WO2023098755A1 - 重组hEGF-CRM197肿瘤治疗性疫苗配制剂 - Google Patents
重组hEGF-CRM197肿瘤治疗性疫苗配制剂 Download PDFInfo
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- WO2023098755A1 WO2023098755A1 PCT/CN2022/135608 CN2022135608W WO2023098755A1 WO 2023098755 A1 WO2023098755 A1 WO 2023098755A1 CN 2022135608 W CN2022135608 W CN 2022135608W WO 2023098755 A1 WO2023098755 A1 WO 2023098755A1
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- growth factor
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Definitions
- the invention relates to the field of biomedicine, in particular to a preparation method and application of a recombinant hEGF-CRM197 tumor therapeutic vaccine preparation.
- tumor vaccines that activate the body's specific anti-tumor immune function to treat cancer has become a research hotspot in the field of malignant tumor treatment.
- Tumor vaccines activate tumor-specific immune responses through tumor-associated antigens to achieve the purpose of killing and eliminating tumor cells. It is a therapeutic active immunotherapy method.
- protein conjugated vaccines or polypeptide conjugated vaccines based on carrier proteins are one of the hotspots in the development of therapeutic vaccines.
- the object of the present invention is to provide a stable recombinant hEGF-CRM197 tumor therapeutic vaccine formulation, which includes recombinant human epidermal growth factor (hEGF) and diphtheria toxin mutant conjugates, phosphate buffered saline with pH in the range of 7.0-8.5 Liquid, and surfactant polysorbate 20, and/or optional monosaccharide or disaccharide, and the method for preparing described preparation.
- hEGF human epidermal growth factor
- diphtheria toxin mutant conjugates phosphate buffered saline with pH in the range of 7.0-8.5 Liquid
- surfactant polysorbate 20 phosphate buffered saline with pH in the range of 7.0-8.5 Liquid
- optional monosaccharide or disaccharide optional monosaccharide or disaccharide
- the first aspect of the present invention provides a recombinant hEGF-CRM197 tumor therapeutic vaccine formulation, said formulation comprising:
- the protein stabilizer includes: surfactant polysorbate 20 and optional monosaccharide or disaccharide.
- the conjugate is obtained by conjugating recombinant human epidermal growth factor hEGF and diphtheria toxin mutant CRM197 through a chemical cross-linking agent glutaraldehyde.
- the conductivity of the buffer is not higher than 3mS/cm.
- the recombinant human epidermal growth factor contained in the conjugate is coupled to the diphtheria toxin mutant in a manner of covalent cross-linking with glutaraldehyde.
- the conjugate has a structure as shown in formula (I):
- hEGF is recombinant human epidermal growth factor hEGF
- CRM197 is a diphtheria toxin mutant CRM197
- L is a linker linking recombinant human epidermal growth factor and diphtheria toxin mutant CRM197, said linker being a covalent bond formed by glutaraldehyde cross-linking;
- the cross-linking molar ratio of recombinant human epidermal growth factor hEGF/diphtheria toxin mutant CRM197 in the conjugate is 6:1.
- the recombinant human epidermal growth factor hEGF is an EGF molecule containing human epidermal growth factor hEGF biological activity or a mutant thereof, and the EGF biological activity means that the EGF molecule can stimulate or induce the human epidermal growth factor hEGF containing EGFR receptors for cell signaling.
- the recombinant human epidermal growth factor hEGF is the full-length sequence of human epidermal growth factor hEGF or a partial fragment thereof.
- the full-length sequence of the human epidermal growth factor hEGF has the amino acid sequence shown in SEQ ID NO:1.
- the recombinant human epidermal growth factor hEGF is based on the full-length sequence of human epidermal growth factor hEGF with 2-3 amino acids truncated at the N-terminal or C-terminal.
- the recombinant human epidermal growth factor hEGF has the amino acid sequence shown in SEQ ID NO:3.
- the diphtheria toxin mutant CRM197 has the amino acid sequence shown in SEQ ID NO:2.
- the basic buffer is a phosphate buffer with a phosphate concentration of 1-40 mmol/L.
- the phosphate buffer includes sodium phosphate buffer (NaH 2 PO 4 &Na 2 HPO 4 ) and potassium phosphate buffer (K 2 HPO 4 &KH 2 PO 4 ).
- the base buffer is 20mmol/L phosphate buffer, pH 8.0.
- the base buffer is 10mmol/L phosphate buffer, pH 7.5.
- the base buffer is 5mmol/L phosphate buffer, pH 7.0.
- the concentration of polysorbate 20 in the preparation is 0.001%-0.1% (v/v).
- the concentration of polysorbate 20 in the preparation is 0.005%-0.02% (v/v).
- the concentration of polysorbate 20 in the preparation is 0.1% (v/v).
- the concentration of polysorbate 20 in the preparation is 0.02% (v/v)
- the concentration of polysorbate 20 in the preparation is 0.01% (v/v).
- the concentration of polysorbate 20 in the preparation is 0.005% (v/v).
- the concentration of polysorbate 20 in the preparation is 0.001% (v/v).
- the optional monosaccharide or disaccharide is selected from the group consisting of sucrose, glucose, mannitol, maltose, or combinations thereof.
- the optional monosaccharide or disaccharide is sucrose
- the preparation contains sucrose at a concentration of 0-4% (mass/volume).
- the preparation contains sucrose at a concentration of 2% (mass/volume).
- the preparation contains sucrose at a concentration of 4% (mass/volume).
- the optional monosaccharide or disaccharide is maltose, and the concentration of maltose in the preparation is 2% (mass/volume).
- the formulation is a liquid formulation.
- the preparation is an injection.
- the content of the conjugate of recombinant human epidermal growth factor hEGF and diphtheria toxin mutant in the preparation is 0.4-3.2 mg/ml.
- the preparation does not contain free amino acids.
- the preparation has the following characteristics: the preparation is stable at a temperature of 37°C for at least 3 months, preferably 6 months.
- the preparation is stable for 6 months at a temperature of 25°C ⁇ 2°C or 37°C ⁇ 2°C.
- stable existence means that the key quality parameters of the preparation, such as appearance, visible foreign matter, protein content, purity, molecular weight distribution, relative potency in vitro, and biological activity in vivo, do not change significantly.
- a second aspect of the present invention provides a method for preparing the recombinant hEGF-CRM197 tumor therapeutic vaccine formulation described in the first aspect of the present invention, the method comprising the following steps:
- amino acid sequence of the recombinant human epidermal growth factor hEGF protein is shown in SEQ ID NO:3.
- amino acid sequence of the diphtheria toxin mutant CRM197 protein is shown in SEQ ID NO:2.
- the reaction condition in step (ii) is room temperature (25°C ⁇ 2°C), and the reaction time is 2 hours to 3 hours.
- the crosslinking agent is glutaraldehyde.
- ultrafiltration is used for purification in step (iii).
- ultrafiltration is used to replace the liquid in step (iv).
- the basic buffer is a phosphate buffer with a pH of 7.0-8.5, and the phosphate concentration is 1-40 mmol/L.
- the conductivity of the base buffer is not higher than 3mS/cm
- the protein stabilizer includes: surfactant polysorbate 20 and optional monosaccharide or disaccharide.
- the concentration of the polysorbate 20 is 0.005%-0.02% (v/v).
- the optional monosaccharide or disaccharide is selected from the group consisting of sucrose, glucose, mannitol, maltose, or combinations thereof.
- the third aspect of the present invention provides a composition, characterized in that the composition comprises (a) the tumor therapeutic vaccine formulation described in the first aspect of the present invention; and (b) a pharmaceutically acceptable carrier.
- the composition is a pharmaceutical composition or a vaccine composition.
- the vaccine composition further contains an adjuvant.
- the adjuvant includes: granular and non-granular adjuvants.
- the particulate adjuvant is selected from the group consisting of aluminum salts, water-in-oil emulsions, oil-in-water emulsions, nanoparticles, microparticles, liposomes, immunostimulatory complexes, or combinations thereof;
- the non-granular adjuvant is selected from the group consisting of muramyl dipeptide and its derivatives, saponins, lipid A, cytokines, derived polysaccharides, bacterial toxins, microorganisms and their products such as Mycobacterium (Mycobacterium tuberculosis, BCG), Brevibacterium, Bacillus pertussis, propolis, or combinations thereof.
- the adjuvant is selected from the group consisting of Montanide ISA 51 VG, aluminum phosphate adjuvant, MF59, AS04, or a combination thereof.
- the composition is prepared as a liquid preparation.
- composition is prepared as injection, suspension, or spray.
- the fourth aspect of the present invention provides a use of the tumor therapeutic vaccine formulation described in the first aspect of the present invention in the preparation of medicaments for treating and/or preventing diseases.
- the disease is tumor or cancer.
- the tumor (or cancer) includes a solid tumor.
- the solid tumor is selected from the group consisting of lung cancer, non-small cell lung cancer, colorectal cancer, breast cancer, liver cancer, gastric cancer, esophageal cancer, pancreatic cancer, melanoma, kidney cancer, prostate cancer, cervical cancer cancer, ovarian cancer, nasopharyngeal cancer, oral cavity cancer, osteosarcoma, glioma, bladder cancer, or a combination thereof.
- Figure 1 shows the detection of antibody titers in mice immunized with recombinant hEGF-CRM197 conjugates.
- Fig. 2 shows the molecular weight distribution (SEC-HPLC) profile of the preparation containing polysorbate 80 (code a7) accelerated for 1 month at 37°C.
- Fig. 3 shows the molecular weight distribution (SEC-HPLC) spectrum of the formulation containing polysorbate 20 (code a13) accelerated at 37°C for 1 month.
- Figure 4 shows the molecular weight distribution (SEC-HPLC) of the recombinant hEGF-CRM197 tumor therapeutic vaccine formulation (0.01% Tween20, 2% sucrose, 20mM PB, pH8.0, 1.6mg/ml) initial detection and 25 °C accelerated 6 Monthly Atlas Comparison.
- Fig. 5 shows the preliminary test of the biological activity of the recombinant hEGF-CRM197 tumor therapeutic vaccine formulation (0.01% Tween20, 2% sucrose, 20mM PB, pH8.0, 1.6mg/ml) and the comparison of accelerated 6 months at 25°C.
- Figure 6 shows the detection of antibody titers induced by recombinant hEGF-CRM197 tumor therapeutic vaccine formulations in cynomolgus monkeys.
- Figure 7 shows the effect of the recombinant hEGF-CRM197 tumor therapeutic vaccine formulation on the EGF content in the serum of cynomolgus monkeys.
- Fig. 8 shows the detection results of antibody titers in mice immunized with rhEGF-P64k conjugate (CIMAvax-EGF) and recombinant hEGF-CRM197 conjugate.
- Figure 9 shows the detection results of rhEGF-P64k conjugate (CIMAvax-EGF) and recombinant hEGF-CRM197 conjugate blocking the combination of EGF and EGFR in vitro.
- the vaccine formulation of the present invention comprises recombinant human epidermal growth factor (hEGF) and diphtheria toxin mutant conjugates, phosphate buffer saline for maintaining pH in the range of 7.0-8.5, surfactant polysorbate 20 (Tween 20), and /or optionally a mono- or di-saccharide.
- hEGF human epidermal growth factor
- Tween 20 surfactant polysorbate 20
- the vaccine formulation of the present invention has a low proportion of polymers and high molecular weight uniformity. , EGF antigen amount, biological activity and other quality attributes have no change.
- the recombinant hEGF-CRM197 tumor therapeutic vaccine formulation of the present invention can be used as a high-efficiency tumor treatment and/or preventive drug that can be rapidly mass-produced and stored for a long time. On this basis, the present invention has been accomplished.
- the term "about” when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value.
- the expression “about 100” includes all values between 99 and 101 and in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).
- treatment refers to the administration of internal or external therapeutic agents to patients, including vaccines and compositions thereof against tumors of the present invention, said patients have one or more disease symptoms, and the therapeutic agents are known to be effective on These symptoms are therapeutic.
- an amount of the therapeutic agent effective to alleviate one or more symptoms of the disease is administered to a patient.
- optional means that the subsequently described event or circumstance can but does not have to occur.
- optional monosaccharide or disaccharide means that a specific monosaccharide or disaccharide can be present but not necessarily, and can be 1, 2 or 3.
- EGF Epidermal Growth Factor
- Epidermal growth factor is a heat-resistant single-chain low-molecular-weight polypeptide consisting of 53 amino acid residues. After EGF specifically recognizes and binds with the EGF receptor on the target cell, a series of biochemical reactions occur, which can finally promote the DNA synthesis and mitosis of the target cell.
- EGF has no sugar-based parts, very stable, heat-resistant and acid-resistant, widely present in body fluids and various glands, mainly synthesized by submandibular glands and duodenum, found in most body fluids of the human body, in milk, urine The content in fluid and semen is specifically increased, but the concentration in serum is low. EGF has a wide range of functions, and is of great significance in estimating the prognosis of tumors and choosing treatment options.
- Epidermal growth factor (EGF) of the present invention is human epidermal growth factor (hEGF), specifically, is a kind of recombinant human epidermal growth factor (hEGF), and described recombinant human epidermal growth factor (hEGF) is human epidermal growth factor (hEGF) ) of the full-length sequence (as shown in SEQ ID NO: 1) based on N-terminal or C-terminal truncation of 2-3 amino acids obtained.
- the recombinant human epidermal growth factor of the present invention is obtained by truncating 2 amino acids at the C-terminus on the basis of the full-length sequence of human epidermal growth factor (hEGF), which has the sequence shown in SEQ ID NO: 3 Amino acid sequence:
- Diphtheria toxin (diphtheria toxin, DT) is an exotoxin produced by Corynebacterium diphtheriae infected with beta bacteriophage, and exists in the clinically used DPT vaccine components. Its safety has been verified by clinical use for many years, and serious adverse reactions are rare, and there is no report of allergic reactions caused by diphtheria components.
- the diphtheria toxin molecule consists of 535 amino acid residues, and is composed of relatively independent catalytic domains (1-193AAs), transmembrane domains (205-378AAs) and receptor binding domains (386-535AAs); the transmembrane domains And the receptor binding domain itself is non-toxic, and its function is to transduce the catalytic domain into the cell through the binding of the cell surface receptor.
- CRM197 is a kind of diphtheria toxin mutant obtained from the bacterial strain of the virulence gene mutant of Bacillus diphtheriae. It has a length of 536 amino acids, specifically, the glycine at the 52nd position of the diphtheria toxin is mutated into glutamic acid, which has such as SEQ ID Amino acid sequence shown in NO:2.
- the mutant toxin A fragment cannot combine with elongation factor II in the nucleus, making it lose the cytotoxic effect, but the antigenicity and immunogenicity are still basically consistent with the natural diphtheria toxin.
- hEGF Human Epidermal Growth Factor
- hEGF-CRM197 Diphtheria Toxin Mutant Conjugate
- the present invention provides a recombinant human epidermal growth factor (hEGF) and diphtheria toxin mutant conjugate, the conjugate has a structure as shown in formula (I):
- hEGF is recombinant human epidermal growth factor (hEGF);
- CRM197 is a diphtheria toxin mutant CRM197
- L is a linker connecting recombinant human epidermal growth factor and diphtheria toxin mutant CRM197, said linker being a covalent bond formed by glutaraldehyde crosslinking;
- the recombinant human epidermal growth factor is an EGF molecule containing human epidermal growth factor (hEGF) biological activity or a mutant thereof, and the EGF biological activity means that the EGF molecule can stimulate or induce Receptor cells carry out signal transduction.
- the recombinant human epidermal growth factor has the amino acid sequence shown in SEQ ID NO:3; the diphtheria toxin mutant CRM197 has the amino acid sequence shown in SEQ ID NO:2 .
- the present invention also provides a recombinant hhEGF-CRM197 vaccine formulation, which comprises: (a) recombinant human epidermal growth factor (hEGF) and diphtheria toxin mutant conjugate; (b) pH7.0-8.5 range Base buffer, wherein the conductivity of the buffer is not higher than 10mS/cm; and (c) protein stabilizer; wherein, the protein stabilizer includes: surfactant polysorbate 20 (Tween 20) and optionally monosaccharides or disaccharides.
- hEGF-CRM197 vaccine formulation which comprises: (a) recombinant human epidermal growth factor (hEGF) and diphtheria toxin mutant conjugate; (b) pH7.0-8.5 range Base buffer, wherein the conductivity of the buffer is not higher than 10mS/cm; and (c) protein stabilizer; wherein, the protein stabilizer includes: surfactant polysorbate 20 (Tween 20) and optionally monosaccharides or disaccharides.
- the basic buffer is a phosphate buffer, and the phosphate concentration is 1-40mmol/L; the concentration of polysorbate 20 in the preparation is 0.001%-0.1% (v/v)
- the optional monosaccharide or disaccharide includes (but not limited to) sucrose, glucose, mannitol, maltose, or a combination thereof.
- the monosaccharide or disaccharide is sucrose, and the sucrose in the preparation The concentration is 0-4%.
- the recombinant hEGF-CRM197 vaccine formulation of the present invention is a tumor therapeutic formulation, therefore, the terms "recombinant hEGF-CRM197 vaccine formulation” and “recombinant hEGF-CRM197 tumor therapeutic vaccine formulation” are used interchangeably, both referring to
- the liquid preparation prepared by the invention contains recombinant human epidermal growth factor (hEGF) and diphtheria toxin mutant conjugate (hEGF-CRM197).
- the present invention also provides a preparation method of the recombinant hEGF-CRM197 vaccine formulation, the method comprising the following steps:
- the invention uses a chemical cross-linking method to couple recombinant human epidermal growth factor (hEGF) protein with diphtheria toxin mutant CRM197 protein, thereby obtaining a novel tumor therapeutic vaccine.
- hEGF human epidermal growth factor
- Coupling hEGF with CRM197 through glutaraldehyde can help hEGF improve immunogenicity and induce the human body to produce anti-hEGF antibodies.
- the preparation solution with the best effect of preparing the tumor therapeutic vaccine into a preparation is screened out through multiple tests.
- the recombinant hEGF-CRM197 vaccine formulation prepared by the above method has low polymer ratio and high molecular weight uniformity. After accelerated treatment at 25°C ⁇ 2°C and 37°C ⁇ 2°C for 6 months, its appearance, visible foreign matter, Quality attributes such as molecular weight distribution, protein content, EGF antigen amount, and biological activity do not change, so it can be stored stably for a long time.
- the present invention also provides a composition
- a composition comprising (a) the tumor therapeutic vaccine formulation described in the first aspect of the present invention; and (b) a pharmaceutically acceptable carrier.
- compositions of the invention include pharmaceutical or vaccine compositions.
- compositions are prepared as liquid formulations, including (but not limited to) injections, suspensions, or sprays.
- the pharmaceutical composition of the present invention comprises (or contains) a therapeutically effective amount of hEGF-CRM197 of the present invention.
- the term "therapeutically effective amount” refers to an amount of a therapeutic agent that treats, alleviates or prevents a target disease or condition, or exhibits a detectable therapeutic or preventive effect. This effect can be detected, for example, by antigen levels. A therapeutic effect also includes a reduction in physical symptoms.
- the precise effective amount for a subject will depend on the size and health of the subject, the nature and extent of the disorder, and the therapeutic agents and/or combination of therapeutic agents chosen for administration. Therefore, it is not useful to prespecify an exact effective amount. However, the effective amount can be determined by routine experimentation for a given situation.
- an effective dosage is about 0.001 mg/kg to 10 mg/kg, preferably about 0.003 mg/kg to 0.1 mg/kg body weight administered to an individual.
- the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.
- pharmaceutically acceptable carrier refers to a carrier used for the administration of a therapeutic agent such as hEGF-CRM197 of the present invention.
- the term refers to pharmaceutical carriers which do not, by themselves, induce the production of antibodies deleterious to the individual receiving the composition and which are not unduly toxic upon administration.
- Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, and the like. These vectors are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable carriers or excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
- compositions can include liquids such as water, saline, glycerol and ethanol.
- auxiliary substances in these carriers such as wetting agents or emulsifying agents, pH buffering substances and the like.
- the compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. Liposomes are also included within the definition of pharmaceutically acceptable carriers.
- the vaccine compositions of the present invention comprise immunogenic antigens (including hEGF-CRM197 of the present invention), and are typically combined with "pharmaceutically acceptable carriers” that include antibodies that do not, by themselves, induce the production of antibodies harmful to the individual receiving the composition.
- any carrier any carrier. Suitable carriers are usually large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, amino acid polymers, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and the like. These vectors are well known to those of ordinary skill in the art. In addition, these carriers can act as immunostimulants ("adjuvants").
- Preferred adjuvants for enhancing the effect of immune compositions include but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations, such as: (a) Montanide ISA 51VG, (b) Montanide ISA 720VG; (C) MF59; (3) saponin adjuvant; (4) Freund's complete adjuvant (CFA) and Freund's incomplete adjuvant (IFA); (5) cytokines, Such as interleukin (such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferon (such as gamma interferon), macrophage colony-stimulating factor ( GM-CSF), etc.; (6) detoxified variants of bacterial ADP-ribosylating toxins (such as Escherichia coli heat-labile toxin LT); and
- Vaccine compositions including immunogenic compositions (eg, may include antigens, pharmaceutically acceptable carriers, and adjuvants), generally contain diluents such as water, saline, glycerol, ethanol, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. More specifically, vaccines, including immunogenic compositions, comprise an immunologically effective amount of the immunogenic composition, and other required components as described above.
- An "immunologically effective amount” refers to an amount administered to a subject as a single dose or a fraction of consecutive doses that is effective for treatment.
- the amount can be based on the health and physiological condition of the individual to be treated, the class of the individual to be treated (e.g., human), the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating physician's assessment of the medical condition, and other related factors. This amount is expected to lie within a relatively wide range and can be determined by routine experimentation.
- vaccine or immunogenic compositions are prepared as injectables, such as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
- the formulation can also be emulsified or encapsulated in liposomes for enhanced adjuvant effect.
- compositions of the invention can be administered directly to a subject.
- the subject to be treated may be a mammal, especially a human.
- the vaccine composition of the present invention can be administered directly to an individual by known methods, usually using the same route of administration as conventional vaccines.
- the routes of administering the pharmaceutical composition or vaccine composition of the present invention include (but are not limited to): subcutaneous, intradermal, intramuscular or other parenteral routes of administration. Routes of administration can be combined, if desired, or adjusted according to disease conditions.
- Vaccine compositions may be administered in single or multiple doses, and may include administration of booster doses to elicit and/or maintain immunity.
- the vaccine composition should be administered in an "effective amount", that is, the amount of the vaccine composition is sufficient to trigger an immune response in the selected route of administration and can effectively improve disease symptoms.
- Representative diseases include, but are not limited to, tumors (or) cancers, especially solid tumors, selected from the group consisting of: lung cancer, non-small cell lung cancer, colorectal cancer, breast cancer, liver cancer, gastric cancer, esophageal cancer, pancreatic cancer , melanoma, kidney cancer, prostate cancer, cervical cancer, ovarian cancer, nasopharyngeal cancer, oral cancer, osteosarcoma, glioma, bladder cancer, etc.
- tumors or cancers, especially solid tumors, selected from the group consisting of: lung cancer, non-small cell lung cancer, colorectal cancer, breast cancer, liver cancer, gastric cancer, esophageal cancer, pancreatic cancer , melanoma, kidney cancer, prostate cancer, cervical cancer, ovarian cancer, nasopharyngeal cancer, oral cancer, osteosarcoma, glioma, bladder cancer, etc.
- each vaccine dose is selected according to the amount that can elicit an immune protective response without obvious side effects.
- each dose of vaccine is sufficient to contain about 0.1 mg-10 mg, preferably 0.4 mg-5 mg.
- the optimal amount for a particular vaccine can be determined using standard research methods, including observation of antibody titers and other responses in subjects.
- the need for a booster dose can be determined by monitoring the level of immunity provided by the vaccine. After assessment of antibody titers in sera, a booster dose of immunization may be required.
- the immune response to the present invention can be enhanced by administering a pharmaceutical or vaccine composition.
- the vaccines of the invention can be administered in conjunction with other immunomodulators, or with other therapeutic agents.
- the main innovation of the present invention is to develop and provide stable formulations containing human epidermal growth factor and diphtheria toxin mutant conjugates, the main advantages of which include:
- the preparation of the present invention contains an innovative component of a conjugate of human epidermal growth factor and diphtheria toxin mutant, which can break immune tolerance and induce the production of anti-human epidermal growth factor antibodies in the human body;
- the preparation of the present invention contains a low-salt and relatively alkaline basic buffer, which is significantly different from conventional preparation formulations, and can make protein conjugate molecules produce a lower proportion of polymers, and the molecular weight distribution in the buffer is better Uniform;
- the present invention finds out the formulation of the recombinant hEGF-CRM197 tumor therapeutic vaccine formulation using polysorbate 20 and excluding polysorbate 80 through experiments, so that the stability of the protein conjugate molecule in the formulation is better.
- the production method of the preparation of the present invention is simple, can realize large-scale production, and can be stored stably for a long time.
- Example 1 Preparation and detection of recombinant hEGF-CRM197 vaccine stock solution
- 1Combination reaction Take an appropriate amount of 25% glutaraldehyde, and prepare 0.5% glutaraldehyde with phosphate buffered saline (PBS: 20mM PB, 150mM NaCl, pH 7.0). Take equal amounts of recombinant human epidermal growth factor and diphtheria toxin mutant CRM197 (molar ratio is about 10:1), and dilute to 2 mg/ml with phosphate buffer. Recombinant human epidermal growth factor and diphtheria toxin mutant CRM197 prepared at corresponding concentrations were respectively added into the reaction vessel, after thorough mixing, 0.5% glutaraldehyde was added dropwise until the final concentration of glutaraldehyde was 0.1%. Put the reaction vessel into a constant temperature shaker at 25° C., set the shaker speed at 130 rpm, and react for 2 hours.
- PBS 20mM PB, 150mM NaCl, pH 7.0
- 2Ultrafiltration purification After the reaction, use a 50kDa ultrafiltration membrane to concentrate first, then dilute with phosphate (PBS), and concentrate again, repeat the concentration and continue to ensure that the dilution factor reaches at least 2187 times (3 to the 7th power).
- PBS phosphate
- 3Basic buffer replacement continue to use the ultrafiltration membrane, dilute and adjust the concentration with phosphate buffer (PB: 20mM PB, pH 8.0), and ensure that the fluid is changed until the conductivity of the filtrate is not higher than 3mS/cm. Finally, the ultrafiltrate was concentrated until the concentration of the recombinant hEGF-CRM197 conjugate reached more than 2 mg/ml, so as to ensure that the preparation requirements were met.
- phosphate buffer PB: 20mM PB, pH 8.0
- mice The emulsion was injected subcutaneously into the mice at a volume of 0.1ml/mouse. A total of 8 mice were injected again at intervals of 1 week. Blood was collected from the orbits at 1, 3, and 5 weeks after the second injection. Anti-EGF antibody titers were detected.
- Anti-EGF antibody titer detection method
- 3Washing After discarding the blocking solution, wash each well with 200 ⁇ L of PBS containing 0.05% (v/v) Tween-20 repeatedly for 3 times, and blot the liquid in the well;
- mice 4 Dilute the collected mouse serum, take 100 ⁇ L of the diluted serum and add it to the leftmost well of the 96-well plate. Serum from immunized mice was used as a control. After adding the serum, incubate at 37°C for 1 hour; after incubation, discard the incubation solution, and wash repeatedly 3 times as described above;
- Termination add 100 ⁇ l 0.5mol/L sulfuric acid to each well to terminate the color development;
- the test results are shown in Figure 1.
- the antibody titers exceeded 16,000 at 1, 3, and 5 weeks after the second immunization, and the geometric averages reached 215,000, 5.31 million, and 15.02 million, respectively.
- the prepared recombinant hEGF-CRM197 vaccine stock solution was taken, and the recombinant hEGF-CRM197 vaccine was respectively replaced into the basic buffer solution described in Table 1 by ultrafiltration.
- the basic buffer can significantly affect the molecular weight distribution of the recombinant hEGF-CRM197 vaccine stock solution.
- the screening goal of this study is to obtain a basic buffer with a low polymer ratio and high molecular weight uniformity. Therefore, the SEC-HPLC analysis method was selected in this study, and the recombinant hEGF-CRM197 vaccine stock solution in different basic buffers was analyzed by gel filtration analysis column.
- the formulations with 100% qualified appearance and visible foreign matter were accelerated at 37°C for 1 month, and were identified by methods such as visible foreign matter, protein content, molecular weight distribution (polymer ratio and main component ratio), and relative potency in vitro.
- Example 3 Preparation, activity identification and stability study of recombinant hEGF-CRM197 tumor therapeutic vaccine formulation (0.01% Tween20, 2% sucrose, 20mM PB, pH8.0, 1.6mg/ml)
- the preparation amount and preparation concentration calculate the contents of stock solution and various auxiliary materials required for the recombinant hEGF-CRM197 tumor therapeutic vaccine after preparation.
- the preparation container add recombinant hEGF-CRM197 tumor therapeutic vaccine stock solution, Tween 20 and sucrose stock solution, and finally add basic buffer solution (20mM PB) to constant volume.
- the finished preparation is aseptically dispensed into vials.
- the examiner adjusts the position so that the test product is located at the distance of the eye, and the distance from the test product to the human eye is 25cm.
- test sample Take the test sample, mix it with loading buffer 3:1, and put it in a boiling water bath for 5 minutes.
- the samples were analyzed with a gel with acrylamide concentration of 12.5%, and electrophoresis was performed at a constant voltage of 200V for 45min.
- the protein bands in the gel can be visualized by Coomassie brilliant blue staining and scanned for imaging. Calculate the ratio of the recombinant hEGF-CRM197 vaccine band with a molecular weight not smaller than the recombinant CRM197 band to all components, which is the proportion of the main component.
- the emulsion was injected subcutaneously into the mice at a volume of 0.1ml/mouse, a total of 8 mice were injected once every other week, and blood was collected from the orbit one week after the second injection, and the anti-EGF antibody titer was detected after the blood was collected .
- Anti-EGF antibody titer detection method
- 3Washing After discarding the blocking solution, wash each well with 200 ⁇ L of PBS containing 0.05% (v/v) Tween-20 repeatedly for 3 times, and blot the liquid in the well;
- mice 4 Dilute the collected mouse serum, take 100 ⁇ L of the diluted serum and add it to the leftmost well of the 96-well plate. Serum from immunized mice was used as a control. After adding the serum, incubate at 37°C for 1 hour; after incubation, discard the incubation solution, and wash repeatedly 3 times as described above;
- Termination add 100 ⁇ l 0.5mol/L sulfuric acid to each well to terminate the color development;
- Example 4 In vivo biological activity detection of recombinant hEGF-CRM197 tumor therapeutic vaccine formulation in cynomolgus monkeys
- the "pre-emulsification” process requires 20 cycles, slowly pushing the syringe back and forth, with an average of about 8 seconds per cycle, and it takes at least 2 minutes to complete the "pre-emulsification”. After the "pre-emulsification” is completed, enter the "rapid emulsification” process.
- the "rapid emulsification” process has a total of 40 cycles, and the syringe needs to be pushed back and forth as quickly as possible.
- the entire emulsification process needs 60 cycles and takes about 3 minutes, and the finally formed water-in-oil emulsion is milky white and viscous.
- test product After the emulsification is complete, push all the water-in-oil emulsion into a 1.5ml centrifuge tube, and place it in an ice box for later use.
- the test product is fully mixed with an equal volume of adjuvant until it is completely emulsified.
- mice 30 cynomolgus monkeys in total, 10 in each group, aged 3 to 5 years, weighing 2 to 4 kg; they were respectively adjuvant control group, low dose group and high dose group.
- Administration route and method subcutaneous injection on the back of the neck;
- Dosing frequency and duration once a week, 5 times in total
- Administration time generally from 08:30 to 12:00 in the morning;
- Dosing concentration 0.5mg/ml
- Administration volume 0.8ml/body in the low-dose group, and 1.6ml/body in other groups.
- the antibody titer assay method is the same as the anti-EGF antibody titer assay method in Example 1.
- the results of antibody titer detection are shown in Figure 6.
- the results show that the recombinant hEGF-CRM197 tumor therapeutic vaccine formulation can induce cynomolgus monkeys to produce up to 5 million anti-EGF antibody titers, indicating that the recombinant hEGF-CRM197 tumor therapeutic vaccine formulation can break the immune tolerance of cynomolgus monkeys, Cynomolgus monkeys were induced to produce high-titer antibodies, and the in vivo biological activity of the recombinant hEGF-CRM197 tumor therapeutic vaccine formulation was verified.
- Add sample and incubate first add 50 ⁇ L/well Assay Diluent RD1-21, then add 200 ⁇ L/well standard, control and sample solutions respectively, flick the edge of the plate for 1 minute to mix the solution evenly, and seal the plate with the sealing film Incubate at room temperature for 2 hours.
- washing Carefully peel off the sealing film, discard the liquid, shake dry, add 400 ⁇ L of washing buffer to each well, discard, repeat this 3 times, and pat dry.
- Termination Add 50 ⁇ l of Stop Solution to each well, and the color changes from blue to yellow. If the color in the well is green or the color changes unevenly, vortex gently to mix.
- Reading 450nm reading.
- Standard solution 250pg/ml, 125pg/ml, 62.5pg/ml, 31.2pg/ml, 15.6pg/ml, 7.8pg/ml, 3.9pg/ml
- data analysis can use computer software to draw four Parametric logistic curve.
- a standard curve can be drawn based on the average absorbance value of each standard, with the absorbance value marked on the Y axis and the concentration marked on the X axis. Then the EGF concentration in the sample was calculated according to the absorbance value of the sample.
- each concentration standard solution must be repeatedly titrated into two small holes in order to calculate the average spectrophotometric value, and the sample also needs to calculate the average absorbance value).
- the final concentration is the actual measured concentration multiplied by the dilution factor.
- Comparative Example 1 Comparison of biological activities of rhEGF-P64k conjugate (CIMAvax-EGF) and recombinant hEGF-CRM197
- the rhEGF-P64k conjugate (CIMAvax-EGF) was formulated with reference to the recombinant hEGF-CRM197 vaccine and the animal immunization method, and the same method was used respectively in Serum was collected 1 week, 3 weeks, and 5 weeks after the second immunization, and the antibody titer was determined according to the same method.
- FITC-EGF and CIMAvax EGF/recombinant hEGF-CRM197 tumor therapeutic vaccine were used to immunize the serum of BALB/c mice 3 weeks after the second immunization, and the serum of BALB/c mice injected with normal saline 3 weeks after the second immunization According to the volume ratio of 1:25, 1:75, 1:100, incubate at room temperature and avoid light for 15mim.
- NCI-H446 cells in the logarithmic growth phase were digested with trypsin and divided into 11 tubes on average.
- the number of cells in each tube was about 10 4 , centrifuged at 5000rpm for 3min, and the supernatant was discarded.
- FITC-EGF and serum were mixed with NCI-H446 cells respectively, supplemented with physiological saline to a total volume of 110 ⁇ l per tube, and incubated at 4°C in the dark for 30 min. Marked as samples 1-9 respectively, samples 1-3 were recorded as normal saline group, samples 4-6 were recorded as EGF-P64K group, and samples 7-9 were recorded as EGF-CRM197 group. Add 110 ⁇ l of normal saline to sample 10 for zero adjustment of the flow cytometer, add 1 ⁇ l of FITC-EGF to sample 11, and add normal saline to a total volume of 110 ⁇ l per tube as a negative control.
- the serum volume of the CIMAvax EGF group was 3 times that of the recombinant EGF-CRM197 group, roughly judging that the recombinant EGF-CRM197 vaccine group induced neutralizing antibodies 3 times that of the CIMAvax EGF group.
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Abstract
本发明提供了一种重组hEGF-CRM197肿瘤治疗性疫苗配制剂,具体地,本发明的制剂包含治疗有效量的重组人表皮生长因子hEGF与白喉毒素突变体CRM197偶联物、pH7.5-8.5范围的磷酸盐基础缓冲液、聚山梨酯20表面活性剂和任选的单糖或二糖。本发明的制剂中的蛋白偶联物分子可以打破免疫耐受,诱导人体内产生抗人表皮生长因子抗体;此外,蛋白偶联物分子产生更低比例的多聚物,在缓冲液中的分子量分布更均一,稳定性更好。因此本发明的制剂可实现规模化生产,并且可长期稳定的保存。
Description
本发明涉及生物医药领域,具体地,涉及一种重组hEGF-CRM197肿瘤治疗性疫苗配制剂的制备方法和用途。
近年来,随着肿瘤学、免疫学以及分子生物学等相关学科的迅速发展和交叉渗透,采用激活机体特异性抗肿瘤免疫功能的肿瘤疫苗治疗癌症成为恶性肿瘤治疗领域的研究热点。肿瘤疫苗是通过肿瘤相关抗原来激活肿瘤特异性的免疫反应,以达到杀伤、清除肿瘤细胞的目的,它是一种治疗性的主动免疫治疗方法。其中基于载体蛋白的蛋白偶联疫苗或多肽偶联疫苗是治疗性疫苗研发的热点之一。
截止目前,基于蛋白化学偶联疫苗的制剂开发研究较为罕见,基于化学偶联的蛋白质偶联物常具有分子形式复杂、结构复杂、疏水性强等特点,这给治疗性化学偶联药物的制剂开发提出了新的挑战。
发明内容
本发明的目的在于提供一种稳定的重组hEGF-CRM197肿瘤治疗性疫苗配制剂,其包括重组人表皮生长因子(hEGF)与白喉毒素突变体偶联物、pH在7.0-8.5范围的磷酸盐缓冲液、和表面活性剂聚山梨酯20、和/或任选单糖或二糖,以及配制所述制剂的方法。
本发明的第一方面,提供了一种重组hEGF-CRM197肿瘤治疗性疫苗配制剂,所述制剂包含:
(a)重组人表皮生长因子hEGF-白喉毒素突变体CRM197偶联物;
(b)pH7.0-8.5范围的磷酸盐基础缓冲液,其中所述缓冲液的电导率不高于10mS/cm;和
(c)蛋白稳定剂;
其中,所述蛋白稳定剂包括:表面活性剂聚山梨酯20和任选的单糖或二糖。
在另一优选例中,所述偶联物是将重组人表皮生长因子hEGF与白喉毒素突变体CRM197通过化学交联剂戊二醛偶联所得。
在另一优选例中,所述缓冲液的电导率不高于3mS/cm。
在另一优选例中,所述偶联物包含的重组人表皮生长因子以戊二醛共价交联的方式与白喉毒素突变体偶联。
在另一优选例中,所述偶联物具有如式(I)所示的结构:
hEGF-L-CRM197 (I)
其中,
hEGF是重组人表皮生长因子hEGF;
CRM197是白喉毒素突变体CRM197;
L是连接重组人表皮生长因子与白喉毒素突变体CRM197的接头,所述接头是通过戊二醛交联形成的共价键;和
“-”是化学键。
在另一优选例中,所述偶联物中重组人表皮生长因子hEGF/白喉毒素突变体CRM197的交联摩尔比为6:1。
在另一优选例中,所述重组人表皮生长因子hEGF为含有人表皮生长因子hEGF生物学活性的EGF分子或其突变体,所述EGF生物学活性是指所述EGF分子可以刺激或诱导含EGFR受体的细胞进行信号传导。
在另一优选例中,所述重组人表皮生长因子hEGF是人表皮生长因子hEGF的全长序列或其部分片段。
在另一优选例中,所述人表皮生长因子hEGF的全长序列具有如SEQ ID NO:1所示的氨基酸序列。
在另一优选例中,所述重组人表皮生长因子hEGF是在人表皮生长因子hEGF的全长序列基础上N端或C端截去2-3个氨基酸。
在另一优选例中,所述重组人表皮生长因子hEGF具有如SEQ ID NO:3所示的氨基酸序列。
在另一优选例中,所述白喉毒素突变体CRM197具有如SEQ ID NO:2所示的氨基酸序列。
在另一优选例中,所述基础缓冲液为磷酸盐缓冲液,磷酸盐浓度为1~40mmol/L。
在另一优选例中,所述磷酸盐缓冲液包含磷酸钠缓冲液(NaH
2PO
4&Na
2HPO
4)和磷酸钾缓冲液(K
2HPO
4&KH
2PO
4)。
在另一优选例中,所述基础缓冲液为20mmol/L磷酸盐缓冲液,pH 8.0。
在另一优选例中,所述基础缓冲液为10mmol/L磷酸盐缓冲液,pH 7.5。
在另一优选例中,所述基础缓冲液为5mmol/L磷酸盐缓冲液,pH 7.0。
在另一优选例中,所述制剂中聚山梨酯20的浓度为0.001%~0.1%(v/v)。
在另一优选例中,所述制剂中聚山梨酯20的浓度为0.005%~0.02%(v/v)。
在另一优选例中,所述制剂中聚山梨酯20的浓度为0.1%(v/v)。
在另一优选例中,所述制剂中聚山梨酯20的浓度为0.02%(v/v)
在另一优选例中,所述制剂中聚山梨酯20的浓度为0.01%(v/v)。
在另一优选例中,所述制剂中聚山梨酯20的浓度为0.005%(v/v)。
在另一优选例中,所述制剂中聚山梨酯20的浓度为0.001%(v/v)。
在另一优选例中,所述任选的单糖或二糖选自下组:蔗糖、葡萄糖、甘露醇、麦芽糖、或其组合。
在另一优选例中,所述任选的单糖或二糖为蔗糖,所述制剂含蔗糖的浓度为0~4%(质量/体积)。
在另一优选例中,所述制剂含蔗糖的浓度为2%(质量/体积)。
在另一优选例中,所述制剂含蔗糖的浓度为4%(质量/体积)。
在另一优选例中,所述任选的单糖或二糖为麦芽糖,所述制剂含麦芽糖的浓度为2%(质量/体积)。
在另一优选例中,所述制剂为液体制剂。
在另一优选例中,所述制剂为注射剂。
在另一优选例中,所述制剂中重组人表皮生长因子hEGF与白喉毒素突变体偶联物的含量为0.4~3.2mg/ml。
在另一优选例中,所述制剂中不含有游离的氨基酸。
在另一优选例中,所述制剂具有以下特征:所述制剂在37℃的温度条件下稳定存在至少3个月,较佳地6个月。
在另一优选例中,所述制剂在25℃±2℃或37℃±2℃的温度条件下稳定存在6个月。
在另一优选例中,“稳定存在”是指所述制剂的外观、可见异物、蛋白含量、纯度、分子量分布、体外相对效力、体内生物学活性等关键质量参数均无显著变化。
本发明的第二方面,提供了一种制备本发明第一方面所述的重组hEGF-CRM197肿瘤治疗性疫苗配制剂的方法,所述方法包括以下步骤:
(i)提供纯化的重组人表皮生长因子hEGF蛋白,和白喉毒素突变体CRM197蛋白;
(ii)将所述hEGF蛋白和CRM197蛋白与化学交联剂戊二醛进行反应,从而得到hEGF-CRM197偶联物;
(iii)纯化所述hEGF-CRM197偶联物,从而得到纯化的hEGF-CRM197偶联物;
(iv)将所述纯化的hEGF-CRM197偶联物换液至添加了蛋白质稳定剂的基础缓冲液中,从而获得所述重组hEGF-CRM197肿瘤治疗性疫苗配制剂。
在另一优选例中,所述重组人表皮生长因子hEGF蛋白的氨基酸序列如SEQ ID NO:3所示。
在另一优选例中,所述白喉毒素突变体CRM197蛋白的氨基酸序列如SEQ ID NO:2所示。
在另一优选例中,在步骤(ii)中反应条件为室温(25℃±2℃),反应时间为2小时至3小时。
在另一优选例中,所述交联剂为戊二醛。
在另一优选例中,在步骤(iii)中采用超滤的方式进行纯化。
在另一优选例中,在步骤(iv)中采用超滤的方式进行换液。
在另一优选例中,所述基础缓冲液是pH7.0-8.5范围的磷酸盐缓冲液,磷酸盐浓度为1~40mmol/L。
在另一优选例中,所述基础缓冲液的电导率不高于3mS/cm
在另一优选例中,所述蛋白稳定剂包括:表面活性剂聚山梨酯20和任选的单糖或二糖。
在另一优选例中,所述聚山梨酯20的浓度为0.005%~0.02%(v/v)。
在另一优选例中,所述任选的单糖或二糖选自下组:蔗糖、葡萄糖、甘露醇、麦芽糖、或其组合。
本发明的第三方面,提供了一种组合物,其特征在于,所述组合物包含(a)本发明第一方面所述的肿瘤治疗性疫苗配制剂;以及(b)药学上可接受的载体。
在另一优选例中,所述的组合物是药物组合物或疫苗组合物。
在另一优选例中,所述的疫苗组合物还含有佐剂。
在另一优选例中,所述佐剂包括:颗粒型和非颗粒型佐剂。
在另一优选例中,所述颗粒型佐剂选自下组:铝盐、油包水乳剂、水包油乳剂、纳米颗粒、微小颗粒、脂质体、免疫刺激复合物,或其组合;
在另一优选例中,所述非颗粒型佐剂选自下组:胞壁酰二肽及其衍生物、皂苷、脂质A、细胞因子、衍生多糖、细菌毒素,微生物及其产物如分枝杆菌(结核杆菌、卡介苗)、短小杆菌、百日咳杆菌、蜂胶、或其组合。
在另一优选例中,所述佐剂选自下组:Montanide ISA 51 VG、磷酸铝佐剂、MF59、AS04、或其组合。
在另一优选例中,所述组合物被制备为液态制剂。
在另一优选例中,所述组合物被制备为注射剂、悬浮液、或喷雾剂。
本发明的第四方面,提供了一种本发明第一方面所述的肿瘤治疗性疫苗配制剂在制备用于治疗和/或预防疾病的药物中的用途。
在另一优选例中,所述疾病为肿瘤或癌症。
在另一优选例中,所述肿瘤(或癌症)包括实体肿瘤。
在另一优选例中,所述实体肿瘤选自下组:肺癌、非小细胞肺癌、结直肠癌、 乳腺癌、肝癌、胃癌、食管癌、胰腺癌、黑色素瘤、肾癌、前列腺癌、宫颈癌、卵巢癌、鼻咽癌、口腔癌、骨肉瘤、脑胶质瘤、膀胱癌、或其组合。
应理解,在本发明范围内中,本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合,从而构成新的或优选的技术方案。限于篇幅,在此不再一一累述。
图1显示了重组hEGF-CRM197偶联物免疫小鼠的抗体效价检测。
图2显示了含聚山梨酯80制剂(编号a7)37℃加速1个月的分子量分布(SEC-HPLC)图谱。
图3显示了含聚山梨酯20制剂(编号a13)37℃加速1个月的分子量分布(SEC-HPLC)图谱。
图4显示了重组hEGF-CRM197肿瘤治疗性疫苗配制剂(0.01%Tween20,2%蔗糖,20mM PB,pH8.0,1.6mg/ml)分子量分布(SEC-HPLC)初检及25℃加速6个月图谱比较。
图5显示了重组hEGF-CRM197肿瘤治疗性疫苗配制剂(0.01%Tween20,2%蔗糖,20mM PB,pH8.0,1.6mg/ml)生物学活性初检及25℃加速6个月比较。
图6显示了重组hEGF-CRM197肿瘤治疗性疫苗配制剂在食蟹猴体内诱导产生的抗体滴度检测。
图7显示了重组hEGF-CRM197肿瘤治疗性疫苗配制剂对食蟹猴血清中EGF含量的影响。
图8显示了rhEGF-P64k偶联物(CIMAvax-EGF)与重组hEGF-CRM197偶联物免疫小鼠的抗体滴度检测结果。
图9显示了rhEGF-P64k偶联物(CIMAvax-EGF)与重组hEGF-CRM197偶联物体外阻断EGF与EGFR结合的检测结果。
本发明人经过广泛而深入的研究,首次意外地开发了一种可稳定存在并保存的重组hEGF-CRM197肿瘤治疗性疫苗配制剂。本发明的疫苗配制剂包括重组人表皮生长因子(hEGF)与白喉毒素突变体偶联物、维持pH在7.0-8.5范围的磷酸盐缓冲液、表面活性剂聚山梨酯20(Tween 20)、和/或任选单糖或二糖。实验证明,本发明的疫苗配制剂多聚物比例低,分子量均一性高,在25℃±2℃和37℃±2℃加速处理6个月后,其外观、可见异物、分子量分布、蛋白含量、EGF抗原量、生物学活性等质量属性均无变化。因此,本发明的重组hEGF-CRM197肿瘤 治疗性疫苗配制剂可以作为一种可快速大量生产,并长期保存的高效肿瘤治疗和/或预防药物。在此基础上,完成了本发明。
术语
为了更容易理解本发明,以下具体定义了某些技术和科学术语。除非在本文中另有明确定义,本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。
如本文所用,在提到具体列举的数值中使用时,术语“约”意指该值可以从列举的值变动不多于1%。例如,如本文所用,表述“约100”包括99和101和之间的全部值(例如,99.1、99.2、99.3、99.4等)。
如本文所用,术语“治疗”指给予患者内用或外用治疗剂,包含本发明的针对肿瘤的疫苗及其组合物,所述患者具有一种或多种疾病症状,而已知所述治疗剂对这些症状具有治疗作用。通常,以有效缓解一种或多种疾病症状的治疗剂的量(治疗有效量)给予患者。
如本文所用,术语“任选”或“任选的”意味着随后所描述的事件或情况可以发生但不是必须发生。例如,“任选的单糖或二糖”是指特定的单糖或二糖可以有但不是必须有,可以是1个、2个或3个。
表皮生长因子(EGF)
表皮生长因子(EGF)是一种由53个氨基酸残基组成的耐热单链低分子多肽。EGF与靶细胞上的EGF受体特异性识别结合后,发生一系列生化反应,最终可促进靶细胞的DNA合成及有丝分裂。EGF无糖基部位,非常稳定,耐热耐酸,广泛存在于体液和多种腺体中,主要由颌下腺、十二指肠合成,在人体的绝大多数体液中均已发现,在乳汁、尿液、精液中的含量特异性地增高,但在血清中的浓度较低。EGF作用广泛,对估计肿瘤预后,选择治疗方案上均有重要意义。
本发明的表皮生长因子(EGF)为人表皮生长因子(hEGF),具体地,是一种重组人表皮生长因子(hEGF),所述重组人表皮生长因子(hEGF)是在人表皮生长因子(hEGF)的全长序列(如SEQ ID NO:1所示)基础上N端或C端截去2-3个氨基酸所获得。
在本发明的一个优选例中,本发明的重组人表皮生长因子是在人表皮生长因子(hEGF)的全长序列基础上C末端截去2个氨基酸所得,其具有SEQ ID NO:3所示的氨基酸序列:
白喉毒素突变体CRM197
白喉毒素(diphtheria toxin,DT)是感染了beta噬菌体的白喉棒状杆菌(Corynebacterium diphtheriae)产生的一种外毒素,存在于临床使用的百白破疫苗成份中。其安全性得到多年临床使用的验证,罕见严重不良反应,目前尚无由白喉成份引起过敏反应的报道。
白喉毒素分子由535个氨基酸残基构成,空间上相对独立的催化结构域(1-193AAs)、跨膜结构域(205-378AAs)和受体结合域(386-535AAs)组成;跨膜结构域和受体结合域本身无毒性,其功能是通过细胞表面受体结合,将催化结构域转导进入细胞内。
CRM197是从白喉杆菌毒性基因突变株的菌株中获得的一种白喉毒素突变体,具有536个氨基酸的长度,具体是白喉毒素第52位点上的甘氨酸突变为谷氨酸,其具有如SEQ ID NO:2所示的氨基酸序列。该突变体毒素A片段不能与细胞核内延长因子II结合,使其丧失了细胞毒作用,但在抗原性与免疫原性仍基本与天然的白喉毒素保持一致。
本发明的白喉毒素突变体CRM197:
重组人表皮生长因子(hEGF)与白喉毒素突变体偶联物(hEGF-CRM197)
本发明提供了一种重组人表皮生长因子(hEGF)与白喉毒素突变体偶联物,所述偶联物具有如式(I)所示的结构:
hEGF-L-CRM197 (I)
其中,
hEGF是重组人表皮生长因子(hEGF);
CRM197是白喉毒素突变体CRM197;
L是连接重组人表皮生长因子与白喉毒素突变体CRM197的接头,所述接头 是通过戊二醛交联形成的共价键;和
“-”是化学键。
其中,所述重组人表皮生长因子(hEGF)为含有人表皮生长因子(hEGF)生物学活性的EGF分子或其突变体,所述EGF生物学活性是指所述EGF分子可以刺激或诱导含EGFR受体的细胞进行信号传导。在本发明的优选例中,所述重组人表皮生长因子(hEGF)具有如SEQ ID NO:3所示的氨基酸序列;所述白喉毒素突变体CRM197具有如SEQ ID NO:2所示的氨基酸序列。
重组hEGF-CRM197疫苗配制剂
本发明还提供了一种重组hhEGF-CRM197疫苗配制剂,所述制剂包含:(a)重组人表皮生长因子(hEGF)与白喉毒素突变体偶联物;(b)pH7.0-8.5范围的基础缓冲液,其中所述缓冲液的电导率不高于10mS/cm;和(c)蛋白稳定剂;其中,所述蛋白稳定剂包括:表面活性剂聚山梨酯20(Tween 20)和任选的单糖或二糖。
在本发明的优选例中,所述基础缓冲液为磷酸盐缓冲液,磷酸盐浓度为1~40mmol/L;所述制剂中聚山梨酯20的浓度为0.001%~0.1%(v/v);所述任选的单糖或二糖包括(但不限于)蔗糖、葡萄糖、甘露醇、麦芽糖、或其组合,在另一优选例中,所述单糖或二糖为蔗糖,制剂中蔗糖的浓度为0~4%。
本发明的重组hEGF-CRM197疫苗配制剂是一种肿瘤治疗性制剂,因此,术语“重组hEGF-CRM197疫苗配制剂”和“重组hEGF-CRM197肿瘤治疗性疫苗配制剂”可互换使用,均指本发明制备的含有重组人表皮生长因子(hEGF)与白喉毒素突变体偶联物(hEGF-CRM197)的液体制剂。
重组hEGF-CRM197疫苗配制剂的制备方法
本发明还提供了所述重组hEGF-CRM197疫苗配制剂的制备方法,所述方法包括以下步骤:
(i)提供纯化的重组人表皮生长因子(hEGF)蛋白,和白喉毒素突变体CRM197蛋白;
(ii)将所述EGF蛋白和CRM197蛋白与交联剂戊二醛在室温下反应2-3小时,从而得到hEGF-CRM197偶联物;
(iii)使用超滤的方式纯化所述hEGF-CRM197偶联物,从而得到纯化的hEGF-CRM197偶联物;
(iv)将所述纯化的hEGF-CRM197偶联物通过超滤换液至添加了蛋白质稳定剂的基础缓冲液中,从而获得所述重组hEGF-CRM197疫苗配制剂。
本发明采用化学交联法,将重组人表皮生长因子(hEGF)蛋白与白喉毒素突变体CRM197蛋白进行偶联,从而得到了一种新型的肿瘤治疗性疫苗。通过戊二 醛将hEGF与CRM197偶联,可以帮助hEGF提高免疫原性,诱导人体产生抗hEGF抗体。通过多次试验筛选出了将所述肿瘤治疗性疫苗制备成制剂的效果最优的配制液。
使用上述方法制备得到的重组hEGF-CRM197疫苗配制剂的多聚物比例低,分子量均一性高,在25℃±2℃和37℃±2℃加速处理6个月后,其外观、可见异物、分子量分布、蛋白含量、EGF抗原量、生物学活性等质量属性均无变化,因此可长期稳定地保存。
组合物和施用方法
本发明还提供了一种组合物,所述组合物包含(a)本发明第一方面所述的肿瘤治疗性疫苗配制剂;以及(b)药学上可接受的载体。
本发明的组合物包括药物组合物或疫苗组合物。
所述组合物被制备为液态制剂,其中包括(但不限于)注射剂、悬浮液、或喷雾剂等。
本发明的药物组合物包含(或含有)治疗有效量的本发明的hEGF-CRM197。
本文所用的术语“治疗有效量”指治疗剂治疗、缓解或预防目标疾病或状况的量,或是表现出可检测的治疗或预防效果的量。该效果可通过例如抗原水平来检测。治疗效果也包括生理性症状的减少。对于某一对象的精确有效量取决于该对象的体型和健康状况、病症的性质和程度、以及选择给予的治疗剂和/或治疗剂的组合。因此,预先指定准确的有效量是没用的。然而,对于某给定的状况而言,可以用常规实验来确定该有效量。
为了本发明的目的,有效的剂量为给予个体约0.001亳克/千克至10毫克/千克,较佳地约0.003亳克/千克至0.1毫克/千克体重。
药物组合物还可含有药学上可接受的载体。术语“药学上可接受的载体”指用于治疗剂(例如本发明的hEGF-CRM197)给药的载体。该术语指这样一些药剂载体:它们本身不诱导产生对接受该组合物的个体有害的抗体,且给药后没有过分的毒性。合适的载体可以是大的、代谢缓慢的大分子,如蛋白质、多糖、聚乳酸(polylactic acid)、聚乙醇酸等。这些载体是本领域普通技术人员所熟知的。在Remington’s Pharmaceutical Sciences(Mack Pub.Co.,N.J.1991)中可找到关于药学上可接受的载体或赋形剂的充分讨论。
组合物中药学上可接受的载体可包括液体,如水、盐水、甘油和乙醇。另外,这些载体中还可能存在辅助性的物质,如润湿剂或乳化剂、pH缓冲物质等。通常,可将组合物制成可注射剂,例如液体溶液或悬液;还可制成在注射前适合配入溶液或悬液、液体赋形剂的固体形式。脂质体也包括在药学上可接受的载体的定义中。
本发明的疫苗组合物包含免疫性抗原(包括本发明的hEGF-CRM197),并且通常与“药学上可接受的载体”组合,这些载体包括本身不诱导产生对接受该组合物的个体有害的抗体的任何载体。合适的载体通常是大的、代谢缓慢的大分子,如蛋白质、多糖、聚乳酸、聚乙醇酸、氨基酸聚合物、氨基酸共聚物、脂质凝集物(如油滴或脂质体)等。这些载体是本领域普通技术人员所熟知的。另外,这些载体可起免疫刺激剂(“佐剂”)作用。
增强免疫组合物效果的优选佐剂包括但不限于:(1)铝盐(alum),如氢氧化铝、磷酸铝、硫酸铝等;(2)水包油型乳剂配方,例如:(a)Montanide ISA 51VG,(b)Montanide ISA 720VG;(C)MF59;(3)皂素佐剂;(4)Freund完全佐剂(CFA)和Freund不完全佐剂(IFA);(5)细胞因子,如白介素(如IL-1、IL-2、IL-4、IL-5、IL-6、IL-7、IL-12等)、干扰素(如γ干扰素)、巨噬细胞集落刺激因子(GM-CSF)等;(6)细菌ADP-核糖基化毒素(如大肠杆菌热不稳定毒素LT)的脱毒变异体;以及(7)作为免疫刺激剂来增强组合物效果的其它物质。
包括免疫原性组合物在内的疫苗组合物(例如,可包括抗原、药学上可接受的载体以及佐剂),通常含有稀释剂,如水,盐水,甘油,乙醇等。另外,辅助性物质,如润湿剂或乳化剂、pH缓冲物质等可存在于这类运载体中。更具体地,包括免疫原性组合物在内的疫苗,包含免疫学有效量的免疫原性组合物,以及上述其它所需的组分。“免疫学有效量”指以单剂或连续剂一部分给予个体的量对治疗是有效的。该用量可根据所治疗个体的健康状况和生理状况、所治疗个体的类别(如人)、个体免疫系统合成抗体的能力、所需的保护程度、疫苗的配制、治疗医师对医疗状况的评估、及其它的相关因素而定。预计该用量将在相对较宽的范围内,可通过常规实验来确定。
通常,可将疫苗组合物或免疫原性组合物制成可注射剂,例如液体溶液或悬液;还可制成在注射前适合配入溶液或悬液、液体赋形剂的固体形式。该制剂还可乳化或包封在脂质体中,以增强佐剂效果。
一旦配制成本发明的组合物,可将其直接给予对象。待治疗的对象可以是哺乳动物,尤其是人。
当用作疫苗时,可用已知的方法将本发明的疫苗组合物直接施用于个体,通常采用与常规疫苗相同的施用途径。给予本发明药物组合物或疫苗组合物的途径包括(但并不限于):皮下、皮内、肌内或其它肠胃外给药途径。如果需要,可以组合给药途径,或根据疾病情况进行调节。疫苗组合物可以单剂量或多剂量给予,且可以包括给予加强剂量以引发和/或维持免疫力。应以“有效量”给予疫苗组合物,即疫苗组合物的量在所选用的给药路径中足以引发免疫应答,能有效改善疾病症状。
代表性的疾病包括(但并不限于)选自下组的肿瘤(或)癌症,尤其是实体瘤: 肺癌、非小细胞肺癌、结直肠癌、乳腺癌、肝癌、胃癌、食管癌、胰腺癌、黑色素瘤、肾癌、前列腺癌、宫颈癌、卵巢癌、鼻咽癌、口腔癌、骨肉瘤、脑胶质瘤、膀胱癌等。
在各疫苗剂份中所选用的药物组合物的量,是按可引发免疫保护性应答而无明显的副作用的量而定。通常,各剂的疫苗足以含有约0.1mg-10mg,较佳地为0.4mg-5mg。可用包括观察对象中的抗体滴定度和其它反应的标准研究方法来确定具体疫苗的最佳用量。可通过监控疫苗提供的免疫力水平来确定是否需要增强剂量。在评估了血清中的抗体滴定度后,可能需要选用增强剂量免疫接种。施用药物组合物或疫苗组合物就可提高对本发明的免疫应答。
此外,本发明的疫苗可以结合其它免疫调节剂一起给予,或者与其他治疗剂一起给予。
与常规制剂配方相比,本发明的主要创新点是开发并提供了稳定的含人表皮生长因子与白喉毒素突变体偶联物的制剂,其主要优点包括:
(1)本发明的制剂含人表皮生长因子与白喉毒素突变体偶联物的创新性成分,可以打破免疫耐受,诱导人体内产生抗人表皮生长因子抗体;
(2)本发明的制剂含有低盐和相对偏碱的基础缓冲液,显著区别于常规制剂配方,可以使蛋白偶联物分子产生更低比例的多聚物,在缓冲液中的分子量分布更均一;
(3)本发明通过实验摸索出使用聚山梨酯20并排除聚山梨酯80配制重组hEGF-CRM197肿瘤治疗性疫苗配制剂的配方,使蛋白偶联物分子在配制剂中的稳定性更好。
(4)本发明的制剂生产方法简单,可实现规模化生产,并且可长期稳定的保存。
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件,例如Sambrook等人,分子克隆:实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。除非另外说明,否则百分比和份数是重量百分比和重量份数。
实施例1:重组hEGF-CRM197疫苗原液制备和检测
在反应容器中,分别加入重组人表皮生长因子、白喉毒素突变体CRM197以及交联剂戊二醛,室温反应2~3小时后,利用超滤纯化方法去除游离戊二醛和未结合人表皮生长因子,从而获得重组hEGF-CRM197疫苗原液。
(1)重组hEGF-CRM197偶联物的制备
①结合反应:取适量的25%戊二醛,用磷酸盐缓冲液(PBS:20mM PB,150mM NaCl,pH 7.0)配制浓度为0.5%的戊二醛。分别取等量的重组人表皮生长因子、白喉毒素突变体CRM197(摩尔比大约为10:1),用磷酸盐缓冲液分别稀释至2mg/ml。分别将配制成对应浓度的重组人表皮生长因子、白喉毒素突变体CRM197加入反应容器中,充分混匀后,滴加0.5%的戊二醛至戊二醛终浓度为0.1%。在将反应容器放入25℃恒温摇床中,设置摇床转速130rpm,反应2小时。
②超滤纯化:反应后,用50kDa超滤膜,先浓缩后用磷酸盐(PBS)稀释,并再次浓缩,反复浓缩并继续,确保稀释倍数至少达到2187倍(3的7次方)。
③基础缓冲液更换:继续利用超滤膜,用磷酸缓冲液(PB:20mM PB,pH 8.0)稀释并浓度,确保换液至滤出液电导率不高于3mS/cm。最后将超滤液浓缩至重组hEGF-CRM197偶联物的浓度达到2mg/ml以上,确保满足制剂配制的要求。
(2)重组hEGF-CRM197偶联物的蛋白交联摩尔比检测
方法:液相串联质谱-质谱法(LC-MS/MS)
①还原烷基化
取切糖后样本50μg,用50mmol/L碳酸氢铵溶液调节pH至7.5~8.0,加入100mmol/L二硫苏糖醇(DTT)使其终浓度为10mmol/L,混匀,56℃孵育1h。加入250mmol/L碘乙酰胺(IAM)使其终浓度为25mmol/L,室温避光反应1h。
②Trypsin酶解
取还原烷基化样本50μg,按酶与蛋白1:50加入Trypsin溶液,充分混匀,37℃酶解12h,加入FA至终浓度为0.1%,终止反应。
③QE-HF数据采集
取适量酶切液,以10000g离心5min,取上清,进行QE-HF分析。
④数据处理
从Mascot检索结果中,提取各样本全部肽段及强度。选取6批样本中相同肽段,按检出肽段数和强度分别计算总rhEGF与CRM197的相对比例,按照rhEGF与CRM197的分子量转换成摩尔比。结果测得所述重组hEGF-CRM197偶联物中rhEGF/CRM197的摩尔比为6:1。
(3)重组hEGF-CRM197偶联物免疫小鼠的抗体效价检测
取重组hEGF-CRM197偶联物原液并将重组hEGF-CRM197疫苗浓度稀释至0.2mg/ml,取0.5ml至2ml注射器中,另取0.5ml液体佐剂Montanide ISA 51 VG至另一支2ml注射器中,将两支注射器用接头连接,先慢速来回推动15轮,后再以尽可能地快速来回推动30次即完成乳化。乳化结束后,将所有乳液推动至一侧的注射器中。将乳化液以0.1ml/只的量注射至小鼠皮下,共8只小鼠,间隔1周再注射1次,分别在第2次注射后1、3、5周眼眶取血,取血后检测抗EGF抗体 滴度。
抗EGF抗体滴度检测方法:
①包被:取ELISA检测96孔板,将rhEGF用Na
2CO
3-NaHCO
3,pH9.6的包被缓冲液稀释至0.5μg/ml,分别取100μL稀释后rhEGF蛋白加至96孔板的各个孔中,2~8℃孵育过夜。
②封闭:次日,弃去包被液,每孔加入150μL含0.05%(v/v)Tween-20、1%BSA的PBS封闭液,37℃孵育封闭1小时;
③洗涤:弃去封闭液后每孔用200μL含0.05%(v/v)Tween-20的PBS反复洗涤3次,并吸干孔中的液体;
④将采集的小鼠血清稀释,取100μL稀释后血清加入96孔板的最左侧孔中,每只小鼠为1行,从左到右倍比稀释,共稀释12个梯度,并以未免疫小鼠血清为对照,加入血清后37℃,孵育1h;孵育后,弃去孵育液,如上所述洗涤方式反复洗涤3次;
⑤用封闭液将二抗HRP酶标抗鼠抗体稀释为1:5000,每孔加HRP酶标抗体100μL,37℃,孵育1h;弃去孵育液,如上所述洗涤方式反复洗涤5次;
⑥显色:每孔加入100μl TMB,室温避光孵育15min;
⑦终止:每孔加入100μl 0.5mol/L硫酸终止显色;
⑧终止后,用微孔板分光光度计检测各个孔OD450的光吸收值。于450nm处测定光吸收值,确定血清样品OD值/生理盐水对照组样品OD值≥2.1的最高稀释倍数。
检测结果如图1所示,二次免疫后1、3、5周抗体滴度均超过1.6万,几何平均值分别达到21.5万、531万和1502万。
实施例2:重组hEGF-CRM197疫苗配制剂筛选
(1)基础缓冲液筛选:
取制备后的重组hEGF-CRM197疫苗原液,分别利用超滤换液的方法,将重组hEGF-CRM197疫苗分别换液至如下表1所述的基础缓冲液中。基础缓冲液可以显著影响重组hEGF-CRM197疫苗原液的分子量分布,本研究的筛选目标是获得多聚物比例低,分子量均一性高的基础缓冲液。因此,本研究选用SEC-HPLC分析方法,利用凝胶过滤分析柱分析在不同基础缓冲液中的重组hEGF-CRM197疫苗原液。
结果如表1所示,较高的pH和较低的盐浓度,以及不含有游离氨基酸的情况下,多聚物比例最低,如编号13所示的缓冲液。
表1.
(2)蛋白稳定剂筛选:
取重组hEGF-CRM197疫苗原液,分别根据如表2所示加入对应量高浓度的蛋白稳定剂储液,配制并除菌过滤后,分装成0.5ml/瓶,每组20瓶,进行37℃极端加速稳定性试验,首先在加速处理2周后利用外观、可见异物进行初步筛选。
结果如表2所示,含聚山梨酯80(Tween80)和聚山梨酯20的制剂可见异物均合格,同时添加糖类也在一定程度上提高稳定性。
表2.
将外观和可见异物100%合格的制剂配方,继续在37℃加速1个月,分别利用可见异物、蛋白含量、分子量分布(多聚物比例和主成分比例)、体外相对效力等方法鉴定。
如下表3所示,可见异物、蛋白含量、分子量分布-主成分、体外相对效力检验指标中,均满足稳定性要求,含聚山梨酯80(Tween 80)配方中加速稳定性检测结果中分子量分布多聚物比例有明显的上升(详见表3,如图2),在含聚山梨酯20(Tween 20)配方中加速稳定性检测结果中分子量分布多聚物比例无显著变化(详见表3,如图3)。该结果表明,含聚山梨酯20的磷酸盐缓冲液中,制剂在极端高温条件下是稳定的。
表3.
实施例3:重组hEGF-CRM197肿瘤治疗性疫苗配制剂(0.01%Tween20,2%蔗糖,20mM PB,pH8.0,1.6mg/ml)制备、活性鉴定及稳定性研究
3.1制剂成品配制
根据配制量和配制浓度,计算配制后重组hEGF-CRM197肿瘤治疗性疫苗所需原液、各种辅料的含量。分别在配制容器中,加入重组hEGF-CRM197肿瘤治 疗性疫苗原液、Tween 20和蔗糖储液,并最终加入基础缓冲液(20mM PB)定容。
利用0.2μm孔径除菌滤芯除菌过滤后,将制剂成品无菌分装至西林瓶中。
3.2制剂成品检验
(1)外观及可见异物
检查人员调节位置,使供试品位于眼部的明视距离处,供试品至人眼距离为25cm。除去容器标签,擦净容器外壁。手持容器颈部,轻轻旋转和翻转容器,使药液中存在的可见异物悬浮,注意不使药液产生气泡,并分别在黑色和白色背景下,目视检查,重复3次,总时限为20s。
检测供试品中,不得检出明显可见异物。如也未检出微细可见异物,判为符合规定。
细微可见异物的说明:第一,白点,系指不能辨清平面或棱角的白色物体;第二,细小蛋白质絮状物或蛋白质颗粒,系指半透明的小于约1mm的絮状沉淀或蛋白质颗粒;第三,少量絮状物或蛋白质颗粒,系指在规定检查时间中,较难计数的蛋白质絮状物或蛋白质颗粒;第四,微量沉积物,系指静置后供试品中的微小沉淀物,轻轻转动后有烟雾状沉淀浮起,轻摇即散失者;第五,摇不散的沉淀,系指久置后蛋白质溶液出现少量沉淀物,轻轻摇动后不能分散消失者。
(2)蛋白含量
分别精密量取0.2mg/ml、0.4mg/ml、0.6mg/ml、0.8mg/ml、1.0mg/ml标准蛋白质溶液6μl置于96孔板中,每种标准品做两个副孔,即每种标准品检测三次;分别精密量取供试品6μl置于96孔板中,每种供试品做两个副孔,即每种供试品检测三次;各孔依次加30μl试剂A,摇匀;各孔依次加180μl试剂B,摇匀,室温放置10min,在波长750nm处测定OD750吸光值。
以标准蛋白质溶液的OD750吸光值绘制标准曲线,取直线回归方程,将供试品的OD750吸光值代入直线回归方程,即得供试品的蛋白质含量。
(3)纯度(SDS-PAGE)
取供试品,与上样缓冲液3:1混合后,沸水浴5min。用12.5%的丙烯酰胺浓度的胶分析样品,200V电压恒压电泳45min。
电泳结束后,可采用考马斯亮蓝染色法对凝胶中的蛋白条带进行显色,并扫描成像。计算分子量不小于重组CRM197条带的重组hEGF-CRM197疫苗条带占所有组分的比例即为主成分占比。
(4)分子量分布(SEC-HPLC)
①供试品预处理
取供试品,10000rpm离心10分钟后取上清。
②样品分析
程序运行时间:40min
数据记录时间:30min
流速:0.5ml/min
取制剂成品上样,根据以上参数设置分析方法进行分析,分析结果利用软件计算多聚物面积占比和主成分面积占比。
(5)体外相对效力
①将对照品及制剂成品用Na2CO3-NaHCO3缓冲液分别稀释至50、25、12.5、6.25、3.13ng/ml,将稀释好的5个梯度的各批次疫苗包被酶标板,每个稀释度2个复孔,每孔100μl,2~8℃孵育过夜。然后PBST洗板、拍干。用200μl封闭液进行封闭,37℃孵育2h。
②取已包被好的酶标板,加入1μg/ml的S-172-4杂交瘤单克隆抗体,37℃孵育1h。洗板后,加入1:10000倍稀释的goat anti-mouse IgG-HRP,每孔100μl,37℃孵育1h。
③洗板后,加入100μl/孔的TMB,避光孵育15min后,加50μl/孔终止液,450nm读数。
④每批样品分别检测3次。
(6)生物学活性(方法同实施例1,不过仅检测二次免疫后1周)
取制剂成品并将重组hEGF-CRM197疫苗浓度稀释至0.2mg/ml,取0.5ml至2ml注射器中,另取0.5ml液体佐剂Montanide ISA 51 VG至另一支2ml注射器中,将两支注射器用接头连接,先慢速来回推动15轮,后再以尽可能地快速来回推动30次即完成乳化。乳化结束后,将所有乳液推动至一侧的注射器中。将乳化液以0.1ml/只的量注射至小鼠皮下,共8只小鼠,间隔1周再注射1次,第2次注射后1周眼眶取血,取血后检测抗EGF抗体滴度。
抗EGF抗体滴度检测方法:
①包被:取ELISA检测96孔板,将rhEGF用Na
2CO
3-NaHCO
3,pH9.6的包被缓冲液稀释至0.5μg/ml,分别取100μL稀释后rhEGF蛋白加至96孔板的各个孔中,2~8℃孵育过夜。
②封闭:次日,弃去包被液,每孔加入150μL含0.05%(v/v)Tween-20、1%BSA的PBS封闭液,37℃孵育封闭1小时;
③洗涤:弃去封闭液后每孔用200μL含0.05%(v/v)Tween-20的PBS反复洗涤3次,并吸干孔中的液体;
④将采集的小鼠血清稀释,取100μL稀释后血清加入96孔板的最左侧孔中,每只小鼠为1行,从左到右倍比稀释,共稀释12个梯度,并以未免疫小鼠血清为对照,加入血清后37℃,孵育1h;孵育后,弃去孵育液,如上所述洗涤方式反复洗涤3次;
⑤用封闭液将二抗HRP酶标抗鼠抗体稀释为1:5000,每孔加HRP酶标抗体 100μL,37℃,孵育1h;弃去孵育液,如上所述洗涤方式反复洗涤5次;
⑥显色:每孔加入100μl TMB,室温避光孵育15min;
⑦终止:每孔加入100μl 0.5mol/L硫酸终止显色;
⑧终止后,用微孔板分光光度计检测各个孔OD450的光吸收值。于450nm处测定光吸收值,确定血清样品OD值/生理盐水对照组样品OD值≥2.1的最高稀释倍数。
3.3制剂成品稳定性研究
选取制剂成品,如3.2所述方法完成初检后,按如下表4所述方案进行25℃加速稳定性研究。所有检测项目的检验方法如3.2所述。
表4.
检测项目 | 1月 | 2月 | 3月 | 6月 |
外观 | √ | √ | √ | √ |
可见异物 | √ | √ | √ | √ |
蛋白含量 | √ | √ | √ | √ |
纯度(SDS-PAGE) | × | × | √ | √ |
分子量分布(SEC-HPLC) | √ | √ | √ | √ |
生物学活性测定 | × | × | × | √ |
体外相对效力 | × | × | √ | √ |
完成6个月稳定性后,总结研究结果,如表5所示。在所述的制剂成品6个月加速稳定性研究中,外观、可见异物、蛋白含量、纯度(SDS-PAGE)、分子量分布(SEC-HPLC)(图4)、体外相对效力、体内生物学活性(图5)等关键质量参数均在质量可控范围内。
表5.
实施例4重组hEGF-CRM197肿瘤治疗性疫苗配制剂食蟹猴体内生物学活性检测
由于人与食蟹猴EGF序列同源性高达98%,利用食蟹猴研究重组hEGF-CRM197肿瘤治疗性疫苗配制剂的生物学活性可以初步评估重组hEGF-CRM197肿瘤治疗性疫苗配制剂在人体内的生物学活性。
供试品的配制:重组EGF-CRM197疫苗与Montanide ISA 51 VG佐剂等体积混合后乳化,乳化过程具体方法如下:
取2支2ml的注射器,分别标注为1号注射器和2号注射器。1号和2号注射器分别吸取等体积的Montanide ISA 51 VG佐剂和重组EGF-CRM197疫苗,2只注射器用双母鲁尔接头连接。整个乳化过程分为“预乳化”和“快速乳化”两个步骤。将液体从1号注射器全部推至2号注射器,再全部推回1号注射器的过程,定义为一个循环。“预乳化”过程需20个循环,缓慢的来回推动注射器,平均每个循环约8秒,完成“预乳化”,至少需要2分钟。“预乳化”完成后,进入“快速乳化”过程,“快速乳化”过程共40个循环,需尽可能快速的来回推动注射器。整个乳化过程需60个循环,耗时约3分钟,最终形成的油包水乳液呈乳白色粘稠状。乳化完成后,将全部的油包水乳液推至1.5ml离心管中,将其置于冰盒内备用。供试品用等体积佐剂充分混合至完全乳化即可。
实验动物:食蟹猴,共30只,每组10只,年龄3~5岁,体重2~4kg;分别为佐剂对照组、低剂量组、高剂量组。
给药途径及方法:颈背部皮下注射;
给药频率及期限:每周给药1次,共给药5次;
给药时间:一般为上午08:30~12:00;
给药浓度:0.5mg/ml;
给药容积:低剂量组0.8ml/只,其他组均为1.6ml/只。
二次给药后1-5周每周取血.
抗体滴度测定方法同实施例1的抗EGF抗体滴度检测方法。
抗体滴度检测结果如图6所示。结果显示,重组hEGF-CRM197肿瘤治疗性疫苗配制剂可以诱导食蟹猴产生高达500万的抗EGF抗体滴度,说明重组hEGF-CRM197肿瘤治疗性疫苗配制剂可以打破食蟹猴的免疫耐受,诱导食蟹猴产生高滴度抗体,验证了重组hEGF-CRM197肿瘤治疗性疫苗配制剂的体内生物学活性。
进一步检测食蟹猴体内EGF的含量,分别对血清中EGF含量的检测方法:
实验准备:使用前,所有试剂放置室温,使用5.0ml的Calibrator Diluent RD6-1将Human EGF standard溶解,终浓度为250pg/ml。用Calibrator Diluent RD6-1将Human EGF standard倍比稀释至125pg/ml、62.5pg/ml、31.2pg/ml、15.6pg/ml、7.8pg/ml及3.9pg/ml,Calibrator Diluent RD6-1作为0pg/ml的标准品,标准品用于绘制标准曲线。
加样,温育:先加入50μL/孔Assay Diluent RD1-21,再分别加入200μL/孔的标准品、对照和样品溶液,轻弹板边缘1分钟,使溶液混合均匀,用封板膜封板后室温孵育2小时。
洗涤:小心揭掉封板膜,弃去液体,甩干,每孔加入400μL的清洗缓冲液,弃去,如此重复3次,拍干。
加酶:每孔加入200μl的EGF Conjugate,用封板膜封板后室温孵育2小时。
同上述洗涤步骤进行洗涤。
显色:每孔加入200μl的底物溶液,避光室温孵育20分钟。
终止:每孔加入50μl的Stop Solution,颜色由蓝色变成黄色,若孔中的颜色为绿色或颜色变化不均一,轻轻震荡混匀。
读数:450nm读数。
将每个标准品、对照、样品的复孔的读数平均后减去空白对照的平均值。标准液(250pg/ml、125pg/ml、62.5pg/ml、31.2pg/ml、15.6pg/ml、7.8pg/ml、3.9pg/ml)可用于制作标准曲线,数据分析可以使用计算机软件绘制四参数逻辑曲线。或者也可以根据每个标准品的平均吸光度值,绘制一条标准曲线,Y轴标注吸光值,X轴标注浓度。然后根据样品吸光值计算出样品中EGF浓度。(操作时每一浓度标准液都要重复滴定到两个小孔中,以便计算平均分光光度值,样品也需计算平 均吸光值)。最终浓度为实际测定浓度乘以稀释倍数。
检测结果如图7所示。结果显示疫苗组(高剂量组和低剂量组)食蟹猴血清中EGF含量均显著下降,而佐剂对照组EGF含量无显著变化。
对比例1:rhEGF-P64k偶联物(CIMAvax-EGF)与重组hEGF-CRM197生物学活性对比
按实施例1的重组hEGF-CRM197偶联物免疫小鼠的抗体效价检测方法,将rhEGF-P64k偶联物(CIMAvax-EGF)参照重组hEGF-CRM197疫苗配制及动物免疫方法,相同方法分别在二次免疫后1周、3周和5周采集血清,并参照相同的方法进行抗体滴度测定。结果如图8所示,重组hEGF-CRM197偶联物的抗体滴度分别在二免后1周、3周、5周均显著高于rhEGF-P64k偶联物至少10倍。
进一步对CIMAvax-EGF与重组hEGF-CRM197偶联物诱导的抗EGF抗体体外阻断EGFR与EGF结合能力比较:
将1μl FITC-EGF分别与CIMAvax EGF/重组hEGF-CRM197肿瘤治疗性疫苗免疫BALB/c小鼠第二次免疫后3周血清,注射生理盐水的BALB/c小鼠第二次免疫后3周血清按体积比1:25、1:75、1:100室温避光孵育15mim。
将处于对数生长期的NCI-H446细胞用胰酶消化后,平均分为11管,至于1.5ml离心管中,每管细胞数量约10
4,5000rpm离心3min,弃上清。
将孵育后FITC-EGF与血清的混合物分别与NCI-H446细胞混合,补充生理盐水至每管总体积为110μl,4℃避光孵育30min。分别标记为样品1-9,样品1-3记为生理盐水组,样品4-6为EGF-P64K组,样品7-9记为EGF-CRM197组。样品10只加入110μl生理盐水,用于流式细胞仪调零,样品11加入1μl FITC-EGF,补充生理盐水至每管总体积为110μl,作为阴性对照。
将11个样品分别加入1ml生理盐水,5000rpm离心3min洗涤,弃上清。
将11个样品分别加入200μl生理盐水,FACS检测各样品FITC-EGF标记的NCI-H446细胞的阳性比例。
结果如图9所示。结果显示,重组EGF-CRM197/CIMAvax EGF疫苗组第二次免疫后3周血清与FITC-EGF按体积比25:1、75:1、100:1混合孵育后标记的NCI-H446细胞FITC-EGF阳性率分别为25.4%vs.59.2%,7.9%vs.26.8%及4.6%vs.15.6%。实验结果提示,重组EGF-CRM197/CIMAvax EGF疫苗免疫小鼠后,诱导产生的抗血清,均可有效的体外阻断EGF与EGFR结合,其中重组EGF-CRM197疫苗组抑制相同量的EGF所需的血清更少,说明重组EGF-CRM197疫苗组诱导抗体结合能力更佳。对比25%左右的抑制率,CIMAvax EGF组血清量是重组EGF-CRM197组的3倍,大致判断重组EGF-CRM197疫苗组诱导中和抗 体是CIMAvax EGF组的3倍。
在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
Claims (10)
- 一种重组hEGF-CRM197肿瘤治疗性疫苗配制剂,其特征在于,所述制剂包含:(a)重组人表皮生长因子hEGF-白喉毒素突变体CRM197偶联物,所述偶联物的含量为0.4~3.2mg/ml,并且所述偶联物是将重组人表皮生长因子hEGF与白喉毒素突变体CRM197通过化学交联剂戊二醛偶联所得,所述偶联物中重组人表皮生长因子hEGF/白喉毒素突变体CRM197的交联摩尔比为6:1;(b)pH7.5-8.5范围的磷酸盐基础缓冲液,其中所述缓冲液的电导率不高于3mS/cm,所述缓冲液中磷酸盐浓度为5~20mmol/L;和(c)蛋白稳定剂;其中,所述蛋白稳定剂包括:0.005%~0.02%的表面活性剂聚山梨酯20,和0%~4%的单糖或二糖,按所述制剂的总体积计。
- 如权利要求1所述的制剂,其特征在于,所述偶联物具有如式(I)所示的结构:hEGF-L-CRM197 (I)其中,hEGF是重组人表皮生长因子hEGF;CRM197是白喉毒素突变体CRM197;L是连接重组人表皮生长因子与白喉毒素突变体CRM197的接头,所述接头是通过戊二醛交联形成的共价键;和“-”是化学键。
- 如权利要求1所述的制剂,其特征在于,所述重组人表皮生长因子hEGF为含有人表皮生长因子hEGF生物学活性的EGF分子或其突变体,所述EGF生物学活性是指所述EGF分子可以刺激或诱导含EGFR受体的细胞进行信号传导。
- 如权利要求1所述的制剂,其特征在于,所述重组人表皮生长因子hEGF的氨基酸序列为如SEQ ID NO:3所示的氨基酸序列。
- 如权利要求1所述的制剂,其特征在于,所述白喉毒素突变体CRM197的氨基酸序列为如SEQ ID NO:2所示的氨基酸序列。
- 如权利要求1所述的制剂,其特征在于,所述单糖或二糖选自下组:蔗糖、葡萄糖、甘露醇、麦芽糖、或其组合。
- 如权利要求1所述的制剂,其特征在于,所述制剂为注射剂。
- 一种制备如权利要求1所述的重组hEGF-CRM197肿瘤治疗性疫苗配制剂的方法,其特征在于,所述方法包括以下步骤:(i)提供纯化的重组人表皮生长因子hEGF蛋白,和白喉毒素突变体CRM197 蛋白;(ii)将所述hEGF蛋白和CRM197蛋白与化学交联剂戊二醛进行反应,从而得到hEGF-CRM197偶联物;(iii)纯化所述hEGF-CRM197偶联物,从而得到纯化的hEGF-CRM197偶联物;(iv)将所述纯化的hEGF-CRM197偶联物换液至添加了蛋白质稳定剂的基础缓冲液中,从而获得所述重组hEGF-CRM197肿瘤治疗性疫苗配制剂。
- 一种组合物,其特征在于,所述组合物包含:(a)如权利要求1所述的肿瘤治疗性疫苗配制剂;以及(b)药学上可接受的载体。
- 一种如权利要求1所述的肿瘤治疗性疫苗配制剂在制备用于治疗和/或预防肿瘤或癌症的药物中的用途。
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030054011A1 (en) * | 2000-12-06 | 2003-03-20 | Sierra Aillette Mulet | Vaccine composition containing transforming growth factor alpha (TGFalpha). it use in malignant diseases therapy |
CN101475640A (zh) * | 2009-01-19 | 2009-07-08 | 百泰生物药业有限公司 | 一种偶联物及其制备方法与应用 |
CN108126193A (zh) * | 2016-12-01 | 2018-06-08 | 上海亨臻实业有限公司 | TNF-α突变蛋白和载体蛋白CRM197化学偶联的产物及其制备方法 |
CN113855792A (zh) * | 2021-12-01 | 2021-12-31 | 上海惠盾因泰生物科技有限公司 | 重组hEGF-CRM197肿瘤治疗性疫苗配制剂 |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1194167B1 (en) * | 1999-06-09 | 2009-08-19 | Immunomedics, Inc. | Immunotherapy of autoimmune disorders using antibodies which target b-cells |
EP2072060A1 (en) * | 2007-12-18 | 2009-06-24 | Institut Curie | Methods and compositions for the preparation and use of toxin conjugates. |
CN104140972B (zh) * | 2013-05-07 | 2018-01-23 | 上海惠盾生物技术有限公司 | 白喉毒素突变体crm197的制备方法 |
CN108310363B (zh) * | 2018-03-26 | 2021-08-13 | 金华市人民医院 | Crm197在制备治疗多囊卵巢综合症的药物中的应用 |
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Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030054011A1 (en) * | 2000-12-06 | 2003-03-20 | Sierra Aillette Mulet | Vaccine composition containing transforming growth factor alpha (TGFalpha). it use in malignant diseases therapy |
CN101475640A (zh) * | 2009-01-19 | 2009-07-08 | 百泰生物药业有限公司 | 一种偶联物及其制备方法与应用 |
CN108126193A (zh) * | 2016-12-01 | 2018-06-08 | 上海亨臻实业有限公司 | TNF-α突变蛋白和载体蛋白CRM197化学偶联的产物及其制备方法 |
CN113855792A (zh) * | 2021-12-01 | 2021-12-31 | 上海惠盾因泰生物科技有限公司 | 重组hEGF-CRM197肿瘤治疗性疫苗配制剂 |
Non-Patent Citations (2)
Title |
---|
XIONG AN-WEN, FANG JUE-MIN, REN SHENG-XIANG, LI WEI, WANG JING, ZHAO YU, CHEN GUO-YOU, XU QING, ZHOU CAI-CUN: "A Novel Combined Conjugate Therapeutic Cancer Vaccine, Recombinant EGF-CRM197, in Patients With Advanced Solid Tumors: A Phase I Clinical Study", FRONTIERS IN ONCOLOGY, vol. 11, XP093071428, DOI: 10.3389/fonc.2021.745699 * |
ZHU, LEI ET AL.: "Construction and Immunogenicity Analysis of Recombinant Anti-Epidermal Growth Factor Vaccine P64K-EGF", CHINESE JOURNAL OF CELLULAR AND MOLECULAR IMMUNOLOGY, vol. 31, no. 12, 31 December 2015 (2015-12-31), XP009546861, ISSN: 1007-8738 * |
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