antigen vaccine molecule,
  • the specific structure of the vaccine molecule is shown in Figure 1B, where LK1 and LK2 both represent covalent link arms.
  • the antigen, protein, and covalent linking arm are all as described above.
  • the anti-tumor vaccine molecule not only produces significant IgG antibodies, but also induces a relatively high level of IgG2a, resulting in the antibody type biased towards Th1 type cellular immunity.
  • the present invention provides a three-in-one protein-binding vaccine strategy with a novel structure, that is, covalently binding an adjuvant to a carrier protein bound to an antigen. This solution is first applied to the field of anti-cancer (ie anti-tumor) vaccines.
  • the present invention also has the following specific advantages:
  • APCs antigen presenting cells
  • the antibodies produced can recognize cancer cells and can initiate the lysis of the recognized cancer cells by activating, for example, the complement-dependent cytotoxicity (CDC) of rabbit serum.
  • CDC complement-dependent cytotoxicity
  • CTL cytotoxic T lymphocyte killing effect
  • the three-in-one protein conjugate (that is, the anti-tumor vaccine molecule) provided by the present invention is an effective strategy for designing high-efficiency immunotherapy anti-cancer vaccines.
  • test methods used unless otherwise specified, are all conventional methods.
  • the materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.
  • TLR7a-BSA BSA (0.3 ⁇ mol, purchased from Wuhan Chucheng Zhengmao Technology Engineering Co., Ltd., brand CC1050003) was dissolved in PBS (2mL), compound 13 (0.006mmol) was added to DMF to dissolve, the two solutions were mixed and placed on a shaker at 25°C Reaction for 48h.
  • the obtained compound 14 (defined as TLR7a-BSA) was purified by centrifugal filtration using an ultrafiltration tube (Millipore UFC910096 15M, 10KD) and lyophilized. Using MALDI-TOF-MS test, the average number of TLR7a and BSA covalently linked is 6-7 after analysis.
  • Compound 8 was synthesized by manual solid phase, and the operation steps are as follows:
  • Kaiser-test judges whether the amino group is deprotected by observing the color development of the resin.
  • the main components of the developer are ninhydrin, phenol and pyridine.
  • Compound 10 was synthesized by artificial solid phase, and the operation steps are as follows:
  • Kaiser-test judges whether the amino group is deprotected by observing the color development of the resin.
  • the main components of the developer are ninhydrin, phenol and pyridine.
  • Diethyl squarate (6.0 equivalents) and DIPEA (6.0 equivalents) were added to dry DMF solvent (2-3 mL), mixed uniformly, and reacted at room temperature for about 2 hours under the protection of nitrogen.
  • Example 5 Preparation of coupling of antigen MUC1 squaraine monoamide and carrier protein BSA
  • Example 6 Preparation of BSA-MUC1 conjugate and TLR7 agonist coupling, and the anti-tumor vaccine molecule is defined as TLR7a-BSA-MUC1.
  • Kaiser-test judges whether the amino group is deprotected by observing the color development of the resin.
  • the main components of the developer are ninhydrin, phenol and pyridine.
  • the specific immunization time course is the first day, the 15th day and the 29th day, respectively, and the injection method is intraperitoneal injection.
  • the blood collection method is tail-end blood collection.
  • Blood collection time Take blank blood before immunization, take blood 2 hours after the first immunization, and take blood 14 days after each immunization injection, of which the last time (day 42) is the orbital blood collection . After the blood was taken out, it was centrifuged in a centrifuge and the serum was taken out and stored at -80°C.
  • Example 9 Determination of cytokines in vivo
  • Antigen coating add 100 ⁇ L of the diluted capture antibody (diluted 200 times with the coating solution) to each well, seal the 96-well plate with plastic wrap and incubate overnight in a refrigerator at 4°C after adding the solution.
  • Substrate color reaction After mixing the color developer A and color reagent B in equal volumes, add 100 ⁇ L per well to a 96-well plate. Place the 96-well plate at room temperature and shake for 30 minutes in the dark. Finally, 100 ⁇ L stop solution should be added to each well. Put the 96-well plate into the microplate reader immediately and measure the absorbance at 450nm. Draw a standard curve with the absorbance value of the standard, and substitute the absorbance value of the serum into the standard curve to obtain the cytokine content in the serum.
  • Figure 2 shows that the serum taken 2h after the first immunization was used for the determination of IFN- ⁇ and IL-6.
  • Each bar represents the average content of 5 mice.
  • the serum of each mouse was repeated three times independently, and the value was taken for three times.
  • the mean value, the error bar indicates the standard error of the mean (SEM).
  • the significant difference is relative to the PBS group: **P ⁇ 0.01; ****P ⁇ 0.0001; ns, the significant difference is not obvious. Comparison between vaccines: **P ⁇ 0.01; ****P ⁇ 0.0001; ns, the significant difference is not obvious.
  • Example 10 Indirect non-competitive ELISA to determine the content of antibodies in the blood
  • Blocking add 100 ⁇ L of 1% casein PBS buffer to each well (the concentration of the PBS buffer is a mass percentage, the same below), incubate in a 37°C incubator for 1 hour, and wash the same as above.
  • Antigen and antibody specific binding Dilute the serum with 0.1% casein PBS buffer, the dilution factor is 200, 400, 800, 1600, 3200, 64000, 128000, 256000, 512000, 1024000, 2048000, 4096000, 8192000, 16384000. Add 100 ⁇ L of diluent to each well, set blank and negative controls. Incubate at 37°C for 1h. Same as above for washing.
  • Color development add 100 ⁇ L color development solution to each well (one board needs substrate buffer 9.5mL+0.5mL F solution (2mg/mL TMB/absolute ethanol) + 32uLG solution (35% hydrogen peroxide urea/water solution) ), shake and mix well, store at room temperature in the dark for 5 minutes.
  • Stop add 50 ⁇ L stop solution (2M sulfuric acid) to each well, put it in the microplate reader and mix well and read, and measure the absorbance value of each well at 450nm wavelength.
  • Figure 3 is an evaluation of the anti-MUC1 titer of the vaccine produced by the serum obtained after the third immunization (IgG antibody titer test result). Each bar represents the average titer of 5 mice, and each value is repeated three times independently. The error bars indicate the standard error of the mean. The significant difference is relative to the PBS group: **P ⁇ 0.01; ***P ⁇ 0.001; ns, the significant difference is not obvious. Comparison between vaccines: **P ⁇ 0.01; ***P ⁇ 0.001; ns, the significant difference is not obvious.
  • Figure 4 shows the evaluation of the anti-MUC1 IgG titer produced by the vaccine for the serum obtained after the first, second, and third immunizations. Each bar represents the average titer of 5 mice, and each value is repeated three times independently. The error bars indicate the standard error of the mean.
  • Figure 5 shows the evaluation of the anti-MUC1 IgM titers produced by the vaccine for the sera obtained after the first, second, and third immunizations. Each bar represents the average titer of 5 mice, and each value is repeated three times independently. The error bars indicate the standard error of the mean.
  • Figure 6 shows the evaluation of the anti-MUC1 antibody subtypes produced by the vaccine for the serum obtained after the third immunization. Each bar represents the average titer of 5 mice, and each value is repeated three times independently. The error bars indicate the standard error of the mean. Significant difference relative to PBS group: ns, significant difference is not obvious; ****P ⁇ 0.0001.
  • Example 11 Indirect non-competitive ELISA to determine the content of antibodies in blood
  • BSA antigen
  • Blocking add 100 ⁇ L of 1% casein PBS buffer to each well, incubate at 37°C for 1 hour, and wash as above.
  • Antigen and antibody specific binding Dilute the serum with 0.1% casein PBS buffer, the dilution factor is 200, 400, 800, 1600, 3200, 64000, 128000, 256000, 512000, 1024000, 2048000, 4096000, 8192000, 16384000. Add 100 ⁇ L of diluent to each well, set blank and negative controls. Incubate at 37°C for 1h. Same as above for washing.
  • Color development add 100 ⁇ L color development solution to each well (one board needs substrate buffer 9.5mL+0.5mL F solution (2mg/mL TMB/absolute ethanol) + 32uL G solution (35% hydrogen peroxide) Urea/water solution)), shake and mix well, and store at room temperature in the dark for 5 minutes.
  • Stop add 50 ⁇ L stop solution (2M sulfuric acid) to each well, put it in the microplate reader and mix well and read, and measure the absorbance value of each well at 450nm wavelength.
  • Figure 7 shows the evaluation of the anti-BSA titer produced by the vaccine for the serum obtained after the third immunization, coated with BSA.
  • Each bar represents the average titer of 5 mice, and each value is repeated three times independently.
  • the error bars indicate the standard error of the mean.
  • Significant difference relative to the PBS group *P ⁇ 0.05; **P ⁇ 0.01; ****P ⁇ 0.0001.
  • Comparison between vaccines *P ⁇ 0.05; ****P ⁇ 0.0001; ns, the significant difference is not obvious.
  • Example 12 Determination of MCF-7 cell viability by MTT method
  • This example aims to explore whether antibodies can mediate complement lysis by activating CDC.
  • rabbit complement (1:50 dilution) 1% BSA/PBS (RC stands for rabbit complement; HIRC stands for high temperature inactivated rabbit complement) (50 ⁇ L/well) and incubate for 4 hours.
  • BSA/PBS rabbit complement
  • HIRC high temperature inactivated rabbit complement
  • Figure 8 shows the MTT experiment to evaluate the survival rate of MCF-7 cells for the serum obtained after the third immunization.
  • Each bar represents the average value of 5 mice, and each value is repeated three times independently.
  • the error bars indicate the standard error of the average.
  • Significant difference relative to the PBS group *P ⁇ 0.05; ***P ⁇ 0.001, ****P ⁇ 0.0001, ns, the significant difference is not obvious.
  • Comparison between vaccines *P ⁇ 0.05; ****P ⁇ 0.0001.
  • Example 13 Determination of antibody binding to MCF-7 cells by flow cytometry (FACS)
  • Figure 9 shows the flow cytometry (FACS) analysis of the binding of antiserum from immunized mice to MCF-7 cancer cells. Take the PBS group (black) as the control. These images are representative of five independent experiments.
  • Example 14 Determination of the binding of antibodies to MCF-7 cells by confocal microscopy
  • MCF-7 cells were stained with mouse serum to determine their potential to recognize MUC1 targets.
  • Cytotoxicity (%) (experimental group-spontaneous target cell/absorbance of cell maximum enzyme activity-spontaneous target cell) ⁇ 100%
  • Figure 10 shows the CTL experiment to evaluate the in vitro cytotoxicity of spleen cells to MCF-7 cells after the third immunization. Each bar represents the average of 5 mice, and the error bars indicate the standard error of the average. Comparison between vaccines: *P ⁇ 0.05; **P ⁇ 0.01.
  • the anti-tumor vaccine molecules provided by the present invention produce significantly high-affinity IgG antibodies against tumor-associated antigens or specific antigens.
  • the antibodies produced can recognize cancer cells and initiate the lysis of the recognized cancer cells by activating the complement-dependent cytotoxicity (CDC) of rabbit serum.
  • CDC complement-dependent cytotoxicity

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Abstract

L'invention concerne une molécule vaccinale antitumorale, son procédé de préparation et son utilisation. La molécule vaccinale anti-tumorale a une structure telle que représentée dans la formule (I). Dans la formule (I), A est un adjuvant, B est un antigène, les m des A sont respectivement liés de manière covalente à une protéine au moyen d'un bras de liaison covalent, et les n des B sont respectivement liés de manière covalente à la protéine au moyen d'un bras de liaison covalent. Le vaccin antitumoral est une nouvelle molécule antitumorale, présente une bonne performance immunitaire, peut produire un anticorps IgG à titre élevé, présente une immunité cellulaire relativement forte, a une bonne stabilité thermique, et est facile à stocker et à transporter. Am-Protéine-Bn Formule (I)
PCT/CN2020/133798 2019-12-04 2020-12-04 Molécule vaccinale antitumorale, son procédé de préparation et son utilisation WO2021110120A1 (fr)

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