WO2023155872A1 - 一种实现生物活性分子其活性控释和缓释的方法及药物应用 - Google Patents

一种实现生物活性分子其活性控释和缓释的方法及药物应用 Download PDF

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WO2023155872A1
WO2023155872A1 PCT/CN2023/076746 CN2023076746W WO2023155872A1 WO 2023155872 A1 WO2023155872 A1 WO 2023155872A1 CN 2023076746 W CN2023076746 W CN 2023076746W WO 2023155872 A1 WO2023155872 A1 WO 2023155872A1
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peg
polyethylene glycol
mpeg
group
modification
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PCT/CN2023/076746
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French (fr)
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马永
杭建花
赵百学
许振�
王和
王俊
曹锫沛
江辰阳
庄宇
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江苏众红生物工程创药研究院有限公司
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Publication of WO2023155872A1 publication Critical patent/WO2023155872A1/zh

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19Cytokines; Lymphokines; Interferons
    • A61K38/20Interleukins [IL]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52Cytokines; Lymphokines; Interferons
    • C07K14/54Interleukins [IL]
    • C07K14/55IL-2

Definitions

  • the invention relates to a method for realizing the controlled release and sustained release of the activity of biologically active molecules and a drug application, in particular to a method for the controlled release and sustained release of the activity of highly active cytokines, such as interleukin-2.
  • Interleukin-2 (Interleukin-2, IL-2) is a member of the interleukin family.
  • IL-2 can promote the differentiation and maturation of T cells, NK cells, and B cells, and activate their biological activity, induce the activation of lymphokine-activated killer cells (LAK), and promote the synthesis and release of many lymphokines such as interferon and tumor necrosis factor, as well as the production of antibodies.
  • LAK lymphokine-activated killer cells
  • the ability of IL-2 to expand lymphocyte populations in vivo and enhance the effector functions of these cells makes it have antitumor effects.
  • high doses of IL-2 were approved for the treatment of metastatic renal cell carcinoma and malignant melanoma.
  • VLS capillary leakage Syndrome
  • rIL Preparation of modified polyethylene glycol of -2 and its anti-hepatocarcinoma effect in vitro and in vivo, 1997).
  • the slow release of drugs in the body and the improvement of drug bioavailability are not ideal through the modification of conventional polyethylene glycol directly linked to drugs.
  • a water-soluble polymer and a drug are connected to form a polymer-drug conjugate through a linker, and the water-soluble polymer falls off from the conjugate, which can achieve the purpose of sustained release and controlled release of drug activity.
  • the drug stays in the lesion (such as cancer) for a longer period of time, which can reduce the frequency of administration and reduce the inconvenience of medication for patients.
  • patent document CN200680029849.5 discloses a conjugate, which includes an aromatic moiety containing ionizable hydrogen atoms such as fluorene, a spacer moiety, and a water-soluble polymer.
  • Patent CN103517718A further discloses the formation of the polymer and rIL-2 conjugate.
  • the conjugate is actually a Nektar company in the United States NKTR-214, a CD122 (IL-2R ⁇ ) biased agonist launched by the company.
  • the conjugate is coupled with six branched PEGs with a molecular weight of 20K and a fluorene ring structure on IL-2 (the amino acid sequence is the same as that of aldesleukin), and the fluorenylmethyl is completed through a ⁇ -elimination reaction under mild alkaline conditions.
  • lysine residues such as K31, K34, K42, K47, K48, K75
  • PEG is located near the key hydrophobic binding site of IL-2/IL-2R ⁇ interaction to reduce the binding to CD25 (IL-2R ⁇ ) and activate CD122 (IL-2R ⁇ )
  • the inventors of the present invention have screened out a specific structure PEG modifier from existing mature polyethylene glycol modifiers through a large number of experiments and researches, and this PEG can be used for the modification of IL-2 or its variants to obtain Excellent active controlled release and sustained release effect, have outstanding advantages in the bioavailability of related similar drugs, reduce the frequency of administration, greatly improve the therapeutic effect of drugs and patient compliance.
  • the present invention and the PEG-IL2 drug NKTR-214 under research also have differences in the most fundamental molecular design level.
  • the present invention firstly provides a method for realizing the controlled release and sustained release of the activity of bioactive molecules.
  • the inventors Through the reaction of specific types and structures of polyethylene glycol and bioactive molecules such as IL-2 to form conjugates, under certain in vitro conditions or in vivo physiological conditions, PEG is gradually shed from the conjugate structure, and the activity of bioactive molecules is gradually improved.
  • the release effect can maintain a certain life
  • the relatively stable effective blood drug concentration of the active molecule of the drug realizes the slow release and controlled release of the drug in vivo and in vitro, and can achieve a dynamic balance of better drug bioavailability, which has the advantages of reducing the frequency of administration, improving the bioavailability of the drug, Clinical application prospects such as better patient compliance and better safety.
  • a novel IL-2 mutein is provided, which is mutated against the polyethylene glycol modification site, so that the PEG modification is more controllable; further, the mutant targets IL-2 and the receptor The binding site is mutated, and the activity of IL-2 is higher than that of wild type.
  • An object of the present invention is to provide a method for realizing the controlled release and sustained release of the activity of biologically active molecules.
  • the method is to carry out amino modification to the biologically active molecules with a polyethylene glycol modifier, and after the PEG in the obtained modification gradually falls off The activity of bioactive molecules is gradually released, and the polyethylene glycol modifier is polyethylene glycol succinimide succinate.
  • the bioactive molecule is protein or polypeptide, preferably interleukin, most preferably IL-2.
  • the PEG modifier is preferably linear polyethylene glycol succinimide succinate.
  • Another object of the present invention is to provide a polyethylene glycol modified product of human IL-2, the PEG modifier used in it is polyethylene glycol succinimide succinate, the average amount of even Link 4.5 to 8.5 PEG.
  • the PEG modifier is linear polyethylene glycol succinimide succinate.
  • the molecular weight of the PEG modifier is 5-20 kDa.
  • the molecular weight is a marked value, and the actual molecular weight may be 90%-110% of the marked value.
  • the molecular weight of the PEG modifier is 5 kDa, an average of 5.5 to 7.5 PEGs are coupled to each human interleukin-2; when the molecular weight of the PEG modifier is 10 to 20 kDa, an average of 6.5 to 8.5 PEGs are coupled to each human interleukin-2 .
  • the structure of the PEG modifier is as follows:
  • n is an integer of 97-494.
  • the actual molecular weight of the PEG modifier is about 4.5k-22k, and the corresponding molecular weight indication value is 5-20kDa.
  • polyethylene glycol modified structure of the human IL-2 is as follows:
  • n is an integer of 97 to 494
  • m is 4.5 to 8.5.
  • Another object of the present invention is to provide a mutant of human IL-2.
  • the mutant does not contain adjacent lysine, and the modified product modified by PEG is more uniform.
  • the inventors found that the 8th, 9th, 48th, and 49th positions of the wild-type human IL-2 shown in SEQ ID NO: 1 from the N-terminus are all lysines, which may be modified by PEG, and the adjacent positions are almost will not be PEG at the same time grooming.
  • saturation modification occurs, the 8th or 9th position of IL-2 is modified, and the 48th or 49th position is modified, which may produce 4 different modification site isomers, resulting in human IL-2 polyethylene glycol modified products uneven.
  • the inventors chose to mutate the 8th and/or 9th, 48th and/or 49th amino acids to greatly reduce the generation of PEG-modified site isomers and facilitate the homogeneity control of PEG-modified products.
  • those skilled in the art were not aware of the possible adverse effects on PEG modification when the adjacent amino acids on the IL-2 sequence were all lysines, so there was no motivation to modify IL-2 with polyethylene glycol. corresponding mutations.
  • the 8th or/and 9th amino acid from the N-terminal is substituted; or relative to the wild-type human IL-2, from the N-terminal
  • the 48th or/and 49th amino acid is substituted; the wild-type human IL-2 amino acid sequence is shown in SEQ ID NO:1.
  • the 8th or/and 9th amino acid, and the 48th or/and 49th amino acid are substituted from the N-terminus.
  • the 8th lysine is mutated into arginine
  • the 9th lysine is mutated into arginine
  • the 48th lysine is mutated into arginine, tryptophan or tyrosine
  • the 49th lysine was mutated to aspartic acid.
  • the examples are for illustration only, and do not limit the scope of the present invention.
  • the purpose of mutating the 8th, 9th, 48th, and 49th amino acids is to solve the problem that there are adjacent lysines in wild-type IL-2, thereby generating a variety of PEG-modified isomers and affecting the homogeneity of PEG-modified products. Therefore, those skilled in the art know that, except for the amino acids exemplified in the examples, mutations to other amino acids other than lysine can achieve the above-mentioned purpose.
  • the mutations at these positions are respectively selected from the following substitution residues: 1st position: A1 deletion; 8th position: K8R; 9th position: K9R; 18th position: L18M; 19th position: L19S; 48th position: K48W and/or , Position 81: R81D; the wild-type human IL-2 amino acid sequence is shown in SEQ ID NO: 1.
  • the above-mentioned mutation is selected from one of the mutation schemes in the following table:
  • the inventors also took into account the need for IL-2 mutants to weaken IL-2R ⁇ bias, maintain or enhance IL-2R ⁇ , and IL-2R ⁇ bias, so that wild-type human IL-2 from N Terminals 1, 18, 19, 27, 35, 38, 41, 42, 43, 45, 54, 64, 65, 72, 78, 79, 80, 81, 82, 83, 87, 92 and/or 97 Amino acid substitutions were carried out at one, two or more positions corresponding to the position, and effective screening was carried out, such as G438P8.
  • IL-2 acts through the IL-2 receptor (IL-2R), which includes three subunits, IL-2R ⁇ (CD25), IL-2R ⁇ (CD122), and IL-2R ⁇ (CD132). Three subunits form three receptor forms: high-binding receptors contain all three subunits IL-2R ⁇ , IL-2R ⁇ , IL-2R ⁇ ; intermediate-binding receptors contain IL-2R ⁇ , IL-2R ⁇ ; low-binding The force receptor is IL-2R ⁇ .
  • IL-2R ⁇ is highly expressed in Treg cells, IL-2R ⁇ is expressed in CD8+ T cells, NK cells, and Treg cells, and IL-2R ⁇ is expressed in all immune cells.
  • IL-2 mediates multiple effects in the immune response by binding to receptors on different cells.
  • IL-2 can stimulate the proliferation and differentiation of T cells and induce the generation of cytotoxic T lymphocytes. Promotes the proliferation and differentiation of B cells and the synthesis of immunoglobulins, and stimulates the production and activation of NK cells, which has been approved as an immunotherapeutic agent for the treatment of cancer and chronic viral infections; on the other hand, IL-2 can promote immunosuppressive Activation and proliferation of CD4+CD25+ regulatory T cells (ie, Treg cells), leading to immunosuppression.
  • CD4+CD25+ regulatory T cells ie, Treg cells
  • Another object of the present invention is to provide a polyethylene glycol modified product of a human IL-2 mutant, the PEG modifier adopted is polyethylene glycol succinimide succinate, and each human IL-2 5-8 PEGs are coupled to it; the human IL-2 mutant is one of the above-mentioned different mutants.
  • Another object of the present invention is to provide an application of polyethylene glycol modified human IL-2 or its mutant in the preparation of drugs for treating tumor diseases.
  • the tumors include, but are not limited to: squamous cell carcinoma, melanoma, colon cancer, breast cancer, ovarian cancer, prostate cancer, gastric cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, thyroid cancer, kidney cancer, cholangiocarcinoma, Brain cancer, cervical cancer, maxillary sinus cancer, bladder cancer, esophageal cancer, Hodgkin's disease and adrenocortical cancer.
  • Another object of the present invention is to provide a composition for treating tumor diseases, said composition comprising the above-mentioned polyethylene glycol modification of human IL-2, human IL-2 mutant, or human IL-2 mutant Polyethylene glycol modifications, including HER2 anti- Body, PD-1 antibody, PD-L1 antibody or CD26 antibody, etc.
  • the HER2 antibodies include Roche's Herceptin, Perjeta and Kadcyla.
  • the PD-1 antibodies include Bristol-Myers Squibb’s Opidivo, Merck’s Keytruda, Junshi’s toripalimab, Innovent’s sintilimab, Hengrui’s camrelizumab, Tislelizumab from BeiGene, etc.
  • the PD-L1 antibodies include Roche’s Tecentriq, AstraZeneca’s Imfinzi, Merck’s Bavencio, etc.
  • the CD26 antibody may be an anti-CD26 antibody such as YS110 (the prior application CN200680034937.4), or the prior application CN202111245489.5.
  • the tumor diseases treated by the above composition include but not limited to: squamous cell carcinoma, melanoma, colon cancer, breast cancer, ovarian cancer, prostate cancer, gastric cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, thyroid cancer, kidney cancer , cholangiocarcinoma, brain cancer, cervical cancer, maxillary sinus cancer, bladder cancer, esophageal cancer, Hodgkin's disease and adrenocortical cancer.
  • Polyethylene glycol succinimide succinate reacts with the ⁇ -amino group of the lysine side chain in IL-2 and its mutants. Polyethylene glycol and IL-2 are linked by amide bonds. The receptor binding site is completely covered, so the modified PEG-IL-2 molecule is inactive. At the same time, different from other modifiers, there is an ester bond in the conjugate after polyethylene glycol succinimide succinate is combined with protein or polypeptide drug, and the ester bond is easily unstable due to its inherent instability. The detachment of the PEG chain due to hydrolysis was usually regarded as a sign that the modifier is easy to detach and cause the instability of the modified molecule in the past.
  • this PEG is gradually replaced by other PEG modifiers that are difficult to detach (such as polyethylene glycol) in application. succinimidyl propionate, etc.), but in the present invention, the hydrolysis property enables IL-2 to possess the property of gradually releasing drug activity in vivo, and can obtain extremely excellent Pharmacological effect.
  • PEG modifiers such as polyethylene glycol
  • Figure 6 The pharmacodynamic evaluation results of IL-2 modified with different structures of PEG-SS (linear or branched) in CT26.WT murine colon cancer cell BALB/c mouse subcutaneous transplantation model.
  • Figure 6a shows the tumor volume growth curves of the animals in each group
  • Figure 6b shows the tumor weight of the animals in each group
  • Figure 6c shows the changes in the body weight of the animals in each group.
  • Figure 7 The pharmacodynamic evaluation results of different IL-2 mutants modified with linear PEG-SS in the subcutaneous transplantation model of B16-F10 murine melanoma cells C57BL/6 mice.
  • Figure 7a shows the tumor volume growth curves of animals in each group, and
  • Figure 7b shows the tumor weight of animals in each group.
  • FIG. 8 Pharmacodynamic evaluation of PEG-SS modified IL-2 mutants with different molecular weights and degrees of modification in the B16-F10 mouse-derived melanoma tumor model.
  • Figure 8a shows the tumor growth curves of animals in each group.
  • Figure 8b shows the tumor weights of animals in each group on day 17 after cell inoculation.
  • Figure 8c shows the change in growth rate of animal body weight.
  • FIG. 9 Pharmacodynamic evaluation of PEG-SS modified IL-2 mutants with different molecular weights and degrees of modification in the CT26.WT murine colon cancer tumor model.
  • Figure 9a is the tumor growth curves of animals in each group.
  • Fig. 9b The tumor weights of animals in each group on day 22 after cell inoculation.
  • Figure 9c Changes in growth rate of animal body weight.
  • FIG. 10 Pharmacodynamic evaluation of PEG-SS modified IL-2 mutants with different molecular weights and modification degrees in A375 human melanoma model.
  • Figure 10a Animal tumor growth curves.
  • Fig. 10b The tumor weights of animals in each group on the 45th day after cell inoculation.
  • Figure 10c Animal body weight growth rate.
  • Figure 11 Evaluation of the efficacy of different doses of PEG-modified IL-2 mutants in the A375 human melanoma model.
  • Figure 11a Animal tumor growth curves.
  • Figure 11b The tumor weights of animals in each group.
  • Figure 11c Animal body weight growth rate.
  • Figure 12 Evaluation of the efficacy of different doses of PEG-modified IL-2 mutants in the A498 human kidney cancer model.
  • Figure 12a Animal tumor growth curves.
  • Figure 12b The tumor weights of animals in each group.
  • Figure 12c Animal body weight growth rate.
  • Interleukin-2 Interleukin-2, IL-2, can be obtained from recombinant or non-recombinant methods, and can be wild-type IL-2, or a mutant.
  • IL-2 can be expressed in bacteria (such as Escherichia coli), mammalian cells (such as CHO), and yeast (such as Pichia pastoris).
  • IL-2 can be derived from human, animal sources, preferably, IL-2 is derived from human.
  • the amino acid sequence of human IL-2 is shown in SEQ ID NO: 1, and the mutant is a mutant obtained by performing operations such as substitution, insertion or deletion of some amino acids on the basis of it.
  • polyethylene glycol modified IL-2 in the specific embodiment of polyethylene glycol modified IL-2, IL-2 and its mutants shown in SEQ ID NO: 1 are used, but those skilled in the art know that the specific embodiment is only used for illustration and not limitation within the scope of the present application, polyethylene glycol modified IL-2 can also be selected from other reported human IL-2 sequences, or the amino acid at the corresponding position of SEQ ID NO: 1 in other reported human IL-2 sequences Mutants obtained with corresponding mutations (mutations without adjacent lysines, mutations with reduced IL-2R ⁇ bias, enhanced IL-2R ⁇ , IL-2R ⁇ bias).
  • Polyethylene glycol usually polymerized by ethylene oxide, has branched, linear and multi-armed types. Generally, those with a molecular weight below 20,000 are called PEG, and those with a higher molecular weight are called PEO. Ordinary polyethylene glycol has a hydroxyl group at both ends, and if one end is blocked with a methyl group, methoxy polyethylene glycol (mPEG) can be obtained.
  • mPEG methoxy polyethylene glycol
  • PEG modification agent refers to polyethylene glycol derivatives with functional groups, which are activated polyethylene glycol and can be used for protein and polypeptide drug modification.
  • the polyethylene glycol modifier used in this application was purchased from Jiangsu Zhonghong Bioengineering Pharmaceutical Research Institute Co., Ltd., Beijing Jiankai Technology Co., Ltd. or Xiamen Sinobanger Biotechnology Co., Ltd.
  • the actual molecular weight of a PEG modifier with a specific molecular weight can be 90% to 110% of the marked value.
  • the actual molecular weight of PEG5K can be 4.5kDa to 5.5kDa
  • the actual molecular weight of PEG20K can be 18kDa to 22kDa.
  • the marked molecular weight of the PEG modifier is 5 to 20kDa , and its actual molecular weight is 4.5-22kDa.
  • the V-PEG-SC-20k used in the examples refers to a branched polyethylene glycol succinimide carbonate modifier with a molecular weight of 20kDa.
  • the PEG modifier is prepared with reference to the patent document CN200680029849.5. According to the patent document, the The PEG modifier reacts PEG and the drug through a linker to form a conjugate, and the release of PEG from the conjugate can achieve the purpose of sustained and controlled release of drug activity, which is consistent with the structure reported by NKTR-214, so the application Humans use this PEG modifier to modify IL-2 as a positive reference.
  • the structure of the V-PEG-SC-20k modifier is as follows:
  • n is an integer of 199-244.
  • V-PEG-SS-20k used in the embodiment refers to a molecular weight of 20kDa branched polyethylene glycol succinimide succinate modifier; the structure of the V-PEG-SS-20k modifier is as follows:
  • n is an integer of 198-244.
  • the mPEG-SS-20k/10k/5k used in the examples refers to the linear molecular weight of 20kDa, 10kDa, 5kDa polyethylene glycol succinimide succinate modification agent;
  • n is an integer of 403-494;
  • n is an integer of 199-244;
  • n is an integer of 97-119.
  • the mPEG-SPA-5k used in the examples refers to the linear molecular weight of 5kDa polyethylene glycol succinimidyl propionate modifier
  • the mPEG-SPA-5k modifier structure is shown below:
  • n is an integer of 98-120.
  • IL-2 in the following table is wild-type IL-2, its amino acid sequence is shown in SEQ ID NO: 1, and other mutants are mutants obtained by amino acid substitution, insertion or deletion based on it, such as the sequence of G438 It is to mutate the 8th and 48th amino acids of the sequence shown in SEQ ID NO: 1, and K8R refers to the mutation of the 8th lysine to arginine.
  • Step 1 According to the amino acid sequence of G438 (the sequence of K8R/K48W mutation on the basis of wild-type IL-2 as described in SEQ ID NO: 1), and optimize for Escherichia coli to obtain a DNA sequence such as SEQ ID NO: 2 As shown, the DNA sequence was cloned into the pBV220 vector to form the recombinant plasmid pBV220-G438, and then the recombinant plasmid was transformed into the Top10 E. coli host to form the expression host Top10-pBV220-G438.
  • Step 2 Inoculate the Top10-pBV220-G438 recombinant expression strain into TB medium (500ml medium, liquid volume: 20%), culture on a shaker at 30°C and 220rpm until the OD600 of the culture solution reaches 1.0 ⁇ 0.1, and then maintain the shaker speed unchanged, improved Shake the culture temperature to 42°C to induce expression of the strain, and induce expression for 4 hours. After induction of expression, the bacterial pellet was collected by centrifugation.
  • Step 3 Use 10mM PBS, pH 7.4 to resuspend the bacterial cell pellet after expression to 100g/L, sonicate with a probe-type ultrasonic breaker (working power 250w, work for 3s and rest for 4s, and break for a total of 30min), and collect the broken product by centrifugation , to obtain a G438 inclusion body precipitate.
  • a probe-type ultrasonic breaker working power 250w, work for 3s and rest for 4s, and break for a total of 30min
  • Step 4 G438 inclusion bodies were resuspended to 50 g/L in PBS+1% TritonX100, stirred and washed 3 times for more than 2 hours each time, and collected by centrifugation to obtain crude G438 inclusion bodies.
  • Step 5 Resuspend the purified G438 inclusion bodies to 1g/100ml in denaturing solution (20mM Tris 8M urea 5mM DTT, pH 10.5), stir and denature for more than 2 hours, and collect the supernatant by centrifugation. The supernatant was purified using Superdex 75.
  • Step 6 Dilute the purified G438 with refolding buffer for refolding (refolding solution: 20mM Tris, 2M urea, 3mM cysteine, 1mM cystine, 0.01% SDS, pH8.0), and the protein concentration in the refolding system is different. Higher than 0.1mg/ml, the renaturation time is more than 36h, and the renaturation temperature is 15°C.
  • Step 7 The renatured G438 is concentrated by ultrafiltration, and the concentrated sample is dialyzed with PBS at pH 7.4 to remove urea and other reagents as pure G438.
  • amino acids of each mutant can be obtained according to the sequence shown in SEQ ID NO: 1 in combination with the mutation scheme listed in Table 1.
  • the mutants obtained from the above-mentioned design schemes were prepared by using the above-mentioned similar method.
  • the biolayer interferometry (BLI) technique was used to detect the affinity of IL-2 mutants to their receptors.
  • Test solution Take protein samples separately, dilute to 300mM with 1 ⁇ Kinetics buffer, mix well, and set aside.
  • Receptor solution Take receptor IL-2R ⁇ (CD25), receptor IL-2R ⁇ (CD122), receptor IL-2R ⁇ / ⁇ samples and dilute them to 15-20 ⁇ g/mL with 1 ⁇ Kinetics buffer, mix well and store in the dark ,spare.
  • mutants G493, G496, G498, G499, G500, G438P4, and G438P5 to the two receptors was significantly reduced or not, which may be due to the large change in protein structure after mutation.
  • Variants such as G495, G438P6, G438P7, G438P8, G438P14, G438P15 exhibit IL-2 ⁇ receptor binding preference.
  • Mutations such as G495, G438P7, G438P8, and G438P14 had little effect on the binding of IL-2 and IL-2R ⁇ / ⁇ , while the binding of G438P15 and IL-2R ⁇ / ⁇ decreased significantly.
  • Mutants that still have the ability to bind to the receptor in vitro were selected for further in vitro CTLL-2 cell proliferation activity tests to evaluate their biological activity.
  • Example 3 Determination of IL-2 and its mutants stimulating CTLL-2 cell proliferation activity
  • CTLL-2 is a mouse-derived cell line, and the biological activity of IL-2 and its mutants and modifications in vitro can be evaluated by detecting the proliferation rate of its cell-dependent strain CTLL-2 cells at different concentrations.
  • the experimental method is the 2020 edition of the Chinese Pharmacopoeia IV General Rule 3524 "Determination of Biological Activity of Human Interleukin-2" (CTLL-2/MTT colorimetric method).
  • RPMI 1640 culture medium Take 1 bag of RPMI 1640 medium powder (specification is IL) and dissolve it in water and dilute to 1000ml, then add 2.1g of sodium bicarbonate to dissolve, mix well, sterilize and filter, and store at 4°C.
  • Basal culture medium Measure 10ml of newborn bovine serum (FBS), add 90ml of RPMI 1640 culture medium. Store at 4°C.
  • Complete culture solution draw 100ml of basal culture solution, add human IL-2 to a final concentration of 400-800IU/ml per 1ml. Store at 4°C.
  • PBS Take 100ml of 10 ⁇ PBS and dilute to 1000ml with sterilized water at 121°C for 20 minutes.
  • Thiacyanine (MTT) solution Weigh 0.1g of MTT, add PBS to dissolve and dilute to 20ml, and filter through a 0.22 ⁇ m filter to sterilize. Store at 4°C protected from light.
  • Lysis solution 15% sodium lauryl sulfate solution, the service life shall not exceed 12 months.
  • CTLL-2 cells Cultivate CTLL-2 cells with complete culture medium at 37°C and 5% carbon dioxide to a sufficient amount, collect CTLL-2 cells by centrifugation, wash 3 times with RPMI 1640 culture medium, and then resuspend in basal culture medium to prepare 1ml
  • the cell suspension containing 6.0 ⁇ 10 5 cells was stored at 37°C and 5% carbon dioxide for later use.
  • Mutants further modified on the basis of G438, such as: G438P1, G438P8, G438P12, G438P20, G438P21, and G438P22 are respectively referred to as GP1, GP8, GP12, GP20, GP21, and GP22, and so on.
  • the concentration is about 20mg/mL, according to the mass ratio of protein:PEG modifier 1 : 20 by weighing PEG, the modification reaction was carried out at normal temperature, after 2 hours of reaction, 1M glycine was added to stop the reaction.
  • modification buffer 100mM disodium hydrogen phosphate-sodium dihydrogen phosphate, pH8.0
  • mobile phase A is 20mM PB+2M NaCl (pH6.0)
  • mobile phase B is 20mM PB (pH6.0).
  • Sample loading After the above-mentioned modified sample was diluted, the sample was loaded at 5 ml/min, and bound to a hydrophobic chromatography column (purchased from GE, Phenyl PH).
  • SEC Size Exclusion Chromatography is a chromatographic technique that separates molecules according to their size.
  • the PEG-IL-2 sample prepared in this example was detected by SEC chromatography, and the results showed that the main peak of the sample was uniform, that is, the degree of modification was uniform, which met the research requirements.
  • V-PEG-SC-20k-rhIL-2 in this preparation example or PEG-SC-20k-rhIL-2 for short.
  • Example 4b Preparation of other PEG-modified samples provided by the present invention:
  • mobile phase B is 20mM NaAc+1M NaCl (pH4.0)
  • mobile phase A is 20mM NaAc (pH4.0).
  • Sample loading After the above-mentioned modified sample was diluted, the sample was loaded at 5 mL/min, and bound to a cation exchange chromatography column (purchased from GE, HPSP).
  • SEC (Size Exclusion) Chromatography is a chromatographic technique that separates molecules according to their size.
  • the PEG-IL-2 samples prepared in this example were detected by SEC chromatography, and the results showed that the main peaks of the samples were uniform, that is, the degree of modification was uniform, which met the research requirements.
  • PEG-IL-2 samples of the same PEG with different modification degrees have significant retention time differences, indicating that the established sample preparation process of the present invention can stably prepare samples with different modification degrees through the control of process parameters.
  • PEG incorporation numbers for specific samples are given in Example 5.
  • Embodiment 5 PEG binding number determination of PEG modified IL-2 (hydrolysis method)
  • 1 PEG standard gradient solution Take 5 ⁇ L, 10 ⁇ L, 20 ⁇ L, 30 ⁇ L, 40 ⁇ L, 50 ⁇ L of V-PEG-SC-20k, V-PEG-SS-20k, mPEG-SS-20k, mPEG-SS-10k, mPEG-SS respectively Add -5k PEG solution (2.5mg/mL) into water to prepare a gradient solution with a final volume of 100 ⁇ L, and mix well to obtain a standard gradient solution. Add 25 ⁇ L 5 ⁇ non-reduced Loding Buffer respectively, mix well, and set aside.
  • 2Iodine staining solution Accurately weigh 17.5g of BaCl 2 , 6g of KI, and 3.9g of I 2 , dissolve in 500mL of double-distilled water, and store in the dark.
  • 10% polyacrylamide gel preparation absorb 1.6mL double distilled water, 1.8mL 30% polyacrylamide solution, 1.3mL 1.5mol/L Tris-HCL pH8.8, 0.53mL 1% SDS solution, Mix 0.033mL of 10% ammonium persulfate solution and 0.033mL of TEMED evenly to make a separating gel.
  • Operating voltage run at 80V for 30 minutes, change to 120V after the bromophenol blue indicator moves to the bottom of the stacking gel, and end when the bromophenol blue indicator runs to the bottom edge of the separating gel.
  • Iodine solution staining after electrophoresis, pry open the glass plate, mark the film and put it in the staining box, fix it with 10% perchloric acid solution for 10 minutes, then recover the 10% perchloric acid and wash it with water for 3 times , and then cover the film with iodine dye solution and stain for 2-3 minutes, the color can be developed in about 1 minute, and then immediately decolorized with water.
  • Embodiment 6 PEG modified IL-2 in vitro slow-release performance assay
  • Example 5 for the pretreatment method, in which the specific operation of the hydrolysis part is as follows: take 100 ⁇ L of the test solution with known protein content, add 200 ⁇ L of 100 mM NaHCO 3 pH9.0 activation buffer, and hydrolyze in a water bath at 37 °C. Take 100 ⁇ L from the hydrolyzed samples at different time points (such as 8h, 16h, and 24h after the start of hydrolysis), dilute to a certain multiple, add 5 ⁇ non-reducing Loding Buffer, mix well, and set aside.
  • Example 7 Determination of PEG-modified IL-2 stimulating CTLL-2 cell proliferation activity
  • Sample activation Take 100 ⁇ L of the test solution with known protein content, add 200 ⁇ l of 100mM NaHCO 3 pH9.0 Or 300 ⁇ l of 100% human serum, incubate in a 37°C water bath for a certain period of time, take out 100 ⁇ l from the hydrolyzed samples at regular intervals, mix well, and set aside. Dilute the sample to an appropriate starting concentration. In a 96-well cell culture plate, make 2-fold serial dilutions, a total of 8 dilutions, and make 2 wells for each dilution. Leave 50 ⁇ l of solution in each well, and discard the excess solution in the well. The above operations were carried out under sterile conditions.
  • Test solution preparation and cell culture are the same as in Example 3.
  • the PEG-SS modification is consistent with the reference product V-PEG-SC-20k-rhIL-2.
  • activation 0h the receptor binding site on the surface is completely covered by PEG, and does not reflect Biological activity, that is, no biological activity (undetectable) without PEG shedding.
  • the biological activity of promoting the proliferation of CTLL-2 cells appeared significantly after being activated for different times under alkaline conditions in vitro.
  • the PEG-SS modification has a tendency of gradually recovering and improving the biological activity with the prolongation of the activation time.
  • PEGs with different molecular weights have different active release effects under different modification degrees, which also constitutes the basis for the controlled release of IL-2 modified with PEG-SS molecules, that is, we can adjust the PEG-SS with different molecular weights and the number of modified PEGs are controlled to explore the optimal release curve of PEG-IL2 in vivo, so that it can achieve the optimal bioavailability of effector molecules in vivo, so that the drug effect is better than the existing ones.
  • Embodiment 8 different structure PEG modified IL-2 is measured to CTLL-2 cell pSTAT5 activity
  • Complete culture medium (RPMI1640 medium + 2mM L-glutamine + 1mM sodium pyruvate + 10% fetal bovine serum + 10% T-STIM, culture supplemented with concanavalin A) for CTLL-2 cells at 37°C , 5% CO 2 and cultured to a density of 2 ⁇ 10 5 cells/mL, washed once with PBSA (PBS, pH 7.2, 1% BSA), adjusted the cell density to 1 ⁇ 10 6 cells/mL, and A volume of 500 ⁇ L was dispensed into flow tubes, and different concentrations of PEG-modified IL-2 formulated with basal culture medium (RPMI1640+2MmL-glutamine+1mM sodium pyruvate+10% fetal bovine serum) were added, After incubating at room temperature for 20 min, paraformaldehyde was immediately added to a final concentration of 1.5%, vortexed and incubated at room temperature for 10 min.
  • basal culture medium RPMI1640+2Mm
  • IL-2 Regardless of which IL-2 binds to IL-2R on any cell, it exerts its biological function through the JAK-STAT pathway.
  • the JAK1 pathway through IL-2R ⁇ binding and the JAK3 pathway through IL-2R ⁇ binding lead to the phosphorylation of key tyrosine residues on the ⁇ and ⁇ subunits of IL-2R, respectively, thereby creating anchor sites for other signaling molecules. Therefore, the biological activity of IL-2 and its modified substances in vitro can be evaluated by detecting the changes in the phosphorylation level of CTLL-2 cells under the action of different concentrations of PEG-modified IL-2.
  • Example 9 PEG-modified IL-2 different mutants are measured for CTLL-2 cell pSTAT5 activity
  • test results are shown in Figure 4, and the results show that: the test results of in vitro activated pSTAT5 levels show that mPEG-SS-20k-GP1 and mPEG-SS-20k-GP8 (in this example, the above-mentioned mPEG-SS-20k-GP8-modified ) after activation in the buffer, the trend of the level of activated phosphorylation is similar, showing the characteristics of gradual activation over time, and can continue to maintain after reaching the peak.
  • Embodiment 10 Comparison of half-life extension effect of PEG modified IL-2 mutants in vivo and activity release of modified products
  • Experiment 1 0.5 mg/kg, 0.1 mg/kg and 0.04 mg/kg of highly modified mPEG-SS-5k-GP8 and 1 mg/kg of mutant GP8 were intravenously injected into SD rats respectively. Serum was collected before administration and at 0.25h, 1h, 2h, 4h, 8h, 24h, 48h, 72h and 96h after administration, and two ELISA detection methods were used to detect the high modification of mPEG-SS-5k-GP8 in serum And the content of mutant GP8.
  • Experiment 2 1 mg/kg of highly modified mPEG-SS-5k-GP8 and positive reference product V-PEG-SC-20k-rhIL-2 were intravenously injected into SD rats respectively. Serum was collected before administration and at 0.25h, 1h, 2h, 4h, 8h, 24h, 48h, 72h and 96h after administration, and the activity at each time point was measured by CTLL-2 cell/MTT colorimetric method, unit as IU/ml.
  • CT26.WT cells were cultured in 1640 medium containing 10% fetal bovine serum.
  • CT26.WT cells in the exponential growth phase were collected and resuspended in PBS to an appropriate concentration.
  • 5 ⁇ 10 5 cells/0.1mL CT26.WT cell suspension was thoroughly mixed and inoculated subcutaneously on the right back of BALB/c mice, each mouse was inoculated with 0.1ml.
  • start the first administration on the day of the grouping (in this experiment, the administration was given once on the 9th day and the 16th day after tumor cell inoculation). See the table below for drug routes.
  • N indicates the number of animals
  • sc indicates administration by subcutaneous injection
  • iv indicates administration by intravenous injection.
  • the day of cell inoculation is D1.
  • Body weight 2-3 times a week.
  • TGI (%) (1-T/C) ⁇ 100%. Recognized that T represents the relative tumor volume at a certain time point of the administration group (ratio of the tumor volume measured at the time and the tumor volume at the time of grouping), and C represents the relative tumor volume at a certain time point of the model group (the tumor volume measured at the time and the tumor volume ratio at the time of grouping). Tumor volume ratio at the time of grouping). On the day of inoculation in the experiment of this application, the groups were grouped according to body weight, and when TGI was calculated, T and C represented the actual measured tumor volumes of the administration group and the model group respectively.
  • Tumor weight After the last test, the animals were euthanized, the tumor mass was peeled off, rinsed with normal saline and blotted dry with filter paper, the tumor mass was weighed, and photographed.
  • Relative tumor inhibition rate TGI (%) (1-T TW /C TW ) ⁇ 100%, T TW represents the average tumor weight at the end of the experiment in the treatment group, and C TW represents the average tumor weight at the end of the experiment in the model group.
  • the average tumor volume of the animals in the model group was 1397.48 ⁇ 289.67mm 3 on the 20th day after cell inoculation.
  • mPEG-SS-20k-GP1 (2mg/kg)
  • mPEG-SS-20k-GP8 (2mg/kg) (in this example, modified in the above mPEG-SS-20k-GP8-) on the 20th day after cell inoculation
  • the average tumor volumes were 282.31 ⁇ 103.02mm 3 and 133.93 ⁇ 105.69mm 3 , which were significantly different from the model group (P ⁇ 0.01), and the relative tumor inhibition rates TGI (%) were 79% and 92%, respectively.
  • the mean tumor volumes of V-PEG-SS-20k-GP1(2mg/kg) and V-PEG-SS-20k-GP8(2mg/kg) on day 20 after cell inoculation were 942.44 ⁇ 209.66mm 3 and 1037.67 ⁇ 332.97mm respectively mm 3 , there was no significant difference compared with the model group, and the relative tumor inhibition rates TGI (%) were 26% and 29%, respectively.
  • V-PEG-SC-20k-rhIL-2 group (2mg/kg) (subsequent examples such as no special instructions, positive reference all use V-PEG-SC-20k-rhIL-2, or abbreviated as PEG-SC- 20k-rhIL-2)
  • the average tumor volume was 211.23 ⁇ 86.02mm 3 on the 20th day after cell inoculation, which was significantly different from the model group (P ⁇ 0.01), and the relative tumor inhibition rate TGI (%) was 84%.
  • the linear PEG modified products mPEG-SS-20k-GP1 and mPEG-SS-20k-GP8 can significantly inhibit tumor growth in the CT26.WT mouse colon cancer model at a dose of 2 mg/kg, and the inhibition rate is comparable to that of the positive reference similar or higher.
  • the branched PEG modification products V-PEG-SS-20k-GP1 and V-PEG-SS-20k-GP8 groups had no effect on tumor growth inhibition in the CT26.WT murine colon cancer model at a dose of 2 mg/kg.
  • both the linear PEG-SS modified product and the positive reference product had body weight loss in the mid-administration period, but after the second administration and during the recovery period, the linear PEG-SS modified product animals The status and weight recovery were significantly better than those in the positive reference group.
  • Example 12 Pharmacodynamic evaluation of different IL-2 mutants modified with linear PEG-SS in the subcutaneous transplantation model of B16-F10 murine melanoma cells C57BL/6 mice
  • B16-F10 cells were cultured in DMEM medium containing 10% fetal bovine serum. B16-F10 cells in the exponential growth phase were collected and resuspended in PBS to an appropriate concentration. 5 ⁇ 10 5 cells/0.1mL B16-F10 cell suspension was thoroughly mixed and inoculated subcutaneously on the right back of C57BL/6 mice, each mouse was inoculated with 0.1ml. When the average tumor volume reaches about 100mm 3 , the group will be grouped according to the tumor volume, and the first administration will be started on the day of the grouping (one administration on D7). The detailed dosing regimen and route of administration are shown in the table below. General clinical observation is identical to embodiment 11.
  • N indicates the number of animals
  • sc indicates administration by subcutaneous injection
  • iv indicates administration by intravenous injection.
  • the day of cell inoculation is D1.
  • the body weight change rate of the animals in the V-PEG-SC-20k-rhIL-2 group was significantly lower on D11 (the animals in all groups survived on D11).
  • the products of different mutants modified by linear PEG-SS mPEG-SS-20k-GP1, mPEG-SS-20k-GP13, mPEG-SS-20k-GP21, mPEG-SS-20k-GP22 The body weight of animals in each group at D11 The decline was not statistically different from that of the model group, but was significantly different from that of the V-PEG-SC-20k-rhIL-2 group.
  • Example 13 Drug efficacy evaluation of linear PEG-SS modified IL-2 mutants with different degrees of modification in B16-F10 murine melanoma tumor model
  • N indicates the number of animals
  • sc indicates administration by subcutaneous injection
  • iv indicates administration by intravenous injection.
  • the day of cell inoculation is D1.
  • the average tumor volume of the animals in the model group was 2614.97 ⁇ 372.77mm 3 on the 17th day after cell inoculation.
  • the average tumor volume on day 17 after cell inoculation was 550.25 ⁇ 100.23mm 3 , 544.90 ⁇ 89.12mm 3 , 574.02 ⁇ 108.15mm 3 , 676.17 ⁇ 128.23mm 3 , 570.45 ⁇ 107.25mm 3 , there were significant differences compared with the model group (P ⁇ 0.01 or P ⁇ 0.05), and the relative tumor inhibition rates TGI (%) were 77%, 80%, 79%, 75% respectively , 79%.
  • the average tumor volume of the PEG-SC-20k-rhIL-2 group was 1215.19 ⁇ 184.70mm 3 on the 17th day after cell inoculation, compared with There was a significant difference between the model group (P ⁇ 0.05), and the relative tumor inhibition rate TGI (%) was 57%.
  • the body weight of the animals in the mPEG-SS-5k-GP8 (medium degree of modification) group did not change significantly during the administration and treatment.
  • mice in the mPEG-SS-20k-GP8 (high modification degree) and mPEG-SS-20k-GP8 (medium modification degree) groups were poorly tolerated at a dose of 2 mg/kg, and mPEG-SS-5k-GP8 ( High degree of modification) group, mPEG-SS-5k-GP8 (low degree of modification) group animals were basically tolerated at a dose of 2 mg/kg. Animals in the mPEG-SS-5k-GP8 (medium degree of modification) group tolerated well at a dose of 2 mg/kg.
  • Example 14 Pharmacodynamic evaluation of linear PEG-SS modified IL-2 mutants with different degrees of modification in CT26.WT murine colon cancer tumor model
  • N indicates the number of animals
  • sc indicates administration by subcutaneous injection
  • iv indicates administration by intravenous injection.
  • the day of cell inoculation is D1.
  • the average tumor volume of the animals in the model group was 1,886.67 ⁇ 341.33 mm 3 on day 22 after cell inoculation.
  • the average tumor volume on day 22 after cell inoculation was 268.15 ⁇ 163.54mm 3 , 132.07 ⁇ 132.07mm 3 , 56.90 ⁇ 36.21mm 3 , 429.12 ⁇ 261.88mm 3 , 87.78 ⁇ 87.78mm 3 , there is a significant difference compared with the model group (P ⁇ 0.01), and the relative tumor inhibition rate TGI (%) is 88%, 90%, 97%, 80%, 95% respectively .
  • the average tumor volume of the PEG-SC-20k-rhIL-2 group was 163.13 ⁇ 73.03mm 3 on the 22nd day after cell inoculation, which was significantly different from the model group (P ⁇ 0.01), and the relative tumor inhibition rate TGI (%) was 92%.
  • Table 22 Tumor volume, relative tumor volume, TGI, T/C (Female, Mean ⁇ SEM) of each group of animals Note: *: P ⁇ 0.05, **: P ⁇ 0.01, vs model group.
  • Table 23 Tumor weight, TGI, T/C (Female, Mean ⁇ SEM) of animals in each group Note: *: P ⁇ 0.05, **: P ⁇ 0.01, vs model group.
  • mPEG-SS-20k-GP8 (moderate degree of modification) 3/6 animals had moderate body weight loss (10% ⁇ weight loss ⁇ 20%) on the 5th day after the first administration, and 1/6 animals died; On the 7th day after administration, 1/6 animals moderately lost body weight (10% ⁇ body weight loss ⁇ 20%); on the 2nd day after the second administration, 1/6 animals experienced moderate body weight loss (10% ⁇ body weight loss ⁇ 20%), did not recover until the end of the experiment.
  • mPEG-SS-5k-GP8 showed moderate body weight loss (10% ⁇ weight loss ⁇ 20%) in 1/6 animals on day 5 after the first administration. Moderate body weight loss (10% ⁇ body weight loss ⁇ 20%) occurred in 1/6 animals on the 2nd day after the second administration, and there was a recovery tendency by the end of the experiment.
  • mPEG-SS-5k-GP8 showed moderate body weight loss (10% ⁇ weight loss ⁇ 20%) in 1/6 animals on the second day after the second administration, and there was a tendency to recover by the end of the experiment.
  • test products mPEG-SS-20k-GP8 (high degree of modification), mPEG-SS-20k-GP8 (medium degree of modification), mPEG-SS-5k-GP8 (high degree of modification), mPEG-SS-5k-GP8 ( Moderate degree of modification), mPEG-SS-5k-GP8 (low degree of modification), at a dose of 2 mg/kg, can inhibit tumor growth in the CT26.WT mouse colon cancer model.
  • mice in mPEG-SS-5k-GP8 high degree of modification
  • mPEG-SS-5k-GP8 medium degree of modification
  • mPEG-SS-5k-GP8 low degree of modification
  • Animals in the mPEG-SS-20k-GP8 (high modification degree) and mPEG-SS-20k-GP8 (medium modification degree) groups were basically tolerated at a dose of 2 mg/kg, and their body weight recovery was better than that of V-PEG-SC-20k - rhIL-2.
  • Example 15 Drug efficacy evaluation of linear PEG-SS modified IL-2 mutants with different degrees of modification in A375 human melanoma tumor model
  • A375 cells were cultured in DMEM medium containing 10% fetal bovine serum, A375 cells in exponential growth phase were collected and resuspended in PBS; In the culture medium, collect PBMCs on the third day after being stimulated by OKT-3 and IL-2 drugs, add PBS to resuspend; 5 ⁇ 10 5 cells/0.1mL A375 cells and 5 ⁇ 10 5 cells/0.1mL PBMC cell suspension According to the ratio of 1:1 (the amount of cell inoculation), it was fully mixed and used for subcutaneous inoculation of NOD/SCID mice, each inoculated with 0.2 mL. On the day after cell inoculation, the group was grouped according to body weight, and the first administration was started on the day of the grouping. The detailed dosing scheme and route of administration are shown in the table below.
  • N indicates the number of animals
  • sc indicates administration by subcutaneous injection
  • iv indicates administration by intravenous injection.
  • the day of cell inoculation is D1.
  • the average tumor volume of the animals in the model group was 1,970.97 ⁇ 298.65 mm 3 on the 45th day after cell inoculation.
  • mice in the mPEG-SS-20k-GP8 (high degree of modification) group, mPEG-SS-20k-GP8 (medium degree of modification) group, and mPEG-SS-5k-GP8 (medium degree of modification) group showed no abnormality on the 45th day after cell inoculation. See tumor growth, compared with the model group, there were significant differences (P ⁇ 0.01).
  • the average tumor volumes of PEG-SC-20k-rhIL-2 group, mPEG-SS-5k-GP8 (high degree of modification), mPEG-SS-5k-GP8 (low degree of modification) were 136.59 ⁇ 134.84mm 3 , 83.34 ⁇ 83.34 mm 3 , 96.62 ⁇ 64.92mm 3 , compared with the model group, there were significant differences (P ⁇ 0.01).
  • the results of tumor weight analysis were close to the tumor volume, and mPEG-SS-20k-GP8 (high degree of modification) group, mPEG-SS-20k-GP8 (medium degree of modification) group, mPEG-SS-5k-GP8 (high degree of modification) group, mPEG-SS-5k-GP8 (medium degree of modification) group, mPEG-SS-5k-GP8 (low degree of modification) group, PEG-SC-20k-rhIL-2 group were calculated according to tumor weight TGI were 100%, 100%, 100%, 100%, 95%, 100%, 95%, 91%.
  • mPEG-SS-20k-GP8 high degree of modification on the 3rd day after the fourth administration showed 1/6 animal moderate body weight loss (10% ⁇ weight loss ⁇ 20%), the animal was given the fourth time On the 6th day after administration, the body weight remained moderately decreased (10% ⁇ weight loss ⁇ 20%), and the animal died 1 day after the last administration. In addition, from the 9th day to the 13th day after the last administration, 1/6 animals experienced moderate body weight loss (10% ⁇ weight loss ⁇ 20%), and the body weight of this animal tended to recover by the end of the experiment.
  • mPEG-SS-20k-GP8 (moderate degree of modification) 1/6 animals experienced severe body weight loss (body weight loss > 20%) on the 3rd day after the second administration, and died on the 6th day after the second administration From the 3rd day after the last administration to the end of the experiment, 1/6 animals experienced moderate body weight loss (10% ⁇ weight loss ⁇ 20%); 1/6 animals died on the 4th day after the last administration.
  • mPEG-SS-5k-GP8 high degree of modification
  • mPEG-SS-5k-GP8 low degree of modification
  • test products mPEG-SS-20k-GP8 (high degree of modification), mPEG-SS-20k-GP8 (medium degree of modification), mPEG-SS-5k-GP8 (high degree of modification), mPEG-SS -5k-GP8 (medium modification degree) and mPEG-SS-5k-GP8 (low modification degree) were slightly better than PEG-SC-20k-rhIL-2.
  • mPEG-SS-20k-GP8 high degree of modification
  • mPEG-SS-20k-GP8 medium degree of modification
  • V-PEG-SC-20k-rhIL-2 animals at a dose of 0.5mg/kg Poor tolerance
  • mPEG-SS-5k-GP8 high degree of modification
  • mPEG-SS-5k-GP8 low degree of modification
  • the animals in the mPEG-SS-5k-GP8 (moderate degree of modification) group were basically tolerated at a dose of 0.5 mg/kg (once/week ⁇ 5 weeks).
  • test products mPEG-SS-5k-GP8 (high degree of modification), mPEG-SS-5k-GP8 (medium degree of modification), mPEG-SS-5k-GP8 (low degree of modification ) was better tolerated than PEG-SC-20k-rhIL-2.
  • Example 16 Drug efficacy evaluation of PEG-modified IL-2 mutants in A375 human melanoma model
  • A375 cells were cultured in DMEM medium containing 10% fetal bovine serum, A375 cells in exponential growth phase were collected and resuspended in PBS; In the culture medium, collect PBMCs on the third day after being stimulated by OKT-3 and IL-2 drugs, add PBS to resuspend; 1 ⁇ 10 6 cells/0.1mL A375 cells and 1 ⁇ 10 6 cells/0.1mL PBMC cell suspension According to the ratio of 1:1 (the amount of cell inoculation), it was fully mixed and used for subcutaneous inoculation of NOD/SCID mice, each inoculated with 0.2 mL. On the day after cell inoculation, the group was grouped according to body weight, and the first administration was started on the day of the grouping. The detailed dosing scheme and route of administration are shown in the table below.
  • N indicates the number of animals; iv: indicates intravenous administration.
  • the day of cell inoculation is D1.
  • the total amount of administration of the positive control group (Quanqi (recombinant human interleukin-2 (125Ser) for injection) was the total amount of administration of the mPEG-SS-5K-GP8-highly modified high-dose group. 1.4 times.
  • the average tumor volume of animals in the negative control group was 1,197.64 ⁇ 143.79 mm 3 on day 45 after cell inoculation.
  • the average tumor volume was 144.87 ⁇ 35.11mm 3 , 296.04 ⁇ 61.10mm 3 and 643.88 ⁇ 153.05mm 3 , compared with the negative control group, there were significant differences (P ⁇ 0.01 or P ⁇ 0.05).
  • the average tumor volume of animals in the positive control group (Quanqi, 1 million IU/kg) was 557.54 ⁇ 104.82 mm 3 on day 45 after cell inoculation, which was significantly different from that in the negative control group (P ⁇ 0.01).
  • the positive control group (Quanqi) in the mPEG-SS-5K-GP8-highly modified high-dose group there was a significant difference in tumor volume (P ⁇ 0.01).
  • the results showed that the total dose of the positive control group (Quanqi) was higher than that of the mPEG-SS-5K-GP8-highly modified high-dose group, but the mPEG-SS-5K-GP8-highly modified drug effect was better.
  • the average tumor volume of animals in the positive reference drug (V-PEG-SC-20k-rhIL-2, imitation NKTR214) group was 224.37 ⁇ 33.28mm 3 on the 45th day after cell inoculation, which was significantly different from the negative control group (P ⁇ 0.01). There was no significant difference in tumor volume between the mPEG-SS-5K-GP8-highly modified middle-dose group and the positive reference group (P>0.05). The results showed that isodose The efficacy of mPEG-SS-5K-GP8-high modification is similar to that of the positive reference drug (V-PEG-SC-20k-rhIL-2, imitation NKTR214).
  • the results of tumor weight analysis were basically consistent with the results of tumor volume analysis.
  • the TGI calculated according to tumor weight in the mPEG-SS-5K-GP8-highly modified high, medium and low dose groups were 87%, 75%, and 46%, respectively.
  • the TGI of the positive control group (Quanqi) and the positive reference group (V-PEG-SC-20k-rhIL-2, imitation NKTR214) calculated according to the tumor weight were 56% and 81%, respectively.
  • mPEG-SS-5K-GP8-highly modified high-dose group 250 ⁇ g/kg
  • 3/8 animals experienced moderate body weight loss during the intravenous injection treatment, and the animals basically tolerated the treatment.
  • Animals in mPEG-SS-5K-GP8-highly modified medium and low dose groups (125 ⁇ g/kg, 62.5 ⁇ g/kg) showed no obvious drug toxicity during intravenous administration and were well tolerated during treatment.
  • Table 33 Table of weight changes of animals in each group (Female, Mean ⁇ SEM) Note: **: P ⁇ 0.01, vs negative control group; *: P ⁇ 0.05, vs negative control group. ⁇ : P ⁇ 0.01, vs positive reference group (imitation of NKTR214).
  • Table 34 The body weight change table of each group of animals (Female, Mean ⁇ SEM) Note: **: P ⁇ 0.01, vs negative control group; *: P ⁇ 0.05, vs negative control group. ⁇ : P ⁇ 0.05, vs positive reference group (imitation of NKTR214).
  • mPEG-SS-5K-GP8-highly modified can significantly inhibit tumor growth in A375 human melanoma model at high and medium doses (250 ⁇ g/kg, 125 ⁇ g/kg), mPEG-SS-5K-GP8-highly modified There is a trend of tumor inhibition at low dose (62.5 ⁇ g/kg), and high, medium and low doses of mPEG-SS-5K-GP8-high modification can show a good dose-effect relationship.
  • the positive reference group (V-PEG-SC-20k-rhIL-2, imitation NKTR214) had a significant effect on tumor growth inhibition on the A375 human melanoma model at a dose of 125 ⁇ g/kg, and the positive control group (Quanqi) at a dose of 1 million IU /kg dose on the A375 human melanoma model There is a tendency towards tumor suppression.
  • Example 17 Pharmacodynamic evaluation of PEG-modified IL-2 mutants in A498 human kidney cancer model
  • A498 cells were cultured in DMEM medium containing 10% fetal bovine serum, A498 cells in exponential growth phase were collected and resuspended in PBS; In the culture medium, collect the PBMCs stimulated by OKT-3 and IL-2 drugs on the third day, add PBS to resuspend; 5 ⁇ 10 6 cells/0.1mL A498 cells and 5 ⁇ 10 6 cells/0.1mL PBMC cell suspension According to the ratio of 1:1 (the amount of cell inoculation), it was fully mixed and used for subcutaneous inoculation of NOD/SCID mice, each inoculated with 0.2 mL. On the day after cell inoculation, the group was grouped according to body weight, and the first administration was started on the day of the grouping. The detailed dosing scheme and route of administration are shown in the table below.
  • N indicates the number of animals; iv: indicates intravenous administration.
  • the day of cell inoculation is D1.
  • the total dose of the positive control group was twice that of the mPEG-SS-5K-GP8-highly modified middle-dose group.
  • the average tumor volume of animals in the negative control group was 1,960.68 ⁇ 398.28 mm 3 on day 61 after cell inoculation.
  • the average tumor volume of animals in the positive control group (Quanqi, 712,500 IU/kg) on the 61st day after cell inoculation was 290.85 ⁇ 145.96mm 3 , compared with the negative control group, there was a significant difference (P ⁇ 0.01).
  • the results showed that only Quanqi 1/2 dose of mPEG-SS-5K-GP8-highly modified middle dose group had a better drug effect than the positive control group (Quanqi).
  • the average tumor volume of animals in the V-PEG-SC-20k-rhIL-2 group was 110.22 ⁇ 63.15mm 3 on the 61st day after cell inoculation, which was significantly different from the negative control group (P ⁇ 0.01) .
  • the results of tumor weight analysis were basically consistent with the results of tumor volume analysis.
  • the TGI calculated according to tumor weight in the mPEG-SS-5K-GP8 medium and low dose groups were 99% and 85%, respectively.
  • the TGI of the positive control group (Quanqi) and the positive reference group (V-PEG-SC-20k-rhIL-2, imitation NKTR214) calculated according to the tumor weight were 83% and 93%, respectively.
  • Table 38 Table of body weight changes of animals in each group (Female, Mean ⁇ SEM) Note: **: P ⁇ 0.01, vs negative control group; *: P ⁇ 0.05, vs negative control group. ⁇ : P ⁇ 0.01, vs positive reference group (imitation of NKTR214).
  • mPEG-SS-5K-GP8-highly modified mPEG-SS-5K-GP8 can significantly inhibit tumor growth in the A498 human renal carcinoma subcutaneous xenograft model at medium and low doses (125 ⁇ g/kg, 62.5 ⁇ g/kg) - Highly modified medium and low doses can show a certain dose-effect relationship.
  • the V-PEG-SC-20k-rhIL-2 group at a dose of 125 ⁇ g/kg, and the positive control group (Quanqi) at a dose of 712,500 IU/kg had significant inhibitory effects on the growth of the A498 human renal carcinoma subcutaneous transplanted tumor model .
  • cynomolgus monkeys in each group were given different doses of mPEG-SS-5K-GP8-high modification (0.01mg/kg group, 0.03mg/kg group) by intravenous injection, and the blank control group was given solvent control, administered weekly Once, a total of 5 administrations (D1, D8, D15, D22, D29), after 30 days from the first administration (D1), peripheral blood was collected, and peripheral blood mononuclear cells (PBMCs) were isolated, and anti- CD25-APC fluorescent antibody (Miltenyi, product number 130-113-842) labeling, FACS detection.
  • D1, D8, D15, D22, D29 peripheral blood mononuclear cells
  • CD25 is a marker of T cell activation in PBMC cells, which proves that the 0.03mg/kg administration group can effectively stimulate the activation of T cells. .
  • Cynomolgus monkeys were used as experimental animals, and different doses of mPEG-SS-5K-GP8-highly modified were given multiple intravenous injections, and the possible toxic reactions during the administration were observed.
  • Four dose groups of 0.03, 0.1, 0.3, and 0.5 mg/kg were designed, among which the 0.3 mg/kg dose group was 0.03 mg/kg, and the animals in the 0.03 mg/kg dose group increased the dose to 0.3 mg/kg after completing 3 administrations. A total of 6 administrations were completed.
  • the first group of animals was given a dose of 0.3 mg/kg on D29-D43;
  • mice were clinically observed every day to monitor changes in animal weight, food intake, body temperature, and blood pressure, and to conduct clinical pathology, immune cell phenotype analysis, cytokines, and general observations.
  • cynomolgus monkeys Under the dose of 0.03mg/kg, cynomolgus monkeys showed no abnormality, and their body weight showed a tendency to increase.
  • the animals had adverse reactions such as lassitude, decreased spontaneous activity, a large number of loose stools, and pale visible mucosa after the first administration.
  • the administration was suspended for one time, and a total of four administrations were completed subsequently.
  • NKTA-214 related literature (NKTA-214, an Engineered Cytokine with Biased IL2 Receptor Binding, Increased Tumor Exposure, and Marked Efficacy in Mouse Tumor Models) reports: in cynomolgus monkeys 14 days apart, 4 doses In the test, the maximum tolerated dose (MTD) was 0.1 mg/kg, at which dose CD25+ increased 24 times, and total lymphocytes increased 4 times.

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Abstract

提供一种实现生物活性分子其活性控释和缓释的方法及药物应用。提供一种特定结构PEG修饰剂,该PEG修饰剂可广泛用于各类生物活性分子的活性控释和缓释。提供所述特定类型、结构的PEG修饰剂与IL-2反应形成的结合物,在一定体外条件或体内生理条件下,PEG从结合物中逐次脱落、IL-2的活性逐步释放,能维持一定生物活性分子较为稳定的有效血药浓度,实现了药物在体内外的活性缓释和控释。

Description

一种实现生物活性分子其活性控释和缓释的方法及药物应用 技术领域
本发明涉及一种实现生物活性分子其活性控释和缓释的方法及药物应用,具体涉及对高活性细胞因子,如:白细胞介素-2的活性控释和缓释的方法。
背景技术
白细胞介素-2(Interleukin-2,IL-2)是白细胞介素大家族中的一员,大量研究表明:IL-2能促进T细胞、NK细胞、B细胞的分化成熟,并激活其生物活性,诱导淋巴因子激活的杀伤细胞(LAK)的活化,还能促进许多淋巴因子如干扰素、肿瘤坏死因子等的合成与释放以及抗体的生成。IL-2在体内扩充淋巴细胞群体和提高这些细胞的效应器功能的能力使得其具有抗肿瘤效果,早在20世纪80年代早期,高剂量IL-2已被批准用于治疗转移性肾细胞癌和恶性黑素瘤。
然而,IL-2在体内的半衰期短,药物利用度低,在临床应用中往往需要频繁多次给药才能维持疗效。另一方面,人IL-2通过结合人IL-2高亲和力受体IL-2Rαβγ(Kd:10-11)不可避免地激活了大量Treg细胞。这两方面共同导致不同程度的副作用发生。最严重的是毛细血管渗漏综合征(Vascular Leakage Syndrome,VLS),导致血管内液体聚集在肝肺等器官中,随后引发肺水肿和肝细胞损伤,使患者不得不中止治疗,极大地降低了病人治疗的依从性,也限制了相关疗法的进一步临床应用。
现有技术中科研人员常利用水溶性聚合物如聚乙二醇修饰连接药物,来延长药物的生理半衰期,降低药物的免疫原性和毒性(Nandini V,Proc.Natl.Acad.Sci,1987;常远,白细胞介素-2的PEG化,1996)。王立夫等人采用纯度为75%、分子量为5000的单甲氧基聚乙二醇活性酯对野生型rIL-2进行修饰,得到的随机修饰产品体外活性仅保留69.7%(王立夫等.rIL-2的聚乙二醇修饰物的制备及其体内外抗肝癌细胞作用,1997)。此外通过常规聚乙二醇直接连接药物的修饰方式来实现药物在体内的缓慢释放、改善药物生物利用度的效果并不理想。现有技术中也有报道通过连接子(linker)将水溶性聚合物和药物连接形成聚合物-药物结合物,水溶性聚合物从结合物上的脱落,可以达到药物活性缓释和控释的目的,药物在病灶(例如患癌)部位停留更长时间,可降低给药频率,减少患者用药不便。如专利文献CN200680029849.5公开了一种轭合物,其中包括含有可电离氢原子的芳香族部分如芴、间隔部分和水溶性聚合物,专利CN103517718A进一步公开了该聚合物与rIL-2形成的缀合物。该缀合物实际为美国Nektar公 司推出的一种CD122(IL-2Rβ)偏向型激动剂NKTR-214。该缀合物在IL-2(氨基酸序列与aldesleukin相同)上偶联了6个分子量为20K同时具有芴环结构的分枝结构PEG,在温和的碱性条件下经β-消除反应完成芴甲氧羰基的脱除,从而实现NKTR-214在生理条件下(pH=7.4,弱碱性)逐步释放蛋白表面的5个PEG,赋予NKTR-214偏向性结合IL-2Rβ受体增强T细胞活化的能力,同时在体内长效循环的功能。在NKTR-214分子制备过程中,通过优化PEG试剂和偶联反应促进在IL-2/IL-2Rα界面处聚集的赖氨酸残基(如K31,K34,K42,K47,K48,K75)特定位点的修饰;其中PEG位于IL-2/IL-2Rα相互作用的关键疏水性结合位点附近,达到降低与CD25(IL-2Rα)的结合,偏向激活CD122(IL-2Rβ)的目的(Charych D H,Hoch U,Langowski J L,et al.NKTR-214,an engineered cytokine with biased IL2 receptor binding,increased tumor exposure,and marked efficacy in mouse tumor models[J].Clinical Cancer Research An Official Journal of the American Association for Cancer Research,2016,22(3):680-690.)。Nektar公司和BMS曾就NKTR214达成36亿美元的合作,但其开展的两项III期临床研究PIVOT IO 001以及PIVOT-09于2022年相继宣告失败,临床治疗数据未达主要终点(2022EMSO,欧洲肿瘤内科学会年会,摘要编号785O、LBA68)。
本发明的发明人经过大量的试验和研究,在现有成熟聚乙二醇修饰剂中筛选到一种特定结构PEG修饰剂,该PEG可用于IL-2或其变体的修饰,使其获得优异的活性控释和缓释效果,在相关同类药物的生物利用度中具有突出性的优势,降低给药频率,大大提高药物的治疗效果和病人的依从性。同时本发明与在研的PEG-IL2类药物NKTR-214在最根本分子设计层面也具有差异,NKTR-214药物中的PEG分子在给药过后,在体内PEG分子的解离行为非常复杂,且不可控,因此理论上无法做到一直能够维持CD122偏向激动的特点,从而会影响药效的稳定发挥;发明人通过原蛋白序列的定点突变实现了CD122的偏向性与PEG修饰位点的可控性,从安全性、有效性、质量可控性三个方面,解决了NKTR-214药物在分子设计方面的潜在不足。并通过动物体内药代、药效等数据明确了这种差异化优势。
发明内容
为克服IL-2等生物活性分子药物利用率低以及其聚乙二醇修饰物活性普遍降低的问题,本发明首先提供了一种实现生物活性分子其活性控释和缓释的方法,发明人通过特定类型、结构的聚乙二醇与IL-2等生物活性分子反应形成结合物,在一定体外条件或体内生理条件下,获得了从结合物结构中PEG逐次脱落、生物活性分子的活性逐步释放的效果,能维持一定生 物活性分子较为稳定的有效血药浓度,实现了药物在体内外的活性缓释和控释,能达到较优药物生物利用度的动态平衡,具有降低给药频率、提高药物的生物利用度、患者的依从性更好和安全性更佳等临床应用前景。另一方面提供了一种新型的IL-2突变蛋白,该突变蛋白针对聚乙二醇修饰位点进行突变,使得PEG修饰的更可控;进一步地,该突变体针对IL-2与受体结合位点进行突变,相较于野生型IL-2活性更高。
本发明的一个目的是提供一种实现生物活性分子其活性控释和缓释的方法,所述方法是以聚乙二醇修饰剂对生物活性分子进行氨基修饰,所得修饰物中PEG逐步脱落后生物活性分子的活性被逐步释放,所述聚乙二醇修饰剂是聚乙二醇琥珀酰亚胺琥珀酸酯。
所述生物活性分子为蛋白质或多肽,优选为白介素,最优选为IL-2。
所述PEG修饰剂优选为直链聚乙二醇琥珀酰亚胺琥珀酸酯。
本发明的另一个目的是提供一种人IL-2的聚乙二醇修饰物,其所采用的PEG修饰剂是聚乙二醇琥珀酰亚胺琥珀酸酯,平均每个人IL-2上偶联4.5~8.5个PEG。
优选的,所述PEG修饰剂是直链聚乙二醇琥珀酰亚胺琥珀酸酯。
再优选的,所述PEG修饰剂分子量为5~20kDa。所述分子量为标示值,实际分子量可以是标示值的90%-110%。当PEG修饰剂分子量为5kDa时,平均每个人白介素-2上偶联5.5~7.5个个PEG;当PEG修饰剂分子量为10~20kDa时,平均每个人白介素-2上偶联6.5~8.5个PEG。
再优选的,所述PEG修饰剂结构如下所示:
其中,n为97~494的整数。n为97~494的整数时,该PEG修饰剂实际分子量约为4.5k-22k,对应的分子量标示值为5~20kDa。
再优选的,所述人IL-2的聚乙二醇修饰物结构如下所示:
其中,n为97~494的整数,m为4.5~8.5。
本发明的另一目的是提供一种人IL-2突变体,第一个方面,该突变体不含相邻的赖氨酸,经PEG修饰的修饰产物更均一。发明人研究发现SEQ ID NO:1所示的野生型人IL-2自N端起第8、9、48、49位均为赖氨酸,均有可能被PEG修饰,而相邻位点几乎不会同时被PEG 修饰。当饱和修饰时,IL-2第8或第9位被修饰、第48或第49位被修饰,就可能产生4种不同修饰位点异构体,造成人IL-2聚乙二醇修饰产物的不均一。据此发明人选择突变第8和/或第9位、第48和/或第49位氨基酸,大大减少PEG修饰位点异构体的产生,便于PEG修饰产物均一性控制。在本申请以前,本领域技术人员并未意识到IL-2序列上相邻氨基酸均为赖氨酸时对PEG修饰可能产生的不利影响,因此没有动机在聚乙二醇修饰IL-2时对相应位点进行突变。
本发明提供的人IL-2突变体,相对于野生型人IL-2,自N端起第8位或/和第9位氨基酸被取代;或者相对于野生型人IL-2,自N端起第48位或/和第49位氨基酸被取代;所述野生型人IL-2氨基酸序列如SEQ ID NO:1所示。
优选地,相对于野生型人IL-2,自N端起第8位或/和第9位氨基酸,以及第48位或/和第49位氨基酸被取代。
具体的实施例例举了第8位赖氨酸突变为精氨酸,第9位赖氨酸突变为精氨酸,第48位赖氨酸突变为精氨酸、色氨酸或酪氨酸,第49位赖氨酸突变为天冬氨酸。但实施例仅用作例举,而非限制本发明的范围。突变第8、9、48、49位氨基酸的目的在于解决野生型IL-2存在相邻赖氨酸,从而生成多种聚乙二醇修饰物异构体、影响PEG修饰产物均一性的问题。因此本领域技术人员知晓除实施例例举的氨基酸外,突变成其他非赖氨酸的氨基酸均能实现上述目的。
第二个方面,在上述不含相邻赖氨酸的基础上,进一步优化获得了体外活性显著优于野生型IL-2的人IL-2突变体。相对于野生型人IL-2,所述突变是以N端起第1、8、9、18、19、48、49、72和/或81位相应的1个、2个或多个位置上进行氨基酸取代;所述野生型人IL-2氨基酸序列如SEQ ID NO:1所示。
再优选地,在这些位置上的突变分别为选自如下的取代残基:1位:A1缺失;8位:K8R;9位:K9R;18位:L18M;19位:L19S;48位:K48W,R;49位:K49R;72位:L72F;81位:R81D;再优选突变体包含选自以下一个或多个突变:8位:K8R;48位:K48W;72位:L72F;和/或,81位:R81D;所述野生型人IL-2氨基酸序列如SEQ ID NO:1所示。
最优选的,上述突变选自下表的各个突变方案中的一种:

第三个方面,发明人也兼顾考虑了IL-2突变体减弱的IL-2Rα偏向性、保持或增强的IL-2Rβ、IL-2Rγ偏向性的需要,因此在野生型人IL-2自N端起第1、18、19、27、35、38、41、42、43、45、54、64、65、72、78、79、80、81、82、83、87、92和/或97位相应的1个、2个或多个位置上进行氨基酸取代,并进行了有效筛选,如G438P8。
IL-2通过IL-2受体(IL-2R)发生作用,IL-2受体包括三个亚基,IL-2Rα(CD25)、IL-2Rβ(CD122)、IL-2Rγ(CD132)。三个亚基形成三种受体形式:高结合力受体包含所有三个亚基IL-2Rα、IL-2Rβ、IL-2Rγ;中结合力受体包含IL-2Rβ、IL-2Rγ;低结合力受体为IL-2Rα。IL-2Rα在Treg细胞中高表达,IL-2Rβ在CD8+T细胞、NK细胞、Treg细胞中均有表达,IL-2Rγ在所有免疫细胞中均有表达。IL-2通过与不同细胞上的受体结合,在免疫反应中介导多重作用,一方面,作为免疫系统刺激剂,IL-2可以刺激T细胞增殖和分化,诱导细胞毒性T淋巴细胞生成,促进B细胞增殖分化和免疫球蛋白合成,并刺激NK细胞生成和活化,由此已被批准作为免疫治疗剂用于癌症和慢性病毒感染的治疗;另一方面,IL-2可以促进免疫抑制性CD4+CD25+调节性T细胞(即Treg细胞)的活化与增殖,从而导致免疫抑制。已有大量研究提出通过改变IL-2对IL-2Rα的偏向性,来降低IL-2用作免疫系统刺激剂时的毒副作用、提高其药效。已有较多通过位点突变改变IL-2受体倾向性的报道,本领域技术人员可以选择其他位点突变的IL-2进行聚乙二醇修饰,而不限于上述具体突变位点的IL-2突变体。
本发明的另一个目的是要提供一种人IL-2突变体的聚乙二醇修饰物,其所采用的PEG修饰剂是聚乙二醇琥珀酰亚胺琥珀酸酯,每个人IL-2上偶联5~8个PEG;其所述人IL-2突变体为上述不同突变体中的一种。
本发明的另一个目的是提供一种人IL-2或其突变体的聚乙二醇修饰物在制备治疗肿瘤疾病药物中的应用。所述肿瘤包括但不限于:鳞状细胞癌、黑色素瘤、结肠癌、乳腺癌、卵巢癌、前列腺癌、胃癌、肝癌、小细胞肺癌、非小细胞肺癌、甲状腺癌、肾癌、胆管癌、脑癌、宫颈癌、上颌窦癌、膀胱癌、食管癌、何杰金氏病以及肾上腺皮质癌。
本发明的另一个目的是提供一种治疗肿瘤疾病的组合物,所述组合物包括上述的人IL-2的聚乙二醇修饰物、人IL-2突变体、或者人IL-2突变体的聚乙二醇修饰物,还包括HER2抗 体、PD-1抗体、PD-L1抗体或CD26抗体等。
所述HER2抗体包括罗氏的Herceptin、Perjeta和Kadcyla。
所述PD-1抗体包括百时美施贵宝的Opidivo、默沙东的Keytruda、君实生物的特瑞普利单抗、信达生物的信迪利单抗、恒瑞医药的卡瑞利珠单抗、百济神州的替雷利珠单抗等。
所述PD-L1抗体包括罗氏的Tecentriq、阿斯利康的Imfinzi、默克的Bavencio等。
所述CD26抗体可以是如YS110(在先申请CN200680034937.4)、或在先申请CN202111245489.5的抗CD26抗体。
上述组合物治疗的肿瘤疾病包括但不限于:鳞状细胞癌、黑色素瘤、结肠癌、乳腺癌、卵巢癌、前列腺癌、胃癌、肝癌、小细胞肺癌、非小细胞肺癌、甲状腺癌、肾癌、胆管癌、脑癌、宫颈癌、上颌窦癌、膀胱癌、食管癌、何杰金氏病以及肾上腺皮质癌。
通过本发明的技术方案,主要实现了以下技术效果:
1、采用聚乙二醇琥珀酰亚胺琥珀酸酯与IL-2及其突变体中的赖氨酸侧链ε-氨基反应,聚乙二醇与IL-2之间以酰胺键相连,由于完全遮蔽了受体结合位点,因而修饰后的PEG-IL-2分子不具活性。同时,与其他修饰剂不同的是,聚乙二醇琥珀酰亚胺琥珀酸酯与蛋白或多肽药物结合后的偶联物中存在一个酯键,而酯键由于其固有的不稳定性,易因水解导致PEG链的脱落,这个性质在过去通常被视为修饰剂易脱落进而造成修饰分子不稳定的表现,因而该PEG在应用上逐步被其他难以脱落的PEG修饰剂(如聚乙二醇琥珀酰亚胺丙酸酯等)所替代,但本发明中,该水解性质使得IL-2具备了在体内逐步释放药物活性的性质,且在特定的分子量及特定修饰数下能获得极其优异的药效作用。
2、通过对IL-2中赖氨酸位点的突变,使其不含相邻赖氨酸,限定了PEG偶联的位置,大大减少PEG修饰位点异构体的产生,便于PEG修饰产物均一性控制。在此基础上,通过进一步的突变位点设计与筛选,获得了具有显著的α受体亲和力降低、β受体亲和力维持的受体结合偏向性的突变体,以及CTLL-2细胞增殖活性显著优于野生型IL-2的突变体。
3、在实际作用时,PEG化IL-2中的活性会随着PEG的逐步脱落不断缓慢释放,而本发明中通过对PEG不同分子量、不同修饰个数的优化和筛选,不仅避免了高修饰度IL-2的低生物利用度,也避免了低修饰度IL-2较高的毒副作用,使得本发明的PEG化IL-2有效实现了生物利用度和安全性的动态平衡。另一方面,也可以进一步通过不同的给药剂量、频率,获得稳定的血药浓度及更好的体内利用度,从而达到更优体内药效。
附图说明
图1.IL-2突变体刺激CTLL-2细胞增殖曲线图
图2.PEG-SS修饰不同突变体蛋白产物碱性条件活化产品刺激CTLL-2细胞增殖活性值
图3.不同结构、分子量PEG-SS修饰IL-2突变体修饰产物CTLL-2磷酸化STAT5水平测定曲线图
图4.PEG-SS修饰IL-2不同突变体修饰产物CTLL-2磷酸化STAT5水平测定曲线图
图5 mPEG-SS-5k-GP8高修饰与原蛋白GP8的体内药代对比
图6.不同结构PEG-SS(直链或分支)修饰IL-2在CT26.WT鼠源结肠癌细胞BALB/c小鼠皮下移植模型中的药效学评价结果。图6a显示了各组动物肿瘤体积增长曲线,图6b显示了各组动物瘤重,图6c显示了各组动物体重变化。
图7.直链PEG-SS修饰不同IL-2突变体在B16-F10鼠源黑色素瘤细胞C57BL/6小鼠皮下移植模型中的药效学评价结果。图7a显示了各组动物肿瘤体积增长曲线,图7b显示了各组动物瘤重。
图8不同分子量、修饰度的PEG-SS修饰IL-2突变体在B16-F10鼠源性黑色素瘤肿瘤模型中的药效评价。图8a显示了各组动物肿瘤生长曲线。图8b显示了细胞接种后第17天各组动物瘤重。图8c显示了动物体重增长率变化。
图9不同分子量、修饰度的PEG-SS修饰IL-2突变体在CT26.WT鼠源性结肠癌肿瘤模型中的药效评价。图9a各组动物肿瘤生长曲线。图9b细胞接种后第22天各组动物瘤重。图9c动物体重增长率变化。
图10不同分子量、修饰度的PEG-SS修饰IL-2突变体在A375人源性黑色素瘤模型中的药效评价。图10a动物肿瘤生长曲线。图10b细胞接种后第45天各组动物瘤重。图10c动物体重增长率。
图11不同剂量PEG修饰IL-2突变体在A375人黑色素瘤模型中的药效评价。图11a动物肿瘤生长曲线。图11b各组动物瘤重。图11c动物体重增长率。
图12不同剂量PEG修饰IL-2突变体在A498人肾癌模型中的药效评价。图12a动物肿瘤生长曲线。图12b各组动物瘤重。图12c动物体重增长率。
具体实施方式
定义:
白细胞介素-2:白介素-2,IL-2,可以源自重组或非重组方法获得,可以是野生型IL-2,或者突变体。IL-2可以在细菌(如大肠杆菌)、哺乳动物细胞(如CHO)、酵母(如毕赤酵母)中表达得到。IL-2可以源自人类、动物来源,优选地,IL-2源自人。在本申请具体的实施例中人IL-2氨基酸序列如SEQ ID NO:1所示,所述突变体是在其基础上对部分氨基酸进行替换、插入或删除等操作得到的突变体。在聚乙二醇修饰IL-2的具体实施例中,采用如SEQ ID NO:1所示的IL-2及其突变体,但本领域技术人员知晓具体的实施例仅用作说明而非限制本申请的范围,聚乙二醇修饰的IL-2还可以选择其他已报道的人IL-2序列,或者在其他已报道的人IL-2序列与SEQ ID NO:1相对应位置的氨基酸作相应突变(不含相邻赖氨酸的突变、减弱的IL-2Rα偏向性、增强的IL-2Rβ、IL-2Rγ偏向性的突变)获得的突变体。
聚乙二醇:PEG,通常经环氧乙烷聚合而成,有分支型,直链型和多臂型。一般情况下,分子量低于20,000的被称为PEG,分子量更大的被称为PEO。普通的聚乙二醇两端各有一个羟基,若一端以甲基封闭则得到甲氧基聚乙二醇(mPEG)。
聚乙二醇修饰剂:PEG修饰剂,指带有官能团的聚乙二醇衍生物,是经过活化的聚乙二醇,可用于蛋白质以及多肽药物修饰。本申请所用聚乙二醇修饰剂购自江苏众红生物工程创药研究院有限公司、北京键凯科技股份有限公司或厦门赛诺邦格生物科技股份有限公司。特定分子量的PEG修饰剂实际分子量可以是标示值的90%~110%,如PEG5K实际分子量可以是4.5kDa~5.5kDa,PEG20K实际分子量可以是18kDa~22kDa,PEG修饰剂标示分子量为5~20kDa时,其实际分子量为4.5~22kDa。
实施例所用V-PEG-SC-20k是指分子量20kDa为分支型聚乙二醇琥珀酰亚胺碳酸酯修饰剂,该PEG修饰剂是参照专利文献CN200680029849.5所制备的,根据专利文献显示该PEG修饰剂是通过连接子(linker)将PEG和药物反应形成结合物,PEG从结合物上的脱落,可以达到药物活性缓释和控释的目的,与NKTR-214报道的结构一致,因此申请人采用该PEG修饰剂对IL-2修饰作为阳性参照品。V-PEG-SC-20k修饰剂结构如下所示:
n为199-244的整数。
实施例所用V-PEG-SS-20k是指分子量为20kDa分支型聚乙二醇琥珀酰亚胺琥珀酸酯修饰剂;V-PEG-SS-20k修饰剂结构如下所示:
n为198-244的整数。
实施例所用mPEG-SS-20k/10k/5k是指直链型分子量分别为20kDa、10kDa、5kDa聚乙二醇琥珀酰亚胺琥珀酸酯修饰剂;
mPEG-SS-20k/10k/5k修饰剂结构如下所示:
mPEG-SS-20k:n为403-494的整数;
mPEG-SS-10k:n为199-244的整数;
mPEG-SS-5k:n为97-119的整数。
实施例所用mPEG-SPA-5k是指直链型分子量为5kDa聚乙二醇琥珀酰亚胺丙酸脂修饰剂;
mPEG-SPA-5k修饰剂结构如下所示:
n为98-120的整数。
实施例1:IL-2突变体设计与制备
1、IL-2突变体设计
下表中IL-2是野生型IL-2,其氨基酸序列如SEQ ID NO:1所示,其他突变体是在其基础进行氨基酸替换、插入或删除等操作得到的突变体,如G438的序列是将SEQ ID NO:1所示序列第8位、第48位氨基酸进行突变,K8R指将第8位赖氨酸突变为精氨酸。
表1突变位点信息表

2、蛋白制备
采用常规的重组蛋白制备方法,而不限于实施例例举的方法。以IL-2突变体G438的制备为例:
步骤一:根据G438的氨基酸序列(在如SEQ ID NO:1所述的野生型IL-2基础上进行K8R/K48W突变的序列),并针对大肠杆菌进行优化得到DNA序列如SEQ ID NO:2所示,将DNA序列克隆到pBV220载体形成重组质粒pBV220-G438,然后重组质粒转化Top10大肠杆菌宿主构成表达宿主Top10-pBV220-G438。
步骤二:Top10-pBV220-G438重组表达菌株接种到TB培养基(500ml培养基,装液量为20%),30℃、220rpm摇床培养至培养液OD600达到1.0±0.1,然后维持摇床转速不变,提升 摇床培养温度到42℃对菌株进行诱导表达,诱导表达4h。诱导表达结束后离心收集菌体沉淀。
步骤三:将表达后菌体沉淀使用10mM PBS,pH7.4重悬至100g/L,探针式超声波破碎仪超声破碎(工作功率250w,工作3s间歇4s,共计破碎30min),离心收集破碎产物,获得G438包涵体沉淀。
步骤四:将G438包涵体使用PBS+1%TritonX100重悬至50g/L,搅拌洗涤3次,每次2h以上,离心收集获得G438粗纯后包涵体。
步骤五:将G438粗纯后包涵体使用变性液(20mM Tris 8M脲5mM DTT,pH10.5)重悬至1g/100ml,搅拌变性2h以上,离心收集上清。上清使用superdex 75进行纯化。
步骤六:将纯化后的G438使用复性缓冲液稀释复性(复性液:20mM Tris 2M脲3mM半胱氨酸1mM胱氨酸0.01%SDS,pH8.0),复性体系中蛋白浓度不高于0.1mg/ml,复性时间36h以上,复性温度为15℃。
步骤七:将复性后的G438使用超滤法浓缩,浓缩后样品使用PBS,pH7.4透析除去脲等试剂作为G438纯品。
各突变体的氨基酸可以根据如SEQ ID NO:1所示的序列,结合根据表1所列突变方案得到。采用上述相似的方法,制备获得上述各设计方案的突变体。
实施例2:IL-2突变体的受体亲和力测定
一、实验方法
采用生物膜层干涉(BLI)技术检测IL-2突变体与其受体的亲和力。
1、样品配制
供试品溶液:分别取蛋白样品,用1×Kinetics buffer分别稀释至300mM,混匀,备用。
受体溶液:取受体IL-2Rα(CD25)、受体IL-2Rβ(CD122)、受体IL-2Rβ/γ样品用1×Kinetics buffer分别稀释至15-20μg/mL,混匀避光保存,备用。
2、样品添加
按方案设计添加样品孔中的样品,每孔加入200uL试剂或样品。
二、实验结果
运行程序并用Fortebio Data Analysis 8.0软件进行数据分析,计算亲和力结合值如下表所示:
表2.IL-2突变体与其受体IL-2Rα(CD25)、受体IL-2Rβ(CD122)的亲和力结合值

注:ND:None Detected,无法检测获得
表3.IL-2突变体与其受体IL-2Rβ/γ的亲和力结合值
三、结果分析
根据受体亲和力测定结果显示:突变体G493、G496、G498、G499、G500、G438P4、G438P5与两种受体结合均明显降低或不结合,可能是突变后造成蛋白结构的较大改变。G495、G438P6、G438P7、G438P8、G438P14、G438P15等变体呈现出IL-2β受体结合偏向性。G495、G438P7、G438P8、G438P14等突变对IL-2与IL-2Rβ/γ的结合影响不大,G438P15与IL-2Rβ/γ的结合显著下降。选取仍然具备体外与受体结合作用的突变体开展进一步的体外CTLL-2细胞增殖活性测试,以评估其生物学活性。
实施例3:IL-2及其突变体刺激CTLL-2细胞增殖活性测定
一、实验方法
CTLL-2为小鼠来源细胞株,可依据在不同浓度下检测其细胞依赖株CTLL-2细胞增殖速率,以评价IL-2及其突变体以及修饰物在体外的生物学活性。本实验方法为2020版中国药典四部通则3524《人白介素-2生物学活性测定法》(CTLL-2/MTT比色法)。
1.试液配制
RPMI 1640培养液:取RPMI 1640培养基粉末1袋(规格为IL)加水溶解并稀释至1000ml,再加碳酸氢钠2.1g溶解后,混匀,除菌过滤,4℃保存。
基础培养液:量取新生牛血清(FBS)10ml,加RPMI 1640培养液90ml。4℃保存。
完全培养液:吸取基础培养液100ml,加人IL-2至终浓度为每1ml含400-800IU/ml。4℃保存。
PBS:吸取10×PBS 100ml,加121℃、20分钟的灭菌水稀释至1000ml。
噻嗟蓝(MTT)溶液:称取MTT 0.1g,加PBS溶解并稀释至20ml,经0.22μm滤膜过滤除菌。4℃避光保存。
裂解液:15%十二烷基硫酸钠溶液,使用期限不得超过12个月。
2.样品制备
取已知蛋白含量的供试品溶液,样品稀释至合适的起始浓度。在96孔细胞培养板中,做2倍系列稀释,共8个稀释度,每个稀释度做2孔。每孔分别留50μl溶液,弃去孔中多余溶液。以上操作在无菌条件下进行。
3.细胞培养
CTLL-2细胞用完全培养液于37℃、5%二氧化碳条件下培养至足够量,离心收集CTLL-2细胞,用RPMI 1640培养液洗涤3次,然后重悬于基础培养液中配制成每1ml含6.0×105个细胞的细胞悬液,于37℃、5%二氧化碳条件下备用。在加有野生型样品和突变体样品的96孔细胞培养板中,每孔加入细胞悬液50μ1,于37℃、5%二氧化碳条件下培养18-24小时;然后每孔加入MTT溶液20μl,于37℃、5%二氧化碳条件下培养4-6小时后,每孔加入裂解液150μl,于37℃、5%二氧化碳条件下保温18-24小时。以上操作均在无菌条件下进行。混匀细胞板中的液体,放入酶标仪,以630nm为参比波长,在波长570nm处测定吸光度,记录测定结果。以样品浓度(ng/ml)为横坐标,OD570检测平均值为纵坐标,用ELISACalc软件进行四参数拟合作供试品反应曲线。
供试品比活(IU/mg)=供试品生物学活性(IU/ml)/20(ng/ml)×106
二、实验结果
相关检测结果如图1、及下表所示:
表3.IL-2突变体刺激CTLL-2细胞增殖活性值

三、结果分析
由突变体生物学活性测定结果可知:新构建的IL-2突变体G438P1、G438P8、G438P12、G438P20、G438P21、G438P22具有显著优于野生型IL-2的CTLL-2细胞增殖活性,具有临床应用的潜力。
以下为表述简化,在G438基础上进一步改造的突变体如:G438P1、G438P8、G438P12、G438P20、G438P21、G438P22分别简称为GP1、GP8、GP12、GP20、GP21、GP22,以此类推。
实施例4:PEG修饰IL-2的制备
实施例4a:阳性参照品V-PEG-SC-20k-rhIL-2的制备:
一、修饰方法
我们选用了专利文献CN200680029849.5公开的一种化合物V-PEG-SC-20k(购自厦门赛诺邦格)作为修饰剂,制备了同样具有缓释效果的PEG-IL-2作为阳性参照品。
将纯化好的野生型IL-2蛋白样品浓缩、置换修饰缓冲液(100mM磷酸氢二钠-磷酸二氢钠,pH8.0),浓度约为20mg/mL,按照蛋白:PEG修饰剂质量比1:20称取PEG,修饰反应在常温下进行,反应2小时后加入1M甘氨酸中止反应。
二、纯化方法
色谱法条件:流动相A为20mM PB+2M NaCl(pH6.0),流动相B为20mM PB(pH6.0)。
上样:上述修饰样品稀释后,以5ml/min上样,结合至疏水层析柱(购自GE公司,Phenyl PH)。
平衡:上样结束后,用A液冲洗15~20个柱体积。
洗脱:0-100%B液洗脱,洗脱体积为10个柱体积,收集主峰样品V-PEG-SC-20k-rhIL-2。
按上述条件对修饰样品进行纯化制备得到所需要样品。
三、纯度分析
对上述PEG化的IL-2及其变体修饰物SEC-HPLC检测结果见下表:
表4.PEG化的IL-2及其变体修饰物SEC-HPLC检测结果
SEC(尺寸排阻)色谱法是一种根据试样分子的尺寸进行分离的色谱技术。本实施例中制备的PEG-IL-2样品经SEC色谱检测,结果显示,样品主峰均一,即修饰程度均一,符合研究需求。
后续各实施例如无特别说明,阳性参考均采用本制备例的V-PEG-SC-20k-rhIL-2,或简写为PEG-SC-20k-rhIL-2。
实施例4b:本发明提供的其他PEG修饰样品制备:
一、修饰方法:
将纯化好的rhIL-2野生型/突变体蛋白样品浓缩、置换修饰缓冲液(100mM磷酸氢二钠-磷酸二氢钠,pH7.5),浓度约为2-15mg/mL,按照下表所示的修饰反应比例称取PEG(PEG类型包括但不仅限于:V-PEG-SS-20k、mPEG-SS-20k、m-PEG-SS-10k、mPEG-SS-5k、mPEG-SPA-5K),修饰反应在常温下进行,反应4小时后加入1M甘氨酸中止反应。不同修饰物的关键反应参数如下表所示:
表5.PEG化的IL-2及其变体修饰物的工艺参数

二、纯化方法
色谱法条件:流动相B为20mM NaAc+1M NaCl(pH4.0),流动相A为20mM NaAc(pH4.0)。
上样:上述修饰样品稀释后,以5mL/min上样,结合至阳离子交换层析柱(购自GE公司,HPSP)。
平衡:上样结束后,用A液冲洗15~20个柱体积。
洗脱:0-100%B液洗脱,洗脱体积为10个柱体积,收集主峰样品。
按上述条件对修饰样品进行纯化制备得到所需要样品。
三、纯度分析
各PEG化的IL-2及其变体修饰物SEC-HPLC检测结果见下表:
表6.PEG化的IL-2及其变体修饰物SEC-HPLC检测结果
SEC(尺寸排阻)色谱法是一种根据试样分子的尺寸进行分离的色谱技术。本实施例中制备的各PEG-IL-2样品经SEC色谱检测,结果显示,样品主峰均一,即修饰程度均一,符合研究需求。同时以GP8为例的同种PEG不同修饰度的PEG-IL-2样品具有显著保留时间差异,表明本发明已建立的样品制备工艺可以稳定的通过工艺参数的控制制备出不同修饰度的样品。具体样品的PEG结合数在实施例5中给出。
实施例5:PEG修饰IL-2的PEG结合数测定(水解法)
一、实验方法
1.试液配制
①PEG标准梯度溶液:分别取5μL、10μL、20μL、30μL、40μL、50μL的V-PEG-SC-20k、V-PEG-SS-20k、mPEG-SS-20k、mPEG-SS-10k、mPEG-SS-5k PEG溶液(2.5mg/mL)加入水中配制成终体积为100μL的梯度溶液,混匀,即得标准梯度溶液。分别加入25μL 5×非还原的Loding Buffer,混匀,备用。
②碘染液:准确称取BaCl2 17.5g、KI 6g、I2 3.9g溶于500mL的双蒸水中,避光保存。
③10%高氯酸溶液的配制:用量筒量取100mL的高氯酸慢慢加入到900mL水中,混匀,即得。
2.凝胶配制
10%的聚丙烯酰胺凝胶配制:分别吸取1.6mL双蒸水,1.8mL的30%聚丙烯酰胺溶液,1.3mL的1.5mol/L Tris-HCL pH8.8,0.53mL的1%SDS溶液,0.033mL的10%过硫酸铵溶液以及0.033mL的TEMED,混合均匀,制成分离胶。
分离胶凝固成型后,分别吸取2.9mL双蒸水,0.9mL的30%聚丙烯酰胺溶液,1.5mL的1.5mol/L Tris-HCL pH6.8,0.6mL的1%SDS溶液,0.047mL的10%过硫酸铵溶液以及0.047mL的TEMED,混合均匀制成浓缩胶。
3.样品制备
水解:取已知蛋白含量的供试品溶液100μL,加入200μL的100mM NaHCO3pH9.0的活化缓冲液于37℃水浴水解24h(其中mPEG-SPA-5k-rhIL-2不发生自发水解,样品经高温处理后,加入胰酶孵育)后,取样加入5×非还原Loding Buffer,混匀,备用。
4.电泳检测
运行电压:80V运行30min,待溴酚蓝指示剂移动至浓缩胶下方改为120V运行,直至溴酚蓝指示剂运行至分离胶底部边缘后结束。
碘液染色:电泳结束后,撬开玻璃板,将胶片做好标记后放在染色盒内,先用10%高氯酸溶液固定10min,然后将10%高氯酸回收,用水洗3遍后,再用碘染液覆盖胶片染色2~3min,1min左右就能显色,再立即用水脱色。
5.凝胶成像与数据处理:
将脱色清晰的凝胶置于凝胶成像仪内进行凝胶成像,使用Quantity One 4.4.0软件进行定 量功能处理。将标准品PEG含量及胶片斑点灰度进行线性回归得到样品中游离PEG的总量,结果按照下列公式进行计算:
二、实验结果
多种修饰产物的体外PEG结合数分析结果如下表所示。
表7.修饰产物的体外PEG结合数分析结果*
*随机修饰各个分子间的PEG连接情况略有不同,因此检测结果非整数值,本表已取整,实际检测结果略有浮动,在本表所述值的±0.5之间,如表7中显示mPEG-SS-5k-GP8-高修饰的PEG结合数是7,实际为6.5~7.5。
实施例6:PEG修饰IL-2体外缓释性能测定
一、实验方法
1、前处理方法参照实施例5,其中水解操作部分具体操作如下:取已知蛋白含量的供试品溶液100μL,加入200μL的100mM NaHCO3pH9.0活化缓冲液于37℃水浴水解。分别于不同时间点(如水解开始后8h、16、24h)从水解样品中分别取出100μL,分别稀释一定倍数后加入5×非还原Loding Buffer,混匀,备用。
2、样品检测及计算方法同实施例5
二、实验结果
多种修饰产物的PEG体外缓释性能分析结果如下表所示。
表8.多种不同修饰产物的体外PEG缓释性能分析*

*不同时间点各个分子间的PEG脱落情况略有不同,因此检测结果非整数值
三、结果分析
本实施例给出的是在体外37℃、100mM NaHCO3pH9.0活化缓冲液中的结果:以上多种PEG-SS不同结构(直链或支链)、不同分子量(5K、10K、20K)的PEG修饰的IL-2或其突变体均具有显著体外缓释性能,阳性参照品也具有理想的体外缓释性能。但其他非PEG-SS结构的常规PEG类型如SPA-PEG,在不引入额外基团(如阳性参照品的芴环结构)的条件下,无法实现PEG的脱落,因而无法达到可缓释的技术效果。不同分子量、不同修饰度的分子在体外显示出了不同的释放速率,但释放出的活性蛋白结构是否能进一步起到药效作用,仍需通过体内外活性进一步评价。
实施例7:PEG修饰IL-2刺激CTLL-2细胞增殖活性测定
一、实验方法
由于PEG修饰对蛋白活性中心的掩蔽效应,修饰后蛋白在体外生物学活性会有所降低,因此需要对样品进行预先活化处理后,可依据在不同浓度下检测其细胞依赖株CTLL-2细胞增殖速率,以评价修饰物在体外活化状态下的生物学活性。
1.样品制备
样品活化:取已知蛋白含量的供试品溶液100μL,分别加入200μl的100mM NaHCO3pH9.0 或300μL 100%人血清,于37℃水浴孵育一定时间,定时从水解样品中分别取出100μl混匀,备用。将样品稀释至合适的起始浓度。在96孔细胞培养板中,做2倍系列稀释,共8个稀释度,每个稀释度做2孔。每孔分别留50μl溶液,弃去孔中多余溶液。以上操作在无菌条件下进行。
2.试液配制、细胞培养同实施例3。
二、实验结果
1.不同聚乙二醇修饰物在100mM NaHCO3pH9.0条件下的活化产物刺激CTLL-2细胞增殖的活性结果如图2及下表所示(相对生物学活性以PEG修饰所用的原蛋白活性值为100%基准进行计算)。
表9-1
表9-2
三、结果分析
由表9可知,PEG-SS修饰物与参照品V-PEG-SC-20k-rhIL-2一致,在未经活化(活化0h)时,表面的受体结合位点被PEG完全遮蔽,不体现生物学活性,亦即PEG无脱落的情况下无生物学活性(无法检出)。在体外碱性条件下分别活化不同时间后均出现显著的促进CTLL-2细胞增殖的生物学活性。此外,PEG-SS修饰物具有随着活化时间延长,生物学活性逐步恢复并且提高的变化趋势。特定修饰度的mPEG-SS-20k-GP8和mPEG-SS-20k-GP12在取样时间点下的生物学活性均高于V-PEG-SC-20k-rhIL-2,在PEG脱落行为相似的情况下,通过调整修饰度可以具备更优的活性释放效果。
此外,不同分子量的PEG在不同修饰度的情况下,具备不同的活性释放效果,这也构成了使用PEG-SS分子修饰的IL-2具备可调控缓释的性能的基础,即我们可以通过调整不同分子量的PEG-SS以及控制不同的修饰PEG个数,探究PEG-IL2在体内的最优的释放曲线,使其达到效应分子在体内最优的生物利用度,从而获得药效优于现有技术的候选分子。
实施例8:不同结构PEG修饰IL-2对CTLL-2细胞pSTAT5活性测定
一、实验方法
CTLL-2细胞用完全培养液(RPMI1640培养基+2mM L-谷氨酰胺+1mM丙酮酸钠+10%胎牛血清+10%T-STIM,培养物补充有伴刀豆蛋白A)于37℃、5%CO2条件下培养至2×105个/mL的密度,用PBSA(PBS,pH7.2,1%BSA)洗涤1次,调整细胞密度至1×106个/mL,按每管500μL的体积分装至流式管中,加入不同浓度的用基础培养液(RPMI1640+2Mm L-谷氨酰胺+1mM丙酮酸钠+10%胎牛血清)调配的PEG修饰的IL-2,室温孵育20min后,立即加入多聚甲醛,至终浓度1.5%,涡旋混匀,室温孵育10min。加入1mL PBS,4℃、1400rpm离心5min,去除多聚甲醛。重悬细胞,加入1mL 4℃预冷的100%甲醇,涡旋混匀,4℃孵育20min。加入3mL PBSA缓冲液,4℃、1400rpm离心5min,洗涤细胞2次。加入Anti-Stat5(pY694)-Alexa647(BD,Cat#612599)室温避光孵育30min。加入3mL PBSA洗涤2次,BD AccuriTM C6上机检测。
二、实验结果
检测结果如图3所示,结果表明:体外活化pSTAT5水平检测结果显示:与V-PEG-SC-20k-rhIL-2和mPEG-SS-20k-GP8(本实施例中为上述mPEG-SS-20k-GP8-中修饰)相比,mPEG-SS-10k-GP8在血清中活化后激活磷酸化水平的增殖曲线同样存在显著差异,磷酸化水平上升相对更缓慢,呈现一种缓慢增加的趋势,到72h时三种样品激活磷酸化水平相当。 与体外PEG脱落释放活性蛋白趋势高度一致。
三、结果分析
IL-2无论和哪种细胞上的IL-2R结合,都是通过JAK-STAT途径发挥其生物学功能的。通过IL-2Rβ结合的JAK1途径和通过IL-2Rγ结合的JAK3途径分别导致IL-2R的β和γ亚基上的关键酪氨酸残基发生磷酸化,从而为其他信号分子产生锚定位点。因此可以通过检测CTLL-2细胞在不同浓度PEG修饰IL-2作用下的磷酸化水平变化来评价IL-2以及修饰物在体外的生物学活性。
因此,通过上述实验结果可以看出,PEG-SS在血清中释放速率直接影响pSTAT5激活和促CTLL-2增殖活性释放的快慢,该结构的PEG具备使IL-2突变体在体内缓释,进而调控免疫激活程度的能力。
实施例9:PEG修饰IL-2不同突变体对CTLL-2细胞pSTAT5活性测定
一、实验方法
同实施例8
二、实验结果
检测结果如图4显示,结果表明:体外活化pSTAT5水平检测结果显示,mPEG-SS-20k-GP1和mPEG-SS-20k-GP8(本实施例中为上述mPEG-SS-20k-GP8-中修饰)在缓冲液中活化后激活磷酸化的水平的趋势相近,呈现随时间推移逐步激活的特点,且达到峰值后能持续保持。
三、结果分析
通过上述实验结果可以看出,本发明中的PEG-SS修饰IL-2的不同突变体(以GP1、GP8为例)在体内具有趋势相近的磷酸化激活水平。
实施例10:PEG修饰IL-2突变体在体内的半衰期延长效果及修饰产物活性释放对比
一、实验方法
实验一:分别向SD大鼠体内静脉注射0.5mg/kg、0.1mg/kg和0.04mg/kg的mPEG-SS-5k-GP8高修饰以及1mg/kg的突变体GP8。分别于给药前,给药后0.25h,1h,2h,4h,8h,24h,48h,72h及96h采取血清,并分别采用两种ELISA检测方法检测血清中mPEG-SS-5k-GP8高修饰及突变体GP8的含量。
实验二:分别向SD大鼠体内静脉注射1mg/kg的mPEG-SS-5k-GP8高修饰及阳性参照品V-PEG-SC-20k-rhIL-2。分别于给药前,给药后0.25h,1h,2h,4h,8h,24h,48h,72h及96h采取血清,并采用CTLL-2细胞/MTT比色法方法测定各时间点的活性,单位为IU/ml。
二、试验结果
实验一:如图5所示,检测结果显示,相较突变体GP8原蛋白,mPEG-SS-5k-GP8高修饰可显著延长药物的半衰期。
实验二:根据检测结果绘制药时曲线,研究可见,mPEG-SS-5k-GP8高修饰在相同给药剂量及给药方式下,具有较阳性参照品V-PEG-SC-20k-rhIL-2更高的AUC(5683809h.IU/ml vs2344730h.IU/ml)(GraphPad Prism 7.00计算)。因此,mPEG-SS-5k-GP8高修饰相较阳性参照品V-PEG-SC-20k-rhIL-2具有更高的生物利用度。
实施例11 不同结构PEG-SS(直链或分支)修饰IL-2在CT26.WT鼠源结肠癌细胞BALB/c小鼠皮下移植模型中的药效学评价
一、实验方法
CT26.WT细胞培养在含10%胎牛血清的1640培养液中。收集指数生长期的CT26.WT细胞,PBS重悬至适合浓度。将5×105cells/0.1mL CT26.WT细胞悬液充分混匀后接种于BALB/c小鼠右侧背部皮下,每只接种0.1ml。待肿瘤体积均值达到100mm3左右,根据肿瘤体积分组,分组当天开始第一次给药(本次实验在肿瘤细胞接种后第9天、第16天分别给药一次),详细给药方案和给药途径见下表。
表10.动物分组、肿瘤细胞接种信息表

注:N:表示动物只数;s.c.:表示皮下注射给药;i.v.:表示静脉注射给药。细胞接种当天为D1。
一般临床观察:检疫期和试验期间每天至少观察一次,包括肿瘤生长及治疗对动物正常行为的影响,具体内容有动物(荷瘤)死亡或濒死,精神状态、行为活动及其它异常情况。
体重:每周检测2~3次。
肿瘤体积:每周检测2~3次,采用游标卡尺分别量取肿瘤长径与短径,肿瘤体积(mm3)=长径*短径2/2。相对肿瘤抑制率:TGI(%)=(1-T/C)×100%。公认地T表示给药组某一时间点的相对肿瘤体积(当次测量的肿瘤体积与分组时肿瘤体积比值),C表示模型组某一时间点的相对肿瘤体积(当次测量的肿瘤体积与分组时肿瘤体积比值)。而本申请实验接种当天根据体重分组,计算TGI时T、C分别表示给药组、模型组当次实际测量的肿瘤体积。
瘤重:末次检测结束后动物实施安乐死,剥离肿瘤块,生理盐水冲洗并用滤纸吸干水分,称量瘤块重量,并拍照。相对肿瘤抑制率TGI(%)=(1-TTW/CTW)×100%,TTW表示治疗组实验终结时平均瘤重,CTW表示模型组实验终结时平均瘤重。
二、实验结果
1、药效:
模型组动物在细胞接种后第20天平均肿瘤体积为1397.48±289.67mm3
mPEG-SS-20k-GP1(2mg/kg)、mPEG-SS-20k-GP8(2mg/kg)(本实施例中为上述mPEG-SS-20k-GP8-中修饰)在细胞接种后第20天平均肿瘤体积分别为282.31±103.02mm3、133.93±105.69mm3,相较模型组有显著性差异(P<0.01),相对肿瘤抑制率TGI(%)分别为79%、92%。
V-PEG-SS-20k-GP1(2mg/kg)、V-PEG-SS-20k-GP8(2mg/kg)在细胞接种后第20天平均肿瘤体积分别为942.44±209.66mm3、1037.67±332.97mm3,相较模型组无显著性差异,相对肿瘤抑制率TGI(%)分别为26%、29%。
阳性参照V-PEG-SC-20k-rhIL-2组(2mg/kg)(后续实施例如无特别说明,阳性参考均采用V-PEG-SC-20k-rhIL-2,或简写为PEG-SC-20k-rhIL-2)在细胞接种后第20天平均肿瘤体积为211.23±86.02mm3,相较模型组有显著性差异(P<0.01),相对肿瘤抑制率TGI(%)为84%。
瘤重分析结果与肿瘤体积分析结果基本吻合。具体实验结果见下表及图6:
表11.各组动物的肿瘤体积、相对肿瘤体积、TGI、T/C(Female,Mean±SEM)


注:*:P<0.05,**:P<0.01,vs模型组。
表12.各组动物的瘤重、TGI、T/C(Female,Mean±SEM)

注:*:P<0.05,**:P<0.01,vs模型组。
2、安全性:
PEG-SS修饰物组动物在给药期间体重降低均小于10%,且于实验后期体重有所恢复,与模型组相比差异不大(D13、D16、D18、D20)。
阳性参照组在第二次给药后第2天(D18)出现1/6只动物中度体重降低(10%<体重降低≤20%),且阳性参照组动物虽然于实验后期体重也有所恢复,但与模型组相比有显著性差异。
表13.各组动物体重增长率(Female,Mean±SEM)
注:*:P<0.05,**:P<0.01,vs模型组。
三、结果分析
直链PEG修饰产物mPEG-SS-20k-GP1、mPEG-SS-20k-GP8在2mg/kg的剂量下对CT26.WT鼠源结肠癌模型均具有显著抑制肿瘤生长的作用,抑制率与阳性参照相近或更高。分支PEG修饰产物V-PEG-SS-20k-GP1、V-PEG-SS-20k-GP8组在2mg/kg的剂量下对CT26.WT鼠源结肠癌模型模型无抑制肿瘤生长的作用。
本次药效评价期间,各组动物在2mg/kg的剂量下耐受良好。在发挥疗效的组别中,直链PEG-SS修饰产物及阳性参照品在给药中期均发现有体重下降情况,但第二次给药后及恢复期内,直链PEG-SS修饰产物动物状态及体重恢复情况显著优于阳性参照组。
本实施例中,令研究者惊讶的是,相同分子量下的直链型mPEG-SS-20K修饰产物PEG与分支型V-PEG-SS-20k修饰产物,在体内实验结果却展现出相当程度的差异,分支PEG-SS修饰产物的体内药效远低于直链PEG-SS修饰产物。因此,发明人确认到了选择直链型mPEG-SS开发IL-2类蛋白药物作为肿瘤免疫激动剂的优势。
实施例12 直链PEG-SS修饰不同IL-2突变体在B16-F10鼠源黑色素瘤细胞C57BL/6小鼠皮下移植模型中的药效学评价
一、实验方法
B16-F10细胞培养在含10%胎牛血清的DMEM培养液中。收集指数生长期的B16-F10细胞,PBS重悬至适合浓度。将5×105cells/0.1mL B16-F10细胞悬液充分混匀后接种于C57BL/6小鼠右侧背部皮下,每只接种0.1ml。待肿瘤体积均值达到100mm3左右,根据肿瘤体积分组,分组当天开始第一次给药(D7给药一次),详细给药方案和给药途径见下表。一般临床观察等同实施例11。
表14.动物分组、肿瘤细胞接种信息表

注:N:表示动物只数;s.c.:表示皮下注射给药;i.v.:表示静脉注射给药。细胞接种当天为D1。
二、实验结果
1、药效:
实验结果见下表及图7
表15.各组动物的肿瘤体积、相对肿瘤体积、TGI、T/C(Female,Mean±SEM)


注:显著差异*:P<0.05,**:P<0.01,vs模型组。
表16.各组动物的瘤重、TGI、T/C(Female,Mean±SEM)

注:*:P<0.05,**:P<0.01,vs模型组。
2、安全性:
V-PEG-SC-20k-rhIL-2组动物于D11时(D11时各组动物均存活)体重变化率与模型组相比有显著性降低。直链PEG-SS修饰的不同突变体的产物mPEG-SS-20k-GP1、mPEG-SS-20k-GP13、mPEG-SS-20k-GP21、mPEG-SS-20k-GP22各组动物于D11时体重下降与模型组相比无统计学差异,但与V-PEG-SC-20k-rhIL-2组相比有统计学差异。表明直链PEG-SS修饰的不同突变体的产物mPEG-SS-20k-GP1、mPEG-SS-20k-GP13、mPEG-SS-20k-GP21、mPEG-SS-20k-GP22对动物体重影响小于V-PEG-SC-20k-rhIL-2。
表.各组动物体重变化率(Female,Mean±SEM)

注:*:P<0.05,**:P<0.01,vs模型组。△△:P<0.01,:P<0.05vs V-PEG-SC-20k-rhIL-2组。
D11之后动物出现死亡(D11时各组动物均存活)。
三、结果分析
直链PEG-SS修饰的不同突变体的产物mPEG-SS-20k-GP1、mPEG-SS-20k-GP13、mPEG-SS-20k-GP21、mPEG-SS-20k-GP22在1mg/kg的剂量下对B16-F10鼠源黑色素瘤模型均有抑制肿瘤生长的作用,抑制率与阳性参照近似,且上述供试品对动物体重影响小于V-PEG-SC-20k-rhIL-2。
实施例13:直链PEG-SS修饰IL-2突变体的不同修饰度产物在B16-F10鼠源性黑色素瘤肿瘤模型中的药效评价
一、实验方法
实验方法、一般临床观察等同实施例11,详细给药方案和给药途径见下。
表17.动物分组、肿瘤细胞接种信息表

注:N:表示动物只数;s.c.:表示皮下注射给药;i.v.:表示静脉注射给药。细胞接种当天为D1。
二、实验结果
1、药效:
模型组动物在细胞接种后第17天平均肿瘤体积为2614.97±372.77mm3
mPEG-SS-20k-GP8(高修饰度)组、mPEG-SS-20k-GP8(中修饰度)组、mPEG-SS-5k-GP8(高修饰度)组、mPEG-SS-5k-GP8(中修饰度)组、mPEG-SS-5k-GP8(低修饰度)组在细胞接种后第17天平均肿瘤体积分别为550.25±100.23mm3、544.90±89.12mm3、574.02±108.15mm3、676.17±128.23mm3、570.45±107.25mm3,相较模型组有显著性差异(P<0.01或P<0.05),相对肿瘤抑制率TGI(%)分别为77%、80%、79%、75%、79%。
PEG-SC-20k-rhIL-2组在细胞接种后第17天平均肿瘤体积为1215.19±184.70mm3,相较 模型组有显著差异(P<0.05),相对肿瘤抑制率TGI(%)为57%。
瘤重分析结果与肿瘤体积分析结果基本吻合。具体实验结果见下表及图8:
表18.各组动物的肿瘤体积、相对肿瘤体积、TGI、T/C(Female,Mean±SEM)

注:*:P<0.05,**:P<0.01,vs模型组。
表19.各组动物的瘤重、TGI、T/C(Female,Mean±SEM)

注:*:P<0.05,**:P<0.01,vs模型组。
2、安全性:
mPEG-SS-20k-GP8(高修饰度)组在首次给药后第4天(细胞接种后第11天)出现1/6只动物中度体重降低(10%<体重降低≤20%);在首次给药后第7天(细胞接种后第14天)出现2/6只动物中度体重降低(10%<体重降低≤20%),出现3/6只动物死亡;在首次给药第9 天(细胞接种后第16天)仍有1/6只动物中度体重降低(10%<体重降低≤20%),出现1/6只动物死亡。至实验结束,剩余2/6只动物体重有恢复趋势。该组动物出现半数以上死亡,推测动物死亡与供试品有关。
mPEG-SS-20k-GP8(中修饰度)组在首次给药后第4天(细胞接种后第11天)出现2/6只动物中度体重降低(10%<体重降低≤20%);在首次给药后第7天(细胞接种后第14天)出现4/6只动物中度体重降低(10%<体重降低≤20%),2/6只动物重度体重降低(体重降低>20%);在首次给药后第9天(细胞接种后第16天)仍有1/6只动物中度体重降低(10%<体重降低≤20%),1/6只动物重度体重降低(体重降低>20%),出现1/6只动物死亡。至实验结束,剩余5/6只动物,其中4/6只动物体重有恢复趋势,1/6只动物体重未恢复。该组动物体重出现中度乃至重度降低,以及1/6只动物死亡,推测上述情况与供试品有关。
mPEG-SS-5k-GP8(高修饰度)组动物在首次给药后第4天(细胞接种后第11天)出现1/6只动物中度体重降低(10%<体重降低≤20%);在首次给药后第7天(细胞接种后第14天)出现1/6只动物中度体重体重(10%<体重降低≤20%)。至实验结束动物体重有恢复趋势。
mPEG-SS-5k-GP8(中修饰度)组动物在给药治疗期间体重无明显变化。
mPEG-SS-5k-GP8(低修饰度)组动物在首次给药后第4天(细胞接种后第11天)出现1/6只动物中度体重降低(10%<体重降低≤20%),至实验结束有恢复趋势。
PEG-SC-20k-rhIL-2组在首次给药后第4天(细胞接种后第11天)出现1/6只动物中度体重降低(10%<体重降低≤20%),至实验结束有恢复趋势;在首次给药后第7天(细胞接种后第14天)死亡3/6只动物。该组动物出现半数死亡,推测动物死亡与供试品有关。
模型组在首次给药后第7天(细胞接种后第14天)出现1/6只动物中度体重降低(10%<体重降低≤20%),至实验结束有恢复趋势。具体结果见下表及图8c。
表20.各组动物体重增长率(Female,Mean±SEM)


注:*:P<0.05,**:P<0.01,vs模型组。
三、结果分析
供试品mPEG-SS-20k-GP8(高修饰度)、mPEG-SS-20k-GP8(中修饰度)、mPEG-SS-5k-GP8(高修饰度)、mPEG-SS-5k-GP8(中修饰度)、mPEG-SS-5k-GP8(低修饰度),在2mg/kg的剂量下对B16-F10鼠源黑色素瘤模型均有抑制肿瘤生长的作用,优于V-PEG-SC-20k-rhIL-2。
治疗期间,mPEG-SS-20k-GP8(高修饰度)、mPEG-SS-20k-GP8(中修饰度)组动物在2mg/kg的剂量下耐受不佳,mPEG-SS-5k-GP8(高修饰度)组、mPEG-SS-5k-GP8(低修饰度)组动物在2mg/kg的剂量下基本耐受。mPEG-SS-5k-GP8(中修饰度)组动物在2mg/kg的剂量下耐受良好。
本实施例中初步证明了不同修饰度产物具有不同的体内药效及安全性,与实施例7中我们根据体外实验猜测的“不同修饰度产物具备不同的性能”结果一致,因此发明人进一步开展了其他不同模型的药效比较工作。
实施例14:直链PEG-SS修饰IL-2突变体的不同修饰度产物在CT26.WT鼠源性结肠癌肿瘤模型中的药效评价
一、实验方法
实验方法、一般临床观察等同实施例11,详细给药方案和给药途径见下表。
表21.动物分组、肿瘤细胞接种信息表

注:N:表示动物只数;s.c.:表示皮下注射给药;i.v.:表示静脉注射给药。细胞接种当天为D1。
二、实验结果
1、药效:
模型组动物在细胞接种后第22天平均肿瘤体积为1,886.67±341.33mm3
mPEG-SS-20k-GP8(高修饰度)组、mPEG-SS-20k-GP8(中修饰度)组、mPEG-SS-5k-GP8(高修饰度)组、mPEG-SS-5k-GP8(中修饰度)组、mPEG-SS-5k-GP8(低修饰度)组在细胞接种后第22天平均肿瘤体积分别为268.15±163.54mm3、132.07±132.07mm3、56.90±36.21mm3、429.12±261.88mm3、87.78±87.78mm3,相较模型组组有显著性差异(P<0.01),相对肿瘤抑制率TGI(%)分别为88%、90%、97%、80%、95%。
PEG-SC-20k-rhIL-2组在细胞接种后第22天平均肿瘤体积为163.13±73.03mm3,相较模型组有显著性差异(P<0.01),相对肿瘤抑制率TGI(%)为92%。
瘤重分析结果与肿瘤体积分析结果基本吻合。具体实验结果见下表及图9:
表22.各组动物的肿瘤体积、相对肿瘤体积、TGI、T/C(Female,Mean±SEM)

注:*:P<0.05,**:P<0.01,vs模型组。
表23.各组动物的瘤重、TGI、T/C(Female,Mean±SEM)


注:*:P<0.05,**:P<0.01,vs模型组。
2、安全性:
mPEG-SS-20k-GP8(高修饰度)在首次给药后第5天出现3/6只动物中度体重降低(10%<体重降低≤20%),1/6只动物死亡。在第二次给药后第2天出现2/6只动物中度体重降低(10%<体重降低≤20%),至实验结束有恢复趋势。
mPEG-SS-20k-GP8(中修饰度)在首次给药后第5天出现3/6只动物中度体重降低(10%<体重降低≤20%),1/6只动物死亡;在首次给药后第7天出现1/6只动物中度体重降低(10%<体重降低≤20%);在第二次给药后第2天出现1/6只动物中度体重降低(10%<体重降低≤20%),至实验结束未恢复。
mPEG-SS-5k-GP8(高修饰度)在首次给药后第5天出现1/6只动物中度体重降低(10%<体重降低≤20%)。在第二次给药后第2天出现1/6只动物中度体重降低(10%<体重降低≤20%),至实验结束有恢复趋势。
mPEG-SS-5k-GP8(中修饰度)在给药期间动物体重未见降低或体重降低小于10%,体重下降的动物于实验后期有所恢复。
mPEG-SS-5k-GP8(低修饰度)在第二次给药后第2天出现1/6只动物中度体重降低(10%<体重降低≤20%),至实验结束有恢复趋势。
PEG-SC-20k-rhIL-2组在首次给药后第5天出现4/6只动物中度体重降低(10%<体重降低≤20%);在第二次给药后第2天有2/6只动物中度体重降低(10%<体重降低≤20%);在第二次给药后第7天仍有1/6只动物中度体重降低(10%<体重降低≤20%),其余动物至实验结束体重有恢复趋势。
表24.各组动物体重增长率(Female,Mean±SEM)

注:*:P<0.05,**:P<0.01,vs模型组。
三、结果分析
供试品mPEG-SS-20k-GP8(高修饰度)、mPEG-SS-20k-GP8(中修饰度)、mPEG-SS-5k-GP8(高修饰度)、mPEG-SS-5k-GP8(中修饰度)、mPEG-SS-5k-GP8(低修饰度),在2mg/kg的剂量下对CT26.WT鼠源结肠癌模型有抑制肿瘤生长的作用。
治疗期间mPEG-SS-5k-GP8(高修饰度)、mPEG-SS-5k-GP8(中修饰度)、mPEG-SS-5k-GP8(低修饰度)组动物在2mg/kg的剂量下耐受良好。mPEG-SS-20k-GP8(高修饰度)、mPEG-SS-20k-GP8(中修饰度)组动物在2mg/kg的剂量下基本耐受,体重恢复情况优于V-PEG-SC-20k-rhIL-2。
实施例15:直链PEG-SS修饰IL-2突变体的不同修饰度产物在A375人源性黑色素瘤肿瘤模型中的药效评价
一、实验方法
A375细胞培养在含10%胎牛血清的DMEM培养液中,收集指数生长期的A375细胞,加入PBS重悬;hu-PBMC(人外周血单个核细胞)培养在含10%胎牛血清的1640培养液中,收集由OKT-3与IL-2药物刺激后第三天的PBMC,加入PBS重悬;5×105cells/0.1mL A375细胞与5×105cells/0.1mL PBMC细胞悬液按照1:1的比例(细胞接种量)充分混合后用于NOD/SCID小鼠皮下接种,每只接种0.2mL。细胞接种后当天,根据体重分组,分组当天开始第一次给药,详细给药方案和给药途径见下表。
表25.动物分组、肿瘤细胞接种信息表

注:N:表示动物只数;s.c.:表示皮下注射给药;i.v.:表示静脉注射给药。细胞接种当天为D1。
一般临床观察、体重、肿瘤体积、瘤重检测同实施例11。
二、实验结果
1、药效:
模型组动物在细胞接种后第45天平均肿瘤体积为1,970.97±298.65mm3
mPEG-SS-20k-GP8(高修饰度)组、mPEG-SS-20k-GP8(中修饰度)组、mPEG-SS-5k-GP8(中修饰度)组动物在细胞接种后第45天未见肿瘤生长,与模型组相比均有显著性差异(P<0.01)。PEG-SC-20k-rhIL-2组、mPEG-SS-5k-GP8(高修饰度)、mPEG-SS-5k-GP8(低修饰度)平均肿瘤体积分别为136.59±134.84mm3、83.34±83.34mm3、96.62±64.92mm3,与模型组相比均有显著性差异(P<0.01)。
瘤重分析结果与肿瘤体积接近,且mPEG-SS-20k-GP8(高修饰度)组、mPEG-SS-20k-GP8(中修饰度)组、mPEG-SS-5k-GP8(高修饰度)组、mPEG-SS-5k-GP8(中修饰度)组、mPEG-SS-5k-GP8(低修饰度)组、PEG-SC-20k-rhIL-2组根据瘤重计算TGI分别为100%、100%、95%、100%、95%、91%。
具体实验结果见下表及图10:
表26细胞接种后第45天的动物肿瘤体积(Female,Mean±SEM)

注:注:**:P<0.01,vs模型组;*:P<0.05,vs模型组。
表27细胞接种后第45天的动物瘤重(Female,Mean±SEM)

注:**:P<0.01,vs模型组;*:P<0.05,vs模型组。
表28动物体重增长率(Female,Mean±SEM)

注:**:P<0.01,vs模型组;*:P<0.05,vs模型组。
表29动物体重增长率(Female,Mean±SEM)
注:**:P<0.01,vs模型组;*:P<0.05,vs模型组。
2、安全性:
mPEG-SS-20k-GP8(高修饰度)在第四次给药后第3天出现1/6只动物中度体重降低(10%<体重降低≤20%),该动物在第四次给药后第6天体重保持中度体重降低(10%<体重降低≤20%),该动物于末次给药后1天死亡。另末次给药后第9天至末次给药后第13天出现1/6只动物中度体重降低(10%<体重降低≤20%),至实验结束,该动物体重有恢复趋势。
mPEG-SS-20k-GP8(中修饰度)在第二次给药后第3天出现1/6只动物重度体重降低(体重降低>20%),于第二次给药后第6天死亡;在末次给药后第3天至实验结束出现1/6只动物中度体重降低(10%<体重降低≤20%);另末次给药后第4天出现1/6只动物死亡。
mPEG-SS-5k-GP8(中修饰度)末次给药后第6天至末次给药后第9天出现1/6只动物中度体重降低(10%<体重降低≤20%),至实验结束,该动物体重有恢复趋势。
mPEG-SS-5k-GP8(高修饰度)、mPEG-SS-5k-GP8(低修饰度)在给药期间动物体重未见降低或体重降低小于10%,体重下降的动物于实验后期有所恢复。
PEG-SC-20k-rhIL-2组在末次给药后第6天出现1/6只动物中度体重降低(10%<体重降低≤20%);末次给药后第9天出现2/6只动物中度体重降低(10%<体重降低≤20%);末次给药后第13天出现2/6只动物中度体重降低(10%<体重降低≤20%),1/6只动物死亡。
三、结果分析
供试品mPEG-SS-20k-GP8(高修饰度)、mPEG-SS-20k-GP8(中修饰度)、mPEG-SS-5k-GP8(高修饰度)、mPEG-SS-5k-GP8(中修饰度)、mPEG-SS-5k-GP8(低修饰度),在0.5mg/kg(1次/周×5周)的剂量下对A375人黑色素瘤模型均有抑制肿瘤生长的作用。从药效角度,供试品mPEG-SS-20k-GP8(高修饰度)、mPEG-SS-20k-GP8(中修饰度)、mPEG-SS-5k-GP8(高修饰度)、mPEG-SS-5k-GP8(中修饰度)、mPEG-SS-5k-GP8(低修饰度)作用略优于PEG-SC-20k-rhIL-2。
治疗期间,mPEG-SS-20k-GP8(高修饰度)、mPEG-SS-20k-GP8(中修饰度)组、V-PEG-SC-20k-rhIL-2动物在0.5mg/kg的剂量下耐受不佳;mPEG-SS-5k-GP8(高修饰度)、mPEG-SS-5k-GP8(低修饰度)组动物在0.5mg/kg(1次/周×5周)的剂量下耐受良好;mPEG-SS-5k-GP8(中修饰度)组动物在0.5mg/kg(1次/周×5周)的剂量下基本耐受。从安全性角度(体重、死亡),供试品mPEG-SS-5k-GP8(高修饰度)、mPEG-SS-5k-GP8(中修饰度)、mPEG-SS-5k-GP8(低修饰度)耐受性优于PEG-SC-20k-rhIL-2。
实施例16:PEG修饰IL-2突变体在A375人黑色素瘤模型中的药效评价
一、实验方法
A375细胞培养在含10%胎牛血清的DMEM培养液中,收集指数生长期的A375细胞,加入PBS重悬;hu-PBMC(人外周血单个核细胞)培养在含10%胎牛血清的1640培养液中,收集由OKT-3与IL-2药物刺激后第三天的PBMC,加入PBS重悬;1×106cells/0.1mL A375细胞与1×106cells/0.1mL PBMC细胞悬液按照1:1的比例(细胞接种量)充分混合后用于NOD/SCID小鼠皮下接种,每只接种0.2mL。细胞接种后当天,根据体重分组,分组当天开始第一次给药,详细给药方案和给药途径见下表。
表30.动物分组、肿瘤细胞接种信息表

注:N:表示动物只数;i.v.:表示静脉注射给药。细胞接种当天为D1。按IL-2活性单位计算,阳性对照组(泉
奇(注射用重组人白介素-2(125Ser)))给药总量为mPEG-SS-5K-GP8-高修饰高剂量组给药总量的1.4倍。
一般临床观察、体重、肿瘤体积、瘤重检测同实施例11。
二、实验结果
1、药效:
阴性对照组动物在细胞接种后第45天平均肿瘤体积为1,197.64±143.79mm3
mPEG-SS-5K-GP8-高修饰高、中、低剂量组(250μg/kg、125μg/kg、62.5μg/kg)动物在细胞接种后第45天平均肿瘤体积分别为144.87±35.11mm3、296.04±61.10mm3、643.88±153.05mm3,与阴性对照组相比均有显著性差异(P<0.01或P<0.05)。
阳性对照组(泉奇,100万IU/kg)动物在细胞接种后第45天平均肿瘤体积为557.54±104.82mm3,与阴性对照组相比有显著性差异(P<0.01)。mPEG-SS-5K-GP8-高修饰高剂量组与阳性对照组(泉奇)相比,肿瘤体积有显著性差异(P<0.01)。结果表明阳性对照组(泉奇)给药总量高于mPEG-SS-5K-GP8-高修饰高剂量组,但mPEG-SS-5K-GP8-高修饰药效更优。
阳性参照药(V-PEG-SC-20k-rhIL-2,仿NKTR214)组动物在细胞接种后第45天平均肿瘤体积为224.37±33.28mm3,与阴性对照组相比有显著性差异(P<0.01)。mPEG-SS-5K-GP8-高修饰中剂量组与阳性参照组相比,肿瘤体积无显著性差异(P>0.05)。结果表明等剂量 mPEG-SS-5K-GP8-高修饰药效与阳性参照药(V-PEG-SC-20k-rhIL-2,仿NKTR214)相近。
瘤重分析结果与肿瘤体积分析结果基本吻合,mPEG-SS-5K-GP8-高修饰高、中、低剂量组根据瘤重计算TGI分别为87%、75%、46%。阳性对照组(泉奇)、阳性参照组(V-PEG-SC-20k-rhIL-2,仿NKTR214)根据瘤重计算TGI分别为56%、81%。
具体实验结果见下表及图11:
表31细胞接种后第45天的动物肿瘤体积(Female,Mean±SEM)

注:**:P<0.01,vs阴性对照组;*:P<0.05,vs阴性对照组。##:P<0.01,vs阳性对照组(泉奇)。按IL-2
活性单位计算,阳性对照组(泉奇)给药总量为mPEG-SS-5K-GP8-高修饰高剂量组给药总量的1.4倍。
表32细胞接种后第46天的动物瘤重(Female,Mean±SEM)

注:**:P<0.01,vs阴性对照组;*:P<0.05,vs阴性对照组。##:P<0.01,vs阳性对照组(泉奇)。按IL-2
活性单位计算,阳性对照组(泉奇)给药总量为mPEG-SS-5K-GP8-高修饰高剂量组给药总量的1.4倍。
2、安全性:
mPEG-SS-5K-GP8-高修饰高剂量组(250μg/kg)动物在静脉注射给药治疗期间有3/8只动物出现中度体重降低情况,动物对治疗基本耐受。mPEG-SS-5K-GP8-高修饰中、低剂量组(125μg/kg、62.5μg/kg)动物在静脉注射给药治疗期间没有表现明显的药物毒性,治疗期间耐受良好。
阳性对照组(泉奇,100万IU/kg)动物在静脉注射给药治疗期间没有表现明显的药物毒性,治疗期间耐受良好。在末次给药后第3天(D32)、末次给药后第16天(D45),与阳性对照组(泉奇,100万IU/kg)相比,mPEG-SS-5K-GP8-高修饰高剂量组(250μg/kg)体重无 统计学差异(P>0.05)。
V-PEG-SC-20k-rhIL-2组(125μg/kg)动物在静脉注射给药治疗期间有3/8只动物出现中度体重降低,1/8只动物出现中度至重度体重降低情况,动物对治疗基本耐受。在末次给药后第3天(D32)、末次给药后第16天(D45),与阳性参照组(V-PEG-SC-20k-rhIL-2,125μg/kg)相比,mPEG-SS-5K-GP8-高修饰中剂量组(125μg/kg)体重有统计学差异(P<0.01或P<0.05),结果表明等剂量的mPEG-SS-5K-GP8-高修饰安全性优于阳性参照组(仿NKTR214)。
表33各组动物体重变化表(Female,Mean±SEM)

注:**:P<0.01,vs阴性对照组;*:P<0.05,vs阴性对照组。△△:P<0.01,vs阳性参照组(仿NKTR214)。
表34各组动物体重变化表(Female,Mean±SEM)

注:**:P<0.01,vs阴性对照组;*:P<0.05,vs阴性对照组。:P<0.05,vs阳性参照组(仿NKTR214)。
三、结果分析
mPEG-SS-5K-GP8-高修饰在高、中剂量(250μg/kg、125μg/kg)下对A375人黑色素瘤模型均有显著抑制肿瘤生长的作用,mPEG-SS-5K-GP8-高修饰在低剂量(62.5μg/kg)下有肿瘤抑制趋势,mPEG-SS-5K-GP8-高修饰高、中、低剂量能体现出良好的量效关系。阳性参照组(V-PEG-SC-20k-rhIL-2,仿NKTR214)在125μg/kg剂量下对A375人黑色素瘤模型有显著抑制肿瘤生长的作用,阳性对照组(泉奇)在100万IU/kg剂量下对A375人黑色素瘤模型 有肿瘤抑制趋势。
mPEG-SS-5K-GP8-高修饰高剂量组(250μg/kg)、V-PEG-SC-20k-rhIL-2组(125μg/kg)动物对治疗基本耐受,mPEG-SS-5K-GP8-高修饰中、低剂量组(125μg/kg、62.5μg/kg)、阳性对照组(泉奇,100万IU/kg)动物对治疗耐受良好。各给药组动物未见死亡。
表明在相近抑瘤效果下,相较于V-PEG-SC-20k-rhIL-2,动物对mPEG-SS-5K-GP8-高修饰有更好的耐受性。在相近安全性下,阳性对照组(泉奇)给药总量高于mPEG-SS-5K-GP8-高修饰高剂量组,但mPEG-SS-5K-GP8-高修饰药效更优。
实施例17:PEG修饰IL-2突变体在A498人肾癌模型中的药效评价
一、实验方法
A498细胞培养在含10%胎牛血清的DMEM培养液中,收集指数生长期的A498细胞,加入PBS重悬;hu-PBMC(人外周血单个核细胞)培养在含10%胎牛血清的1640培养液中,收集由OKT-3与IL-2药物刺激后第三天的PBMC,加入PBS重悬;5×106cells/0.1mL A498细胞与5×106cells/0.1mL PBMC细胞悬液按照1:1的比例(细胞接种量)充分混合后用于NOD/SCID小鼠皮下接种,每只接种0.2mL。细胞接种后当天,根据体重分组,分组当天开始第一次给药,详细给药方案和给药途径见下表。
表35.动物分组、肿瘤细胞接种信息表

注:N:表示动物只数;i.v.:表示静脉注射给药。细胞接种当天为D1。按IL-2活性单位计算,阳性对照组(泉
奇)给药总量是mPEG-SS-5K-GP8-高修饰中剂量组的2倍。
一般临床观察、体重、肿瘤体积、瘤重检测同实施例11。
二、实验结果
1、药效:
阴性对照组动物在细胞接种后第61天平均肿瘤体积为1,960.68±398.28mm3
mPEG-SS-5K-GP8-高修饰中、低剂量组(125μg/kg、62.5μg/kg)动物在细胞接种后第61天平均肿瘤体积分别为18.99±18.99mm3、248.19±209.86mm3,与阴性对照组相比均有显著性差异(P<0.01)。
阳性对照组(泉奇,71.25万IU/kg)动物在细胞接种后第61天平均肿瘤体积为 290.85±145.96mm3,与阴性对照组相比有显著性差异(P<0.01)。结果表明仅有泉奇1/2剂量的mPEG-SS-5K-GP8-高修饰中剂量组的药效优于阳性对照组(泉奇)。
V-PEG-SC-20k-rhIL-2组(125μg/kg)动物在细胞接种后第61天平均肿瘤体积为110.22±63.15mm3,与阴性对照组相比有显著性差异(P<0.01)。mPEG-SS-5K-GP8-高修饰中剂量组与阳性参照组相比,肿瘤体积无显著性差异(P>0.05)。结果表明等剂量mPEG-SS-5K-GP8-高修饰药效较阳性参照药(V-PEG-SC-20k-rhIL-2,仿NKTR214)更优。
瘤重分析结果与肿瘤体积分析结果基本吻合,mPEG-SS-5K-GP8中、低剂量组根据瘤重计算TGI分别为99%、85%。阳性对照组(泉奇)、阳性参照组(V-PEG-SC-20k-rhIL-2,仿NKTR214)根据瘤重计算TGI分别为83%、93%。
具体实验结果见下表及图12:
表36细胞接种后第61天的动物肿瘤体积(Female,Mean±SEM)

注:**:P<0.01,vs阴性对照组;*:P<0.05,vs阴性对照组。
表37细胞接种后第61天的动物瘤重(Female,Mean±SEM)

注:**:P<0.01,vs阴性对照组;*:P<0.05,vs阴性对照组。
2、安全性:
mPEG-SS-5K-GP8-中剂量组(125μg/kg)在静脉注射给药治疗期间有1/8只动物出现1次中度体重降低情况,动物对治疗基本耐受。mPEG-SS-5K-GP8-高修饰低剂量组(62.5μg/kg)动物在静脉注射给药治疗期间没有表现明显的药物毒性,治疗期间耐受良好。
阳性对照组(泉奇,71.25万IU/kg)动物在静脉注射给药治疗期间没有表现明显的药物毒性,治疗期间耐受良好。
V-PEG-SC-20k-rhIL-2组(125μg/kg)动物在静脉注射给药治疗期间有3/8只动物出现中度体重降低,1/8只动物出现中度至重度体重降低情况,动物对治疗基本耐受。在末次给药后第4天(D33),与V-PEG-SC-20k-rhIL-2组(125μg/kg)相比,mPEG-SS-5K-GP8-高修饰中剂量组(125μg/kg)体重有统计学差异(P<0.01),结果表明等剂量的mPEG-SS-5K-GP8-高修饰安全性优于阳性参照组(仿NKTR214)。
表38各组动物体重变化表(Female,Mean±SEM)

注:**:P<0.01,vs阴性对照组;*:P<0.05,vs阴性对照组。△△:P<0.01,vs阳性参照组(仿NKTR214)。
表39各组动物体重变化表(Female,Mean±SEM)
三、结果分析
mPEG-SS-5K-GP8-高修饰在中、低剂量(125μg/kg、62.5μg/kg)下对A498人肾癌皮下移植瘤模型均有显著抑制肿瘤生长的作用mPEG-SS-5K-GP8-高修饰中、低剂量能体现出一定的量效关系。V-PEG-SC-20k-rhIL-2组在125μg/kg剂量下、阳性对照组(泉奇)在71.25万IU/kg剂量下对A498人肾癌皮下移植瘤模型有显著抑制肿瘤生长的作用。
mPEG-SS-5K-GP8-高修饰中剂量组(125μg/kg)、V-PEG-SC-20k-rhIL-2组(125μg/kg)动物对治疗基本耐受,mPEG-SS-5K-GP8-高修饰低剂量组(62.5μg/kg)、阳性对照组(泉奇,71.25万IU/kg)动物对治疗耐受良好。各给药组动物未见死亡。
表明在相近抑瘤效果下,相较于V-PEG-SC-20k-rhIL-2,动物对mPEG-SS-5K-GP8-高修饰有更好的耐受性。
实施例18 PEG修饰IL-2对食蟹猴PBMC细胞激活的影响
食蟹猴每组3只,采用静脉注射给予不同剂量mPEG-SS-5K-GP8-高修饰(0.01mg/kg组、0.03mg/kg组),空白对照组给以溶剂对照,每周给药一次,共给药5次(D1、D8、D15、D22、D29),距离第一次给药(D1)30天后,取外周血,分离其中的外周血单个核细胞(PBMC),用anti-CD25-APC荧光抗体(Miltenyi,货号130-113-842)标记,FACS检测。
表40 FACS检测食蟹猴PBMC细胞CD25的表达
*表示与空白对照组比较,P<0.05,有显著性差异
从上述数据可知,0.03mg/kg组CD25表达量与空白对照组比较有显著性差异,CD25是PBMC细胞中T细胞激活的标志物,证明0.03mg/kg给药组有效刺激了T细胞的激活。
实施例19重复静脉注射给药食蟹猴4周的剂量探索试验
以食蟹猴作为实验动物,多次静脉注射给予不同剂量mPEG-SS-5K-GP8-高修饰,观察给药期间可能出现的毒性反应。设计了0.03、0.1、0.3、0.5mg/kg等4个剂量组,其中0.3mg/kg剂量组是0.03mg/kg剂量组动物在完成3个次给药后将剂量提高至0.3mg/kg,共完成6次给药。
一、分组与实验设计
按照下表根据体重将6只动物按性别随机分成4组
表41试验设计及分组安排
a.第2组首次给药后间隔21天在进行第1、3组动物首次给药;
b.第1组动物于D29-D43给药剂量为0.3mg/kg;
c.第2次给药未执行,共完成4次给药。
试验期间每天对动物进行临床观察,监测动物体重、食量、体温、血压的变化,并进行临床病理、免疫细胞表型分析、细胞因子、大体观察等检测。
二、结果
在0.03、0.1、0.3、0.5mg/kg等4个剂量组的食蟹猴均未出现死亡。
在0.03mg/kg剂量下食蟹猴未表现出异常,体重呈增长趋势。
在0.1mg/kg剂量下,可见雄性脾脏变大,CD56+细胞比例降低,体重呈增长趋势。
在0.3mg/kg剂量下,可见动物采食量的下降,但体重呈增长趋势;大体解剖可见脾脏变大;临床病理检查可见白细胞计数上升,红细胞计数下降、红细胞比容下降、血小板计数、总蛋白下降。
在0.5mg/kg剂量下,动物在首次给药后出现精神不振,自发活动减少,大量稀便,可视粘膜苍白等不良反应,暂停1次给药,后续共完成4次给药,动物采食量和体重下降;大体解剖可见脾脏变大、胸腺变小、腹部膨胀、全身皮肤松弛等症状;临床病理检查可见白细胞计数、单核细胞(绝对值)、中性粒细胞(绝对值)、嗜酸性粒细胞(绝对值)、嗜碱性粒细胞(绝对值)、尿素、肌酸激酶、纤维蛋白原上升,红细胞计数下降、红细胞比容下降、血小板计数、总蛋白下降;CD56+细胞比例降低。
初步研究结果显示在上述实验条件,0.5mg/kg的剂量上,在食蟹猴表现出严重不良反应,但也仍未出现死亡现象。
此外,根据NKTA-214相关文献(NKTA-214,an Engineered Cytokine with Biased IL2 Receptor Binding,Increaesd Tumor Exposure,and Marked Efficacy in Mouse Tumor Models)报道:在食蟹猴上间隔14天、4次给药的试验中,最大耐受剂量(MTD)为0.1mg/kg,该剂量下CD25+升高了24倍,总淋巴细胞升高了4倍。与NKTA-214相比,mPEG-SS-5K-GP8-高 修饰在更短的给药间隔(7天)、更多的给药次数(5次)、更高的给药剂量(0.3mg/kg)下未表现出严重不良反应,提示在安全性和耐受性方面存在优势。

Claims (18)

  1. 一种实现生物活性分子其活性控释和缓释的方法,所述方法是以聚乙二醇修饰剂对生物活性分子进行氨基修饰,所得修饰物中PEG逐步脱落后生物活性分子的活性被逐步释放,所述聚乙二醇修饰剂是聚乙二醇琥珀酰亚胺琥珀酸酯。
  2. 如权利要求1所述的方法,所述生物活性分子为白介素-2。
  3. 如权利要求1所述的方法,所述PEG修饰剂优选为直链聚乙二醇琥珀酰亚胺琥珀酸酯。
  4. 一种人白介素-2的聚乙二醇修饰物,其所采用的PEG修饰剂是聚乙二醇琥珀酰亚胺琥珀酸酯,平均每个人白介素-2上偶联4.5~8.5个PEG。
  5. 如权利要求4所述的聚乙二醇修饰物,所述PEG修饰剂是直链聚乙二醇琥珀酰亚胺琥珀酸酯修饰剂。
  6. 如权利要求4所述的聚乙二醇修饰物,所述PEG修饰剂分子量为5~20kDa。
  7. 如权利要求4所述的聚乙二醇修饰物,当PEG修饰剂分子量为5kDa时,平均每个人白介素-2上偶联5.5~7.5个PEG;当PEG修饰剂分子量为10~20kDa时,平均每个人白介素-2上偶联6.5~8.5个PEG。
  8. 一种人白介素-2的聚乙二醇修饰物,所述PEG修饰剂结构如下图所示:
    其中,n为97~494的整数。
  9. 如权利要求4所述的聚乙二醇修饰物,所述人IL-2的聚乙二醇修饰物结构如下所示:
    其中,n为97~494的整数,m为4.5~8.5。
  10. 一种人白介素-2突变体,其特征在于:不含相邻的赖氨酸。
  11. 权利要求9所述的突变体,其特征在于:相对于野生型人IL-2,自N端起第8位或/和第9位氨基酸被取代;或者相对于野生型人IL-2,自N端起第48位或/和第49位氨基酸被取代;所述野生型人IL-2氨基酸序列如SEQ ID NO:1所示。
  12. 一种人白介素-2突变体,其特征在于:相对于野生型IL-2,所述突变是以N端起第1、8、9、18、19、48、49、72和/或81位相应的1个、2个或多个位置上进行氨基酸取代;所述野生型人白介素-2氨基酸序列如SEQ ID NO:1所示。
  13. 如权利要求9所述的突变体,各突变分别为选自如下的取代残基:
    1位:A1缺失;8位:K8R;9位:K9R;18位:L18M;19位:L19S;48位:K48W,R;49位:K49R;72位:L72F;81位:R81D。
  14. 如权利要求9所述的突变体,所述突变选自下表的各个突变方案中的一种:
  15. 一种人白介素-2突变体的聚乙二醇修饰物,其所采用的PEG修饰剂能够与氨基反应的官能团是琥珀酰亚胺琥珀酸酯,平均每个人白介素-2上偶联4.5~8.5个PEG;其所述人白介素-2突变体为权利要求10-14中任一项所述的不同突变体中的一种。
  16. 一种人白介素-2的聚乙二醇修饰物或人白介素-2突变体的聚乙二醇修饰物在制备治疗肿瘤疾病药物中的应用。
  17. 如权利要求16的应用,其中所述肿瘤包括鳞状细胞癌、黑色素瘤、结肠癌、乳腺癌、卵巢癌、前列腺癌、胃癌、肝癌、小细胞肺癌、非小细胞肺癌、甲状腺癌、肾癌、胆管癌、脑癌、宫颈癌、上颌窦癌、膀胱癌、食管癌、何杰金氏病以及肾上腺皮质癌。
  18. 一种治疗肿瘤疾病的组合物,所述组合物包括权利要求4-8中任一项所述的人白介素-2的聚乙二醇修饰物、权利要求10-14中的人白介素-2突变体、或者权利要求15所述的人白介素-2突变体的聚乙二醇修饰物,还包括HER2抗体、PD-1抗体、PD-L1抗体或CD26抗体。
PCT/CN2023/076746 2022-02-18 2023-02-17 一种实现生物活性分子其活性控释和缓释的方法及药物应用 WO2023155872A1 (zh)

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Citations (6)

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US4766106A (en) * 1985-06-26 1988-08-23 Cetus Corporation Solubilization of proteins for pharmaceutical compositions using polymer conjugation
WO2000053223A1 (en) * 1999-03-11 2000-09-14 Human Genome Sciences, Inc. Apoptosis inducing molecule ii and methods of use
US20020081309A1 (en) * 1998-06-22 2002-06-27 Dean K. Pettit Site specific protein modification
US20050142106A1 (en) * 2003-07-18 2005-06-30 Wittrup K. D. Mutant interleukin-2 (IL-2) polypeptides
US20050186174A1 (en) * 2003-12-10 2005-08-25 Bossard Mary J. Compositions comprising two different populations of polymer-active agent conjugates
CN103517718A (zh) * 2010-11-12 2014-01-15 尼克塔治疗公司 Il-2部分与聚合物的缀合物

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4766106A (en) * 1985-06-26 1988-08-23 Cetus Corporation Solubilization of proteins for pharmaceutical compositions using polymer conjugation
US20020081309A1 (en) * 1998-06-22 2002-06-27 Dean K. Pettit Site specific protein modification
WO2000053223A1 (en) * 1999-03-11 2000-09-14 Human Genome Sciences, Inc. Apoptosis inducing molecule ii and methods of use
US20050142106A1 (en) * 2003-07-18 2005-06-30 Wittrup K. D. Mutant interleukin-2 (IL-2) polypeptides
US20050186174A1 (en) * 2003-12-10 2005-08-25 Bossard Mary J. Compositions comprising two different populations of polymer-active agent conjugates
CN103517718A (zh) * 2010-11-12 2014-01-15 尼克塔治疗公司 Il-2部分与聚合物的缀合物

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