A preparation of recombinant immune cytokine

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to a preparation of a recombinant immunocytokine. The present invention provides a preparation of a recombinant immunocytokine, comprising a BiPI protein and excipients at a specific concentration, which improves the stability of the recombinant immunocytokine targeting PD-L1 and IL-2 receptors during storage, prolongs its shelf life, and has broad application prospects and good economic value.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a preparation of recombinant immune cytokines. Background Art

[0002] The "immune escape" of tumor cells is considered to be the main mechanism of tumor occurrence and development. Tumor immunotherapy is to eliminate tumors by activating the body's own immunity. Recombinant monoclonal antibody drugs have the advantages of targeted specificity and definite efficacy. Therefore, antibody-based tumor immunotherapy strategies have become the main strategy for clinical tumor treatment in the past 30 years.

[0003] According to the "tumor immunoediting" theory, the disease progression of tumors is a dynamic process of interaction between the immune system and cancer cells, in which the body's immune system plays a dual role in clearing tumors and promoting progression. Under normal physiological conditions, the body can detect and eliminate tumor cells by activating the innate immune and adaptive systems to identify tumor-specific antigens. However, some tumor cells resist the attack of the body's immune system through various means such as "immune editing", and eventually tumor immune escape occurs. Tumor immune escape mechanisms include antigen modulation of tumor cells, decreased antigen presentation ability of dendritic cells, loss of T cell response ability, and inhibition of CD8+T cell activation. In addition, tumor cells can also secrete or express immunosuppressive factors (such as: TGF-β, IL-10, CTLA-4, PD-1, etc.) to create a tumor microenvironment that is suitable for tumor occurrence, development, and invasion.

[0004] Tumor immunotherapy is to enhance the body's ability to recognize cancer cells and use the body's own immune ability to eliminate tumors. Compared with traditional therapies such as surgery, chemotherapy, and radiotherapy, tumor immunotherapy has the advantages of good tumor targeting, lasting efficacy, and few clinical side effects. At present, the main tumor immunotherapies used in clinical practice include: cell therapy (ProvengeTM), cytokines (IL-2, IFN-α, etc.) and therapeutic monoclonal antibody drugs. Among them, recombinant monoclonal antibody drugs have the advantages of specific targeting and definite efficacy, and are the most widely used. However, due to the strong genetic variability in cancer patients and the fact that cancer cells often have multiple pathways for immune escape, the existing antibody-based immunotherapy has an efficacy of about 30-50% for advanced metastatic solid tumors. Although it is significantly higher than chemotherapy, it is still not ideal for most patients.

[0005] Fusion proteins (bispecific antibodies or bifunctional fusion proteins) that can simultaneously bind to two different targets prepared by genetic recombination technology are immunotherapy drugs with a new generation of molecular structure. Through clever design, they can achieve new or better pharmacological functions, such as playing a dual role of marking tumor cells and recruiting immune cells, or simultaneously regulating two signal pathways to synergistically kill tumor cells. They are expected to have higher efficacy and better disease-improving effects, making up for the shortcomings of single-target immunotherapy drugs.

[0006] "A recombinant immune cytokine targeting PD-L1 and IL-2 receptors" (referred to as BiPI, patent application number CN201610878334.8) is a recombinant protein that expresses interleukin 2 fused to the C-terminus of the heavy chain of the anti-PD-L1 antibody. It can bind to both the PD-L1 molecule and the IL-2 receptor (IL-2R).

[0007] PD-L1 (Programmed cell death 1 ligand 1), also known as surface antigen cluster of differentiation 274 (cluster of differentiation 274, CD274) or B7 homolog 1 (B7-H1), is a 40kDa transmembrane protein and one of the two ligands of PD-1 (Programmed Cell Death Protein 1). It is highly expressed in the heart, skeletal muscle, placenta, and lungs, and is lowly expressed in the thymus, spleen, kidney, and liver. It is also expressed in activated T cells, B cells, dendritic cells and other immune cells, and is widely expressed on a variety of tumor cells. After binding to PD-1, PD-L1 can transmit immunosuppressive signals and reduce T cell proliferation. This "immune checkpoints" mechanism can be used by cancer cells to achieve immune escape, so PD-1 and PD-L1 have become targets for cancer immunotherapy.

[0008] IL-2 (Interleukin-2) is a cytokine of the chemokine family, mainly produced by T cells (especially CD4 +IL-2 is synthesized by T cells) after being stimulated by antigens or mitogens; B cells, NK cells and monocytes-macrophages can also produce IL-2. IL-2's target cells include T cells, NK cells, B cells and monocytes-macrophages, all of which can express IL-2 receptors (IL-2R) on their surfaces. The biological activities of IL-2 include stimulating T cell growth and inducing cytotoxicity. In addition, it promotes the growth and differentiation of B cells, and enhances the antigen presentation ability, bactericidal ability and cytotoxicity of monocytes-macrophages. However, the biggest limitation of the widespread use of IL-2 is the serious adverse reaction of vascular leakage syndrome characterized by hypertension and fever, and a half-life of only 5 to 7 minutes; another disadvantage is that it widely binds to receptors expressed on immune cells, causing regulatory T cells (Treg) to proliferate, thereby hindering the body's anti-cancer immunity.

[0009] BiPI, a recombinant immune cytokine targeting PD-L1 and IL-2 receptors, uses anti-PD-L1 antibodies to block the PD-1 / PD-L1 signaling pathway and activate T cell autoimmunity. On the other hand, it fuses with IL-2 to give IL-2 targeting, allowing more IL-2 to be recruited at the tumor site, combining the body's adaptive immunity and innate immunity to increase the tumor inhibition effect.

[0010] However, this dual-target drug has a unique structure. To fully exert its effect, it is necessary to ensure that both components of the drug molecule in the formulation (anti-PD-L1 antibody and IL-2 fused to its C-terminus) stably maintain good biological activity. Targeted research on the formulation is needed to achieve the optimal effect. Summary of the invention

[0011] In order to improve the storage stability and extend the shelf life of "a recombinant immune cytokine targeting PD-L1 and IL-2 receptors" (referred to as BiPI, patent application number CN201610878334.8), the present invention provides a preparation formula, and the technical solution is as follows:

[0012] The BiPI protein has a light chain sequence of SEQ ID NO: 1 and a heavy chain sequence of SEQ ID NO: 2, and the light chain and the heavy chain are linked by a disulfide bond.

[0013] The formulation comprises the following dosages or concentrations of BiPI protein and excipients:

[0014] BiPI, 20 mg / mL to 50 mg / mL;

[0015] Histidine, 1.55 ± 0.5 mg / mL;

[0016] Polysorbate 80 (PEG80), 0.1 ± 0.01 mg / mL;

[0017] trehalose, 50 ± 5 mg / mL;

[0018] Hydrochloric acid, adjust the solution pH to 6.0 ± 0.3.

[0019] More specifically, the formulation includes the following doses or concentrations of BiPI protein and excipients:

[0020] BiPI, 50 mg / mL;

[0021] Histidine, 1.55 mg / mL;

[0022] PEG80, 0.1 mg / mL;

[0023] Trehalose, 50 mg / mL;

[0024] Hydrochloric acid, adjust the solution pH to 6.0.

[0025] The solvent used is water, which should meet the quality standards for water for injection.

[0026] The quality and production process of the excipients histidine, polysorbate 80, trehalose, hydrochloric acid, etc. should comply with the requirements of administrative agencies for pharmaceutical excipients and meet the quality standards specified in the Chinese Pharmacopoeia.

[0027] The preparation formula is suitable for intravenous injection.

[0028] The beneficial effect of the present invention is that it can significantly improve the storage stability of BiPI protein at a concentration of 20 to 50 mg / mL and reduce the generation of aggregates and charged heterogeneous bodies during storage. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Results of testing the bispecific binding ability of the BiPI recombinant protein after storage. DETAILED DESCRIPTION

[0030] Example 1: A formulation of a recombinant immune cytokine targeting PD-L1 and IL-2 receptors

[0031] "A recombinant immune cytokine targeting PD-L1 and IL-2 receptors" (referred to as BiPI, patent application number CN201610878334.8) is dissolved in the solution of the formula shown in Table 1 to form an injection preparation for intravenous injection.

[0032] Table 1 BiPI formulations

[0033] Components Final concentration Concentration Unit BiPI 50 mg / mL Histidine 1.55 mg / mL PEG80 0.1 mg / mL Trehalose 50 mg / mL HCl Adjust the solution pH to 6.0

[0034] The method for implementing the formulation of the preparation is as follows: after the cell culture fluid containing BiPI is purified and virus-free through a series of chromatography, filtration and other steps, the obtained solution containing the target protein is concentrated, then dialyzed, diluted, and prepared into a buffer solution configured according to the above formula.

[0035] Example 2: Effects of different buffers and pH on the stability of target protein

[0036] BIPI protein was dissolved in 20 mM phosphate buffer (PBS), citrate buffer (CBS), acetate buffer (ABS), and histidine buffer (HBS) at different pH values ​​at a concentration of 20 mg / mL and placed at 45°C for 28 days. The protein distribution in the solution was then analyzed by reverse phase chromatography (RP-HPLC), size exclusion chromatography (SE-HPLC), and cation exchange chromatography (CEX-HPLC). The initial sample without 45°C incubation was used as a control. The analysis results are listed in Table 2.

[0037] Table 2 Effects of different buffers on the stability of BiPI recombinant protein

[0038]

[0039] The results showed that the use of 20 mM histidine buffer at pH 6.0 was most conducive to the stability of BiPI recombinant protein in terms of the formation of aggregates and charge heteromers.

[0040] In order to further optimize the concentration of the buffer, the above analysis steps were repeated using 20 mM, 10 mM, and 5 mM pH 6.0 histidine buffers, respectively. The analysis results are shown in Table 3.

[0041] Table 3 Effects of different histidine buffers on the stability of BiPI recombinant protein

[0042]

[0043] The results showed that 20 mM and 10 mM concentrations had similar results, while the results of 5 mM concentration were significantly reduced, so 10 mM pH 6.0 histidine buffer was selected as the buffer for BiPI recombinant protein preparation.

[0044] Example 3: Effects of different surfactants on the stability of target protein

[0045] BIPI protein was dissolved at a concentration of 50 mg / mL in 10 mM pH 6.0 histidine buffer supplemented with different concentrations of polysorbate 20 (PEG20) and polysorbate 80 (PEG80) and placed at 45°C for 28 days. The protein distribution in the solution was then analyzed by reverse phase chromatography (RP-HPLC), size exclusion chromatography (SE-HPLC), and cation exchange chromatography (CEX-HPLC). The initial sample without 45°C incubation and the sample without polysorbate addition were used as controls. The analysis results are listed in Table 4.

[0046] Table 4 Effects of different surfactants on the stability of BiPI recombinant protein

[0047]

[0048] The concentration units of PEG20 and PEG80 in the above table are mg / mL.

[0049] The results showed that the addition of non-ionic surfactants such as PEG20 and PEG80 helped to enhance the stability of BiPI at a concentration of 50 mg / mL, but there was no significant difference in the effects of different concentrations of PEG20 and PEG80. Therefore, 0.1 mg / mL polysorbate 80 (PEG80) was selected as the preparation component.

[0050] Example 4: Effects of different sugars on the stability of target protein

[0051] BIPI protein was dissolved at a concentration of 50 mg / mL in 10 mM, pH 6.0 histidine buffer supplemented with different concentrations of sugars and 0.1 mg / mL polysorbate 80 and placed at 45°C for 28 days. The protein distribution in the solution was then analyzed by reverse phase chromatography (RP-HPLC), size exclusion chromatography (SE-HPLC), and cation exchange chromatography (CEX-HPLC). The initial samples without 45°C incubation and the samples without sugar addition were used as controls. The analysis results are listed in Table 5.

[0052] Table 5 Effects of different sugars on the stability of BiPI recombinant protein

[0053]

[0054] The results showed that the addition of trehalose had the strongest effect on improving the stability of BiPI, but there was no significant difference between the effects of 10% and 5% trehalose concentrations, so 5% (equivalent to 50 mg / mL) trehalose was selected as the formulation component.

[0055] Example 5. Testing of the stability of BiPI recombinant protein preparations at different storage temperatures

[0056] The BIPI protein was prepared into a preparation according to the formulation and method described in Example 1, and then stored at -80°C, -20°C, 4°C, and 25°C for 6 months. After being taken out, the protein distribution in the solution was analyzed by reverse phase chromatography (RP-HPLC), molecular exclusion chromatography (SE-HPLC), and cation exchange chromatography (CEX-HPLC). The analysis results are listed in Table 6

[0057] Table 6 Stability test of BiPI recombinant protein preparation at different storage temperatures

[0058]

[0059] The results showed that the molecular distribution of the preparation remained basically unchanged when stored at -80℃, -20℃, and 4℃ for 6 months, the protein purity of SE-HPLC analysis was still above 95%, and the proportion of aggregates, acidic peaks, and basic peaks did not change significantly. However, the molecular distribution of the preparation stored at 25℃ for 6 months showed a slight change worthy of attention.

[0060] Example 6: Flow cytometry detection of bispecific binding ability of BiPI recombinant protein after storage

[0061] The BiPI samples stored at -80°C, -20°C, 4°C, and 25°C for 6 months in Example 5 were subjected to flow cytometry detection according to the following method:

[0062] (1) Collect CT26 cells (mouse colon cancer cells) with good growth status into a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard the upper culture medium, and resuspend the cells in PBS solution containing 1% FBS;

[0063] (2) Count the cells and calculate the number of cells to 2×10 6 Add 100 μL of cells / tube into the flow tube, and set up triplicate wells;

[0064] (3) BiPI and IgG were diluted to a starting concentration of 10 μg / mL, and then diluted 2-fold. Each group of three replicates was diluted to a total of 12 concentrations. 100 μL of the diluted experimental drug and control were added vertically to the flow tube, respectively, and shaken for 3 seconds to fully mix the cells and antibodies. The tube was covered with tin foil and placed in a 4°C refrigerator for 1 hour.

[0065] (4) Centrifuge the flow cytometry tube at 1000 rpm for 5 min, quickly pour off the upper layer of liquid, do not shake to avoid cell loss at the bottom of the tube, then add PBS solution containing 1% FBS at a volume of 200 μL / tube, shake to mix, and centrifuge again. Repeat this step twice to wash away unbound antibodies on the cell surface;

[0066] (5) Add PE-labeled rabbit anti-human IL-2 secondary antibody (1:300 dilution), 100 μL / tube into the flow cytometry tube, shake to mix, and incubate at 4 °C in the dark for 45 min;

[0067] (6) Repeat step (4), wash the cells twice, resuspend the cells in 200 μL of PBS solution containing 1% FBS, shake and mix, and then measure the fluorescence reading on the microscope. Then, plot the fluorescence reading against BiPI concentration (see Figure 1 ).

[0068] Through curve analysis, the EC50 of BiPI samples stored at -80°C, -20°C, 4°C, and 25°C for 6 months were 0.486 mg / mL, 0.501 mg / mL, 0.522 mg / mL, and 0.619 mg / mL, respectively, indicating that after using the formulation listed in Example 1, the BiPI protein well preserved the binding activity against PD-L1 and IL-2 during the low-temperature storage process at -20°C to 4°C. Sequence Listing <110> Shanghai Biomab Pharmaceutical Co., Ltd. <120> A preparation of recombinant immune cytokine <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 214 <212> PRT <213> Artificial Sequence <400> 1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 2 <211> 599 <212> PRT <213> Artificial Sequence <400> 2 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly 435 440 445 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 450 455 460 Gly Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu 465 470 475 480 Glu His Leu Leu Leu Thr Leu Gln Met Ile Leu Asn Gly Ile Asn Asn 485 490 495 Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met 500 505 510 Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu 515 520 525 Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe 530 535 540 His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu 545 550 555 560 Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu 565 570 575 Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln 580 585 590 Ser Ile Ile Ser Thr Leu Thr 595

Claims

1. A preparation of a recombinant immunocytokine, characterized in that: The invention comprises the following doses or concentrations of recombinant immunocytokines and excipients: Recombinant immune cytokines, 20 mg / mL to 50 mg / mL; Histidine, 1.55 ± 0.5 mg / mL; Polysorbate 80, 0.1 ± 0.01 mg / mL; trehalose, 50 ± 5 mg / mL; Hydrochloric acid, adjust the solution pH to 6.0 ± 0.3; The recombinant immune cytokine is formed by linking a peptide chain having SEQ ID NO: 1 and a peptide chain having SEQ ID NO: 2 via a disulfide bond.

2. The preparation of recombinant immunocytokine according to claim 1, characterized in that: The invention comprises the following doses or concentrations of recombinant immunocytokines and excipients: Recombinant immunocytokine, 50 mg / mL; Histidine, 1.55 mg / mL; Polysorbate 80, 0.1 mg / mL; Trehalose, 50 mg / mL; Hydrochloric acid, adjust the solution pH to 6.0; The recombinant immune cytokine is formed by linking a peptide chain with SEQ ID NO: 1 and a peptide chain with SEQ ID NO: 2 through a disulfide bond.

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