WO2024077773A1 - 一种抗人il-2单克隆抗体及其应用 - Google Patents

一种抗人il-2单克隆抗体及其应用 Download PDF

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WO2024077773A1
WO2024077773A1 PCT/CN2022/141438 CN2022141438W WO2024077773A1 WO 2024077773 A1 WO2024077773 A1 WO 2024077773A1 CN 2022141438 W CN2022141438 W CN 2022141438W WO 2024077773 A1 WO2024077773 A1 WO 2024077773A1
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human
antibody
seq
amino acid
acid sequence
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French (fr)
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娄竞
陈建鹤
苏冬梅
靳征
吕云英
张若兰
裴若辰
欧艳梅
曲啸
谢写
张静
曾淋
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深圳市百士通科技开发有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons

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  • the present invention belongs to the technical field of antibody engineering, and relates to an anti-human IL-2 monoclonal antibody and application thereof, and specifically to an anti-human IL-2 monoclonal antibody, the preparation of an antibody complex and application thereof.
  • Interleukin-2 is a cytokine with strong immune activity. It has an effect on T cell activation and growth, and is involved in anti-tumor effects and transplant rejection reactions.
  • drugs targeting IL-2 such as aldesleukin, which have been used clinically to treat malignant tumors and have a long history.
  • aldesleukin drugs targeting IL-2
  • toxic side effects have been found in the clinical application of IL-2, especially toxicity to the liver and lungs, which limits its application.
  • IL-2 it was found that it can stimulate Treg proliferation and cause activation-induced death (AICD) of immune cells, which further affects its efficacy.
  • AICD activation-induced death
  • IL-2 works by binding to the IL-2 receptor, which is composed of three subunits: ⁇ , ⁇ , and ⁇ .
  • ⁇ is a high-affinity receptor
  • ⁇ and ⁇ are low-affinity receptors, but they mediate the signal transduction after IL-2 binds to the receptor.
  • the anti-tumor activity of IL-2 is mainly achieved by activating CD8-positive T cells and NK cells, but it also amplifies CD4-positive Treg cells, weakening or even completely eliminating its anti-tumor effect.
  • CD4-positive Treg cells highly express IL-2R ⁇ (CD25). Since CD25 is a high-affinity receptor for IL-2, when IL-2 concentration is low, it will preferentially bind to Treg cells, bind to CD25 and exert anti-tumor immunosuppressive effects.
  • CD8-positive T cells and NK cells do not express or low-express ⁇ receptors, but highly express ⁇ and ⁇ receptors, so they are affected by IL-2 at high concentrations. Therefore, reducing the binding of IL-2 to ⁇ receptors or enhancing the binding of IL-2 to ⁇ / ⁇ receptors in some way so that IL-2 acts preferentially on CD8-positive T cells and NK cells is an effective way to achieve the anti-tumor effect of IL-2.
  • the present invention provides a preparation and application of an anti-human IL-2 monoclonal antibody and an antibody complex.
  • the present invention uses recombinant human IL-2 protein as an immunogen, prepares a mouse anti-human IL-2 monoclonal antibody m22F8 by hybridoma technology, performs binding activity analysis on the mouse antibody, determines the affinity for IL-2, analyzes the effect of the mouse antibody in blocking IL-2 binding to CD25, and prepares an anti-human IL-2 chimeric antibody by determining the mouse hybridoma antibody gene.
  • CDRs transplantation technology and CDR region mutation design are used to construct a humanized anti-human IL-2 monoclonal antibody, detect the affinity of the humanized antibody for IL-2, analyze its effect of blocking IL-2 binding to CD25, the anti-tumor activity of the anti-human IL-2 monoclonal antibody m22F8 and the antibody complex, and the synergistic anti-tumor activity of the antibody complex and PD1 monoclonal antibody, which has good application prospects for anti-tumor drugs.
  • the present invention provides an anti-human IL-2 monoclonal antibody having:
  • the heavy chain variable region has an amino acid sequence of SEQ ID NO:4, and the light chain variable region has an amino acid sequence of SEQ ID NO:9.
  • nucleotide encoding the anti-human IL-2 monoclonal antibody has:
  • the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO:3; the nucleotide sequence of the light chain variable region is shown in SEQ ID NO:8.
  • the antibody is derived from mouse antibody.
  • the present invention provides an anti-human IL-2 chimeric antibody having:
  • the heavy chain sequence has an amino acid sequence of SEQ ID NO:15
  • the light chain sequence has an amino acid sequence of SEQ ID NO:16.
  • the present invention also provides an Fc mutated anti-human IL-2 chimeric antibody, which has: a heavy chain sequence with an amino acid sequence of SEQ ID NO: 18, and a light chain sequence with an amino acid sequence of SEQ ID NO: 16.
  • the preparation method is as follows: using gene site-directed mutagenesis technology or gene synthesis technology, the 310th H of the human IgG4 constant region is converted into A and the 435th amino acid H is converted into Q to form a mutated human IgG4 constant region, and the amino acid sequence is shown in SEQ ID NO: 17; the heavy chain variable region of the anti-human IL-2 monoclonal antibody is recombined with the mutated human IgG4 constant region to form the heavy chain of the Fc-mutated anti-human IL-2 chimeric antibody, and the amino acid sequence is shown in SEQ ID NO: 18; the light chain variable region sequence of the anti-human IL-2 monoclonal antibody is recombined with the human kappa chain constant region with the amino acid sequence shown in SEQ ID NO: 14 to form the light chain of the anti-human IL-2 chimeric antibody, and the amino acid sequence is shown in SEQ ID NO: 16; then, it is constructed into a pcDNA3.4 expression vector,
  • the present invention also provides a humanized anti-human IL-2 monoclonal antibody, which is constructed on the basis of the anti-human IL-2 monoclonal antibody by using CDRs transplantation technology and CDR region mutation design.
  • the humanized anti-human IL-2 monoclonal antibody has: a heavy chain variable region with an amino acid sequence of SEQ ID NO: 24, and a light chain variable region with an amino acid sequence of SEQ ID NO: 25.
  • the present invention also provides a humanized anti-human IL-2 antibody having:
  • the heavy chain sequence has an amino acid sequence of SEQ ID NO:27, and the light chain sequence has an amino acid sequence of SEQ ID NO:28.
  • the present invention also provides a humanized anti-human IL-2 antibody with Fc mutation, which has:
  • the heavy chain sequence has an amino acid sequence of SEQ ID NO:26, and the light chain sequence has an amino acid sequence of SEQ ID NO:28.
  • the preparation method is as follows: the heavy chain variable region of the humanized anti-human IL-2 monoclonal antibody is recombined with the mutant IgG4 constant region with an amino acid sequence as shown in SEQ ID NO:17 to obtain the heavy chain of the humanized anti-human IL-2 antibody with Fc mutation, and the amino acid sequence is as shown in SEQ ID NO:26; the light chain variable region sequence of the anti-human IL-2 monoclonal antibody is recombined with the human kappa chain constant region with an amino acid sequence as shown in SEQ ID NO:14 to obtain the light chain of the humanized anti-human IL-2 chimeric antibody with an amino acid sequence as shown in SEQ ID NO:28; then, it is constructed into a pcDNA3.4 expression vector, transfected into Expi-293F cells, and the Fc mutation humanized anti-human IL-2 antibody is obtained by Protein G purification.
  • the present invention also provides an antibody complex, which is prepared by mixing IL-2 (interleukin-2) and the anti-human IL-2 chimeric antibody or Fc mutated anti-human IL2 chimeric antibody or humanized anti-human IL-2 antibody or Fc mutated humanized anti-human IL-2 antibody obtained by the present invention in a mass ratio of 1:7.
  • IL-2 interleukin-2
  • the present invention also provides the use of the antibody complex in preparing anti-tumor drugs.
  • the anti-human IL-2 monoclonal antibody prepared by the present invention retains the binding with IL2R ⁇ / ⁇ (CD122/132), and can also block the binding of IL2 with IL2R ⁇ (CD25), and can effectively inhibit the growth of transplanted tumors in mice.
  • the present invention introduces mutations in the Fc of the IL-2 monoclonal antibody, and after the Fc-mutated monoclonal antibody forms a complex with IL-2, it can not only retain the anti-tumor activity of the IL-2/antibody complex in vivo, but also greatly reduce side effects and improve drug safety.
  • IL-2 is interleukin-2, a cytokine of the chemokine family.
  • IL-2 has a molecular weight of 15KD and is a glycoprotein containing 113 amino acid residues. In humans, it is encoded by a gene on chromosome 4.
  • antibody herein is intended to include full-length antibodies and any antigen-binding fragments (i.e., antigen-binding portions) or single chains thereof.
  • a full-length antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains, the heavy chains and light chains being linked by disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (VH for short) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, namely CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (VL for short) and a light chain constant region (CL for short).
  • monoclonal antibody or “mAb” refers to an antibody molecule preparation of a single molecular composition.
  • a monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.
  • EC50 also known as half maximal effect concentration, refers to the concentration that can induce 50% of the maximal effect.
  • IC50 also known as half inhibitory concentration, refers to the concentration of a drug or inhibitor required to inhibit a specified biological process or a component of the biological process (such as an enzyme, receptor, cell, etc.) by half.
  • FIG1 is a graph showing the affinity of the mouse antibody m22F8 for the target antigen IL-2;
  • FIG2 is a diagram showing the blocking of human IL-2 binding to CD25-ECD by murine antibody m22F8;
  • FIG3 is a graph showing the anti-tumor activity of the IL-2/m22F8 complex
  • Figure 4 shows the effect of IL-2/ch22F8mu complex on survival at 3 mg/kg dose for 2 doses of IL-2.
  • Figure 5 shows the effect of IL-2/ch22F8mu complex on survival at 1 mg/kg dose for 6 times.
  • FIG6 is a graph showing the in vivo anti-tumor activity of the IL-2/ch22F8mu complex
  • Figure 7 is a graph showing the binding of ch22F8mu and hu22F8mu to human IL-2;
  • FIG8 is a diagram showing the blocking of IL-2 binding to CD25 by hu22F8mu and ch22F8mu;
  • Figure 9 is a diagram showing IL-2/hu22F8mu complex stimulating CTLL2 cell proliferation
  • Figure 10 is a diagram showing the effects of IL-2/hu22F8mu complex and IL-2/hu22F8 binding to human FcRn/ ⁇ 2M;
  • Figure 11 is a diagram showing the effects of IL-2/hu22F8mu complex and IL-2/hu22F8 binding to mouse FcRn/ ⁇ 2M;
  • FIG12 is a graph showing the in vivo anti-tumor activity of the IL-2/hu22F8mu complex
  • FIG13 is a diagram showing the anti-tumor effect of SPGD01 (IL-2/hu22F8mu);
  • FIG14 is a graph showing the effect of SPGD01 (IL-2/hu22F8mu) on serum ALT in experimental mice;
  • FIG15 is a diagram showing the effect of SPGD01 (IL-2/hu22F8mu) on the composition of CD4+/CD8+ lymphocytes in experimental animals;
  • FIG16 is a diagram showing the effect of SPGD01 (IL-2/hu22F8mu) on the composition of CD4+/CD25+ lymphocytes in experimental animals;
  • FIG17 is a stability diagram of SPGD01 (IL-2/hu22F8mu) and hu22F8mu;
  • FIG. 18 is a stability diagram of SPGD01 (IL-2/hu22F8mu) and hu22F8mu
  • FIG. 19 is a stability diagram of SPGD01 (IL-2/hu22F8mu) and hu22F8mu
  • FIG. 20 is a graph showing the stability of SPGD01 (IL-2/hu22F8mu) and hu22F8mu.
  • Step 1 Human IL-2 protein expressed by prokaryotic cell Escherichia coli (purchased from Sino Biological Company, product number GMP-11848-HNAE, amino acid sequence as shown in SEQ ID NO: 1) was used for routine immunization of Balb/c mice (purchased from Shanghai Lingchang Biotechnology Co., Ltd.); on the first day, after the IL-2 protein was emulsified with Freund's complete adjuvant, Balb/c mice were subcutaneously injected at multiple points (human IL-2 protein, 50 ⁇ g/mouse/0.5ml); on the 21st day, after the human IL-2 protein was emulsified with Freund's incomplete adjuvant, Balb/c mice were subcutaneously injected (human IL-2 protein, 50 ⁇ g/mouse/0.5ml); on the 41st day, human IL-2 protein, 50 ⁇ g/mouse/0.2ml, was stimulated by intraperitoneal injection. After 3 to 4 days, the spleen of the mice was taken
  • Step 2 3 to 4 days after the last immunization of the mice, the mouse spleen cells and mouse myeloma cells SP2/0 were electrofused by an electrofusion instrument (purchased from BTX) using conventional hybridoma technology.
  • the fused cells were evenly suspended in complete culture medium (RPMI1640 and DMEM F12 culture medium were mixed in a ratio of 1:1 and then 1% Glutamine, 1% Sodium pyruvate, 1% MEM-NEAA (minimum essential medium-non-essential amino acid solution), 1% Penicillin-streptomycin, 50 ⁇ M ⁇ -mercaptoethanol and 20% FBS (fetal bovine serum) were added; all products were purchased from Gibco) and incubated for 10 min.
  • complete culture medium RPMI1640 and DMEM F12 culture medium were mixed in a ratio of 1:1 and then 1% Glutamine, 1% Sodium pyruvate, 1% MEM-NEAA (minimum essential medium-non-essential amino
  • hybridoma wells with positive human IL-2 binding and blocking IL-2 binding to CD25 were subjected to the first and second rounds of subcloning by limiting dilution method to obtain a hybridoma cell line named SPGD01-22F8.
  • the method of screening the hybridoma wells with positive human IL-2 binding activity by indirect enzyme-linked immunosorbent assay is as follows: dilute the recombinant human IL-2 protein to 1 ⁇ g/ml with coating solution (50mM carbonate coating buffer, pH 9.6), add 100 ⁇ l/well to the ELISA plate, and coat overnight at 4°C. Wash the plate 3 times with PBST, add 200 ⁇ l/well blocking solution (2% BSA-PBST), place at 37°C for 1 hour, and then wash the plate once with PBST for use. Add the collected hybridoma supernatant to the blocked ELISA plate in sequence, 100 ⁇ l/well, and place at 37°C for 1 hour.
  • the plate was washed three times with PBST, and HRP-labeled goat anti-mouse IgG secondary antibody (purchased from Millipore, catalog number AP181P) was added and placed at 37°C for 30 min. After washing the plate five times with PBST, the residual droplets were patted dry on absorbent paper as much as possible, 100 ⁇ l of TMB (purchased from BD, catalog number 555214) was added to each well, and the plate was placed at room temperature (20 ⁇ 5°C) in the dark for 5 min. 50 ⁇ l of 2M H2SO4 stop solution was added to each well to terminate the substrate reaction, and the OD value was read at 450 nm by a microplate reader to analyze the binding ability of the test antibody to the target antigen IL-2.
  • HRP-labeled goat anti-mouse IgG secondary antibody purchased from Millipore, catalog number AP181P
  • the method for detecting hybridoma wells blocking IL-2 binding to CD25 is as follows: recombinant human CD25-ECD (purchased from Beijing Yiqiao Shenzhou Company, item number 50292-M02H) is diluted to 1 ⁇ g/ml with coating solution (50mM carbonate coating buffer, pH 9.6), 100 ⁇ l/well is added to the ELISA plate, and coated overnight at 4°C. Wash the plate 3 times with PBST, add 200 ⁇ l/well blocking solution (2% BSA-PBST), place it at 37°C for 1h, and then wash the plate once with PBST for use.
  • coating solution 50mM carbonate coating buffer, pH 9.6
  • biotin-labeled IL-2 purchased from sino biological company, item number 11848-HNAE-B
  • biotin-labeled IL-2 purchased from sino biological company, item number 11848-HNAE-B
  • diluted to 10ng/ml are incubated at 37°C for 30min, and then added to the blocked ELISA plate, 100 ⁇ l/well, and placed at 37°C for 1h.
  • the plate was washed 3 times with PBST, and HRP-labeled SA (SA-HRP, Pierce) was added, and the plate was placed at 37°C for 30 min.
  • the hybridoma cell lines obtained by screening were amplified in complete culture medium (as described in Example 1), and the medium was changed to serum-free culture medium SFM medium (purchased from Life Technologies, Catalog No. 12045-076) by centrifugation to a cell density of 1-2 ⁇ 10 7 /ml.
  • SFM medium purchased from Life Technologies, Catalog No. 12045-076
  • the cells were cultured at 5% CO 2 and 37°C for 1 week, and the culture supernatant was obtained by centrifugation and purified by Protein G affinity chromatography to obtain the mouse anti-human IL-2 monoclonal antibody m22F8.
  • Example 3 Determination of the affinity of mouse anti-human IL-2 monoclonal antibody m22F8 for target antigen IL2
  • the affinity of mouse anti-human IL-2 monoclonal antibody m22F8 to recombinant human IL-2 protein was determined by ELISA.
  • the experimental method is as follows:
  • Recombinant human IL-2 protein was diluted to 1 ⁇ g/ml with coating solution (50 mM carbonate coating buffer, pH 9.6), and 100 ⁇ l/well was added to the ELISA plate and coated overnight at 4°C.
  • the plate was washed 3 times with PBST, and 200 ⁇ l/well blocking solution (2% BSA-PBST) was added. After standing at 37°C for 1 hour, the plate was washed once with PBST for use.
  • Mouse anti-human IL-2 monoclonal antibody m22F8 was diluted to 5000/1000/200/40/8/1.6/0.32/0 ng/ml with diluent (1% BSA-PBST), and added to the blocked ELISA plate in sequence, 100 ⁇ l/well, and stood at 37°C for 1 hour. The plate was washed three times with PBST, and HRP-labeled goat anti-mouse IgG secondary antibody (purchased from Millipore, catalog number AP181P) was added and placed at 37°C for 30 min.
  • diluent 1% BSA-PBST
  • HRP-labeled goat anti-mouse IgG secondary antibody purchased from Millipore, catalog number AP181P
  • the results are shown in FIG1 .
  • the EC50 of the mouse anti-human IL-2 monoclonal antibody m22F8 binding to human IL-2 is 6.90 ng/ml, ie, 0.05 nM, showing good affinity.
  • Example 4 Murine anti-human IL-2 monoclonal antibody m22F8 blocks human IL2 binding to CD25-ECD
  • Recombinant hCD25 (amino acid sequence as shown in SEQ ID NO: 2) was diluted to 1 ⁇ g/ml with coating solution (50 mM carbonate coating buffer, pH 9.6), 100 ⁇ l/well was added to the ELISA plate, and coated overnight at 4°C. The plate was washed 3 times with PBST, 200 ⁇ l/well blocking solution (2% BSA-PBST) was added, and the plate was placed at 37°C for 1 hour and then washed once with PBST for use.
  • coating solution 50 mM carbonate coating buffer, pH 9.6
  • the plate was washed 3 times with PBST, 200 ⁇ l/well blocking solution (2% BSA-PBST) was added, and the plate was placed at 37°C for 1 hour and then washed once with PBST for use.
  • Mouse anti-human IL-2 monoclonal antibody m22F8 was diluted to 10000/2000/400/80/16/3.2/0.64/0 ng/ml with diluent (1% BSA-PBST), mixed with the same volume of bio-IL2 diluted to 20 ng/ml, incubated at 37°C for 30 minutes, and then added to the blocked ELISA plate, 100 ⁇ l/well, and placed at 37°C for 1 hour. The plate was washed three times with PBST, and HRP-labeled SA (SA-HRP, Pierce) was added, and the plate was placed at 37°C for 30 min.
  • HRP-labeled SA SA-HRP, Pierce
  • the results are shown in FIG2 .
  • the mouse anti-human IL-2 monoclonal antibody m22F8 can effectively inhibit IL-2 binding to CD25, with an IC50 of 253.1 ng/ml, or 1.69 nM.
  • Example 5 IL-2/m22F8 complex inhibits the growth of MC38 cell transplanted tumors in mice
  • Mouse colon cancer MC38 cells cultured in vitro were collected, and the concentration of the cell suspension was adjusted to 1 ⁇ 10 7 /ml.
  • the right flank of C57BL/6 mice was shaved. Under sterile conditions, 100 ⁇ l of the cell suspension was inoculated subcutaneously on the right flank of C57 mice.
  • the diameter of the subcutaneous transplanted tumor of mice was measured with a vernier caliper. After the average tumor volume grew to 100-200 mm 3 , the animals were randomly divided into 8 groups per group.
  • IL-2 and m22F8 were mixed at a mass ratio of 1:7, and the IL-2/m22F8 complex was prepared after incubation at room temperature for 15 minutes.
  • the IL-2 was administered at a dose of 1 mg/kg, and the control group was given an equal amount of PBS, which was intraperitoneally injected twice a week for 2 consecutive times. During the entire experiment, the diameter of the transplanted tumor was measured twice a week, and the mice were weighed at the same time.
  • the calculation formula for tumor volume (TV) is:
  • RTV Vt/V0.
  • V0 the tumor volume measured at the time of group administration (i.e. d0)
  • Vt the tumor volume at each measurement. Evaluation index of antitumor activity
  • TGI tumor inhibition rate %
  • TGI% 100% - T/C (%)
  • Relative tumor proliferation rate T/C (%) (TRTV/CRTV) ⁇ 100
  • TRTV treatment group RTV
  • CRTV negative control group RTV
  • the IL-2/m22F8 complex exhibited excellent anti-tumor activity, and the tumor inhibition rate was close to 100% after two administrations.
  • the animals generally showed clinical symptoms including decreased activity, decreased food intake, loose hair and decreased body temperature, and 2/8 animals died. This shows that although the IL-2/m22F8 complex has a good anti-tumor effect in vivo, it has safety issues, including obvious clinical symptoms and even death.
  • the heavy chain variable region and light chain variable region of the hybridoma m22F8 were obtained by molecular biology related methods, and were further used to construct chimeric antibodies.
  • RNA of hybridoma cells was extracted by Trizol and the mRNA was reverse transcribed to obtain cDNA. Subsequently, the cDNA was used as a template and PCR was performed with degenerate primers of the heavy chain and light chain of mouse antibodies (Antibody Engineering, Volume 1, Edited by Roland Kontermann and Stefan Dübel, the sequence of the combined primers is from page 323). The PCR products were sequenced and analyzed through the kabat database to determine that the obtained sequence was the variable region sequence of the mouse antibody.
  • the m22F8 heavy chain variable region gene sequence is 351bp in length, encoding 117 amino acid residues.
  • the nucleotide sequence is shown in SEQ ID NO:3, and the amino acid sequence is shown in SEQ ID NO:4.
  • the m22F8 monoclonal antibody light chain variable region gene sequence is 318bp in length, encoding 106 amino acid residues.
  • the nucleotide sequence is shown in SEQ ID NO:8, and the amino acid sequence is shown in SEQ ID NO:9.
  • the obtained heavy chain variable region sequences of each hybridoma were recombined with the human IgG4 constant region (including the S228P mutation) (amino acid sequence as shown in SEQ ID NO:13) to form a chimeric ch22F8 monoclonal antibody heavy chain (amino acid sequence as shown in SEQ ID NO:15); the light chain variable region sequences were recombined with the human kappa chain constant region (amino acid sequence as shown in SEQ ID NO:14) to form a chimeric ch22F8 monoclonal antibody light chain (amino acid sequence as shown in SEQ ID NO:16).
  • the heavy chain variable region (amino acid sequence as shown in SEQ ID NO: 19) and light chain variable region (amino acid sequence as shown in SEQ ID NO: 20) of NARA1 monoclonal antibody were synthesized according to the literature (patent US 2017/0183403 Al).
  • the heavy chain variable region of NARA1 monoclonal antibody was recombined with the human IgG4 constant region to form the NARA1 monoclonal antibody heavy chain (amino acid sequence as shown in SEQ ID NO: 21), and the light chain variable region of NARA1 monoclonal antibody was recombined with the human kappa chain constant region to form the NARA1 monoclonal antibody light chain (amino acid sequence as shown in SEQ ID NO: 23).
  • the heavy chain and light chain genes were constructed into pcDNA3.4 expression vectors, respectively, and paired for transfection into Expi-293F cells.
  • Chimeric antibodies ch22F8 and NARA1 were obtained by Protein A purification.
  • SDS-PAGE electrophoresis and SEC-HPLC determined that the molecular weight of each expressed antibody was about 150 kD, and the antibody purity was >95%. They were quantified, packaged, and stored at -80°C for later use.
  • the 310th H of the human IgG4 constant region (amino acid sequence as shown in SEQ ID NO: 13) was converted to A and the 435th amino acid H was converted to Q, and the other amino acid sequences remained unchanged to form a mutated human IgG4 constant region (amino acid sequence as shown in SEQ ID NO: 17), and the m22F8 heavy chain variable region was recombined with the mutated human IgG4 constant region to form an anti-human IL-2 chimeric antibody heavy chain (amino acid sequence as shown in SEQ ID NO: 18); the heavy chain variable region of the NARA1 monoclonal antibody was recombined with the mutated human IgG4 constant region to form a mutant NARA1 (NARA1mu) heavy chain (amino acid sequence as shown in SEQ ID NO: 22).
  • mutant heavy chain genes were respectively constructed into the pcDNA3.4 expression vector, paired with the respective light chains prepared in Example 6 to transfect Expi-293F cells, and anti-human IL-2 chimeric antibodies (ch22F8mu) and NARA1mu were obtained by Protein G purification.
  • the molecular weight of each expressed antibody was determined to be around 150 kD by SDS-PAGE electrophoresis and SEC-HPLC, and the antibody purity was >95%.
  • the antibodies were quantified, packaged, and frozen at -80°C for later use.
  • IL-2 was mixed with each test antibody (ch22F8, ch22F8mu, NARA1, NARA1mu) at a mass ratio of 1:7, and the complex was formed after incubation at room temperature for 15 minutes.
  • the dose of 1 mg/kg and/or 3 mg/kg and/or 6 mg/kg of IL-2 was intraperitoneally injected into C57BL/6 mice (Viton River Company) twice on the 1st and 4th days. The death of the experimental mice was observed on the 7th day, and the control group was administered with the same volume of PBS.
  • Example 9 IL-2/ch22F8mu reduces liver damage in experimental animals
  • IL-2 was mixed with the test antibodies ch22F8mu and ch22F8 at a mass ratio of 1:7, incubated at room temperature for 15 minutes to form a complex, and injected intraperitoneally into C57BL/6 mice (Viton River) twice on the 1st and 4th days at a dose of 3 mg/kg for IL-2; and injected intraperitoneally into C57BL/6 mice (Viton River) 6 times twice a week at a dose of 1 mg/kg for IL2. On the day after the last administration, blood was collected from surviving mice to obtain serum for ALT determination.
  • Example 10 In vivo antitumor activity of IL-2/ch22F8mu complex
  • the experimental method was the same as in Example 5.
  • IL-2 was mixed with each test antibody (ch22F8, ch22F8mu) at a mass ratio of 1:7, and a complex was formed after incubation at room temperature for 15 minutes.
  • the doses were 0.3 mg/kg and 1 mg/kg for IL-2, and 1 mg/kg for IL-2 alone as a control.
  • the drug was administered by intraperitoneal injection 3 times a week for 2 consecutive weeks.
  • Example 11 Preparation of humanized anti-human IL-2 monoclonal antibody and humanized anti-human IL-2 chimeric antibody
  • the amino acid sequences of the light chain variable region and the heavy chain variable region of the candidate mouse antibody of Example 1 were analyzed, and the three antigen complementary determining regions (CDRs) and four framework regions (FRs) of the mouse antibody were determined according to the Kabat rule.
  • the amino acid sequence of the heavy chain complementary determining region of 22F8 is HCDR1: GFNIKNTY (amino acid sequence as shown in SEQ ID NO: 5), HCDR2: IDPANGNT (amino acid sequence as shown in SEQ ID NO: 6), HCDR3: GRSRGYAMDY (amino acid sequence as shown in SEQ ID NO: 7), and the amino acid sequence of the light chain complementary determining region is LCDR1: DHINNW (amino acid sequence as shown in SEQ ID NO: 10), LCDR2: GATSLET (amino acid sequence as shown in SEQ ID NO: 11) and LCDR3: QQYWSTPT (amino acid sequence as shown in SEQ ID NO: 12).
  • the humanized templates that best matched the non-FR regions of the above-mentioned mouse antibodies were selected from the Germline database.
  • the CDR region of the mouse antibody was then transplanted onto the selected humanized template to replace the CDR region of the human template.
  • the heavy chain variable region was then recombined with the human IgG4 constant region (including the S228P mutation), and the light chain variable region was recombined with the human kappa chain constant region.
  • a humanized anti-human IL-2 monoclonal antibody (hu22F 8)
  • the heavy chain variable region (amino acid sequence as shown in SEQ ID NO:24) was recombined with the human IgG4 constant region to obtain the recombinant humanized anti-human IL-2 monoclonal antibody heavy chain (amino acid sequence as shown in SEQ ID NO:27), and was recombined with the mutated human IgG4 constant region to obtain the humanized anti-human IL-2 antibody (hu22F8mu) heavy chain (amino acid sequence as shown in SEQ ID NO:26), and the humanized anti-human IL-2 monoclonal antibody light chain variable region (amino acid sequence as shown in SEQ ID NO:25
  • the heavy and light chains of each humanized antibody were constructed into the pcDNA3.4 expression vector and transfected into Expi-293F cells.
  • Humanized anti-human IL-2 monoclonal antibody (hu22F8) and mutated humanized anti-human IL-2 antibody (hu22F8mu) were obtained by Protein G purification. SDS-PAGE electrophoresis and SEC-HPLC were used to determine whether the molecular weight of each antibody was correct and the purity was >95%.
  • Example 12 ch22F8mu and hu22F8mu bind to human IL-2
  • the results are shown in Figure 7.
  • the EC50 values of hu22F8 and hu22F8mu binding to human IL-2 detected by ELISA were 6.76 ng/ml and 6.01 ng/ml, i.e. 0.05 nM and 0.04 nM, respectively.
  • the EC50 values of ch22F8 and ch22F8mu binding to human IL-2 were 6.29 ng/ml and 7.24 ng/ml, i.e. 0.04 nM and 0.05 nM, respectively.
  • Example 13 hu22F8mu and ch22F8mu block IL-2 binding to CD25
  • the experimental method is the same as that of Example 4.
  • the results are shown in Figure 8.
  • the IC50 of hu22F8 and hu22F8mu blocking human IL-2 binding to CD25 are 218.6 ng/ml and 227.9 ng/ml, i.e., 1.46 nM and 1.52 nM, respectively;
  • the IC50 of ch22F8 and ch22F8mu blocking human IL-2 binding to CD25 are 256.7 ng/ml and 236.6 ng/ml, i.e., 1.71 nM and 1.58 nM, respectively.
  • Example 14 IL-2/hu22F8mu complex (SPGD01) stimulates CTLL2 cell proliferation
  • This example uses a CTLL2 cell proliferation assay to illustrate the in vitro biological activity of the complexes IL-2/hu22F8mu (SPGD01), IL-2/hu22F8, IL-2/ch22F8, and IL-2/ch22F8mu.
  • the method is as follows:
  • CTLL2 cells were diluted to 1E5/ml with 1640 culture medium containing 10% FBS, and 100ul/well was added to the cell culture plate.
  • IL-2 was mixed with hu22F8mu, hu22F8, ch22F8 and ch22F8mu at a mass ratio of 1:7, and placed at room temperature for 30 minutes to form complexes SPGD01 (IL2/hu22F8mu), IL-2/hu22F8, IL-2/ch22F8 and IL-2/ch22F8mu.
  • IL-2 and the above complexes were diluted to 50ng/ml with 1640 culture medium containing 10% FBS, and then 2-fold dilutions were added to the above culture plates containing CTLL2 cells for a total of 8 gradients, and cultured in a cell culture incubator at 37°C and 5% CO2 for 72h. After dilution, the relative cell number of each well was determined by CCK8, and the EC50 was calculated to determine the activity of the sample.
  • IL-2, SPGD01 (IL-2/hu22F8mu), IL-2/hu22F8, IL-2/ch22F8 and IL-2/ch22F8mu can all stimulate the proliferation of CTLL2 cells.
  • the EC50 of IL-2 is 0.74 ng/ml
  • the EC50 of SPGD01 and IL-2/hu22F8 are 1.01 ng/ml and 1.09 ng/ml, respectively
  • the EC50 of IL-2/ch22F8 and IL-2/ch22F8mu are 0.98 ng/ml and 1.04 ng/ml, respectively, indicating that SPGD01 and IL-2/hu22F8 have consistent biological activities, and IL-2/ch22F8mu and IL-2/ch22F8 also have the same biological activity, further indicating that the mutation of Fc will not lead to the biological activity of the IL-2/antibody complex.
  • Example 15 The ability of SPGD01 to bind to FcRn is significantly weakened
  • the experimental method is the same as that in Example 8.
  • Example 17 In vivo anti-tumor activity of SPGD01 (IL-2/hu22F8mu)
  • the experimental method is the same as that in Example 5.
  • the IL-2/hu22F8 complex exhibited good anti-tumor activity, with the TGI of the 0.5 mg/kg and 0.25 mg/kg groups reaching 95.4% and 85.6%, respectively; SPGD01 (IL-2/hu22F8mu) also exhibited excellent anti-tumor activity, with the TGI of the 2 mg/kg, 1 mg/kg and 0.5 mg/kg groups reaching 95.8%, 84.5% and 75.4%, respectively.
  • Statistical analysis showed that there was no significant difference in the anti-tumor effects of the two (p>0.05).
  • Example 18 Synergistic anti-tumor activity of SPGD01 (IL-2/hu22F8mu) and PD1 monoclonal antibody in vivo
  • the MC38 transplant tumor model was used to evaluate the synergistic anti-tumor effect of SPGD01 (IL-2/hu22F8mu) and anti-PD1 monoclonal antibody in vivo, and the experimental method was the same as Example 5.
  • the dose of SPGD01 was 4 mg/kg based on IL-2, and the dose of rat anti-mouse PD1 monoclonal antibody (purchased from Bio X Cell, catalog number BP0146) was 5 mg/kg, and the drug was administered by intraperitoneal injection 3 times/week for a total of 6 times. Three days after the last administration, the mice were killed, and the mouse serum and mouse spleen were obtained.
  • the enzyme activity method was used to determine the alanine aminotransferase (ALT) content in the mouse serum, and the conventional flow cytometry method (FACS) was used to analyze the spleen cells CD4 positive cells, CD8 positive cells and CD4, CD25 positive cells.
  • FACS flow cytometry method
  • IL-2 and hu22F8mu were mixed at a mass ratio of 1:7 to form SPGD01 (IL-2/hu22F8mu), and after being kept at 4°C with hu22F8mu for a corresponding period of time, molecular sieve high performance liquid chromatography (SEC-HPLC) was used to detect the changes in the purity of SPGD01 and hu22F8mu after being placed for different periods of time to investigate the stability of SPGD01 and hu22F8mu.
  • SEC-HPLC molecular sieve high performance liquid chromatography
  • TSKgel G3000SWXL column (TSK) was used on HPLC Ultimate 3000 (Thermo) chromatograph.
  • the mobile phase was PBS (pH 7.4), the constant flow rate was 0.8 ml/min, and the sample load was 100 ug/100 ul.
  • the 280 nM absorption peak integration method was used to calculate the content (%) of the target protein in the total protein to indicate purity.

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Abstract

提供一种抗人IL-2 单克隆抗体、抗体复合物及其应用。该抗人IL-2单克隆抗体，结合IL2后，保留IL2与IL2Rβ/γ(CD122/132)的结合，阻断IL-2 与IL-2Rα(CD25)的结合，可以有效抑制小鼠体内移植瘤的生长。还提供了Fc恒定区突变的抗人IL-2 单克隆抗体，可降低抗体的副作用，提高药物安全性。

Description

一种抗人IL-2单克隆抗体及其应用 技术领域
本发明属于抗体工程技术领域，涉及一种抗人IL-2单克隆抗体及其应用，具体涉及一种抗人IL-2单克隆抗体，抗体复合物的制备及其应用。
背景技术
白细胞介素-2(IL-2)是一种具有很强的免疫活性的细胞因子，对T细胞激活及生长有作用，并参与抗肿瘤效应和移植排斥反应。针对IL-2的药物较多，如aldesleukin等，在临床上用于治疗恶性肿瘤,具有悠久的历史。但在IL-2的临床应用中发现了较多的毒副作用，特别是对肝、肺的毒性，限制了其应用。而且，在IL-2应用过程中发现，其可以刺激Treg增殖且会引起免疫细胞出现激活诱导的死亡(AICD)，也进一步影响了其疗效。
IL-2通过与IL-2受体结合起作用，IL-2受体由α、β、γ三个亚基组成，其中α为高亲和力受体，β、γ虽然为低亲和力受体，但介导了IL-2结合受体后的信号传导。IL-2的抗肿瘤活性主要通过激活CD8阳性T细胞和NK细胞实现，但其同时扩增CD4阳性的Treg细胞，减弱甚至完全去除了其抗肿瘤作用。
CD4阳性的Treg细胞高表达IL-2Rα(CD25)，由于CD25是IL-2的高亲和力受体，故IL-2低浓度时，会优先作用于结合Treg细胞，与CD25结合并发挥抗肿瘤免疫抑制作用。CD8阳性的T细胞和NK细胞，因为不表达或低表达α受体，但高表达β和γ受体，故在IL-2高浓度时受其作用。因此，通过某种方式，降低IL-2与α受体的结合，或者增强IL-2与β/γ受体的结合，使IL-2偏向作用于CD8阳性的T细胞和NK细胞，是实现IL-2抗肿瘤作用的有效方式。
然而，目前研究发现，不管是IL-2单抗、IL-2与Fc形成的融合蛋白还是抗体-IL-2双功能分子，在用于治疗恶性肿瘤时，都存在一定的毒副作用，包括肝脏毒性。因此，研究和开发出一种毒副作用低，抗肿瘤活性高的IL-2药物是目前亟需解决的难题。
发明内容
为了解决现有技术的缺陷，本发明提供了一种抗人IL-2单克隆抗体、抗体复合物的制备及其应用。本发明以重组人IL-2蛋白作为免疫原，通过杂交瘤技术制备鼠源抗人IL-2单克隆抗体m22F8，对鼠源抗体进行结合活性分析、对IL-2的亲和力测定，分析鼠源抗体阻断IL-2结合CD25的作用，通过小鼠杂交瘤抗体基因的测定，制备出抗人IL-2嵌合抗体。在鼠源抗体、嵌合抗体性能分析的基础上，采用CDRs移植技术、CDR区突变设计构建人源化抗人IL-2单克隆抗体，检测人源化抗体对IL-2的亲和力测定，分析其阻断IL-2结合CD25的作用，抗人IL-2单克隆抗体m22F8和抗体复合物的抗肿瘤活性，以及抗体复合物与PD1单抗协同抗肿瘤活性的研究，对抗肿瘤药物具有良好的应用前景。
本发明提供了一种抗人IL-2单克隆抗体，其具有：
氨基酸序列为SEQ ID NO:4的重链可变区，和氨基酸序列为SEQ ID NO:9的轻链可变区。
进一步地，编码所述的抗人IL-2单克隆抗体的核苷酸，其具有：
如SEQ ID NO:3所示的重链可变区的核苷酸序列；如SEQ ID NO:8所示的轻链可变区的核苷酸序列。
进一步地，所述抗体来自鼠源抗体。
本发明提供了一种抗人IL-2嵌合抗体，其具有：
氨基酸序列为SEQ ID NO:15的重链序列，和氨基酸序列为SEQ ID NO:16的轻链序列。
本发明还提供了一种Fc突变的抗人IL-2嵌合抗体，其具有：氨基酸序列为SEQ ID NO:18的重链序列，和氨基酸序列为SEQ ID NO:16的轻链序列。
其制备方法为：采用基因定点突变技术或基因合成技术，将人IgG4恒定区第310位H转换成A和第435位氨基酸H转换成Q，形成突变的人IgG4恒定区，氨基酸序列如SEQ ID NO:17所示；将所述的抗人IL-2单克隆抗体的重链可变区与突变的人IgG4恒定区重组，形成Fc突变的抗人IL-2嵌合抗体的重链，氨基酸序列如SEQ ID NO:18所示；将所述的抗人IL-2单克隆抗体的轻链可变区序列与氨基酸序列如SEQ ID NO:14所示的人的kappa链恒定区重组，形成抗人IL-2嵌合抗体的轻链，氨基酸序列如SEQ ID NO:16所示；接着构建至pcDNA3.4 表达载体，转染Expi-293F细胞，通过Protein G纯化获得Fc突变的抗人IL-2嵌合抗体。
进一步地，本发明还提供了一种人源化抗人IL-2单克隆抗体，其是在所述的抗人IL-2单克隆抗体的基础上，采用CDRs移植技术、CDR区突变设计构建的人源化抗人IL-2单克隆抗体。
进一步地，所述的人源化抗人IL-2单克隆抗体，其具有：氨基酸序列为SEQ ID NO:24的重链可变区，和氨基酸序列为SEQ ID NO:25的轻链可变区。
本发明还提供了一种人源化抗人IL-2抗体，其具有：
氨基酸序列为SEQ ID NO:27的重链序列，和氨基酸序列为SEQ ID NO:28的轻链序列。
本发明还提供了一种Fc突变的人源化抗人IL-2抗体，其具有：
氨基酸序列为SEQ ID NO:26的重链序列，和氨基酸序列为SEQ ID NO:28的轻链序列。
其制备方法为：将所述的人源化抗人IL-2单克隆抗体的重链可变区与氨基酸序列如SEQ ID NO:17所示的突变IgG4恒定区重组，获得Fc突变的人源化抗人IL-2抗体的重链，氨基酸序列如SEQ ID NO:26所示；将所述的抗人IL-2单克隆抗体的轻链可变区序列与氨基酸序列如SEQ ID NO:14所示的人的kappa链恒定区重组获得人源化抗人IL-2嵌合抗体的轻链，氨基酸序列如SEQ ID NO:28所示；接着构建至pcDNA3.4表达载体，转染Expi-293F细胞，通过Protein G纯化获得Fc突变的人源化抗人IL-2抗体。
本发明还提供了一种抗体复合物，其由IL-2(白细胞介素-2)与本发明获得的抗人IL-2嵌合抗体或Fc突变的抗人IL2嵌合抗体或人源化抗人IL-2抗体或Fc突变的人源化抗人IL-2抗体按质量比1:7混合制得。
此外，本发明还提供了所述的抗体复合物在制备治疗抗肿瘤药物中的用途。
与现有技术相比，本发明制备的抗人IL-2单克隆抗体，其保留与IL2Rβ/γ(CD122/132)的结合，同时还可以阻断IL2与IL2Rα(CD25)的结合，可以有效抑制小鼠体内移植瘤的生长。同时，本发明在IL-2单抗的Fc中引入突变，将Fc突变的单抗与IL-2形成复合物后，既可以保留IL-2/抗体复合物的体内的抗肿瘤活性，还可以极大降低副作用，提高药物安全性。
为更好理解本发明，首先定义一些术语。其他定义则贯穿具体实施方式部分而列出。
术语“IL-2”是白细胞介素-2，是趋化因子家族的一种细胞因子，IL-2分子量为15KD，是含有113个氨基酸残基的糖蛋白，在人类由第4号染色体上的一个基因编码。
本文中的术语“抗体”意在包括全长抗体及其任何抗原结合片段(即，抗原结合部分)或单链。全长抗体是包含至少两条重(H)链和两条轻(L)链的糖蛋白，重链和轻链由二硫键连接。各重链由重链可变区(简称VH)和重链恒定区构成。重链恒定区由三个结构域构成，即CH1、CH2和CH3。各轻链由轻链可变区(简称VL)和轻链恒定区(简称CL)构成。
术语“单克隆抗体”或“单抗”是指单一分子组成的抗体分子制品。单克隆抗体组成呈现出对于特定表位的单一结合特异性和亲和力。
术语“EC50”，又叫半最大效应浓度，是指是指能引起50％最大效应的浓度。
术语“IC50”，又叫半抑制浓度，是指对指定的生物过程或该生物过程中的某个组份(例如酶、受体、细胞等)抑制一半时所需的药物或抑制剂的浓度。
附图说明：
图1为鼠源抗体m22F8对靶抗原IL-2的亲和力图；
图2为鼠源抗体m22F8阻断人IL-2结合CD25-ECD图；
图3为IL-2/m22F8复合物的抗肿瘤活性图；
图4为IL-2/ch22F8mu复合物按IL-2计，3mg/kg剂量，给药2次，对存活
小鼠的血清ALT测定图；
图5为IL-2/ch22F8mu复合物按IL-2计，1mg/kg剂量，给药6次，对存活
小鼠的血清ALT测定图；
图6为IL-2/ch22F8mu复合物的体内抗肿瘤活性图；
图7为ch22F8mu和hu22F8mu结合人IL-2图；
图8为hu22F8mu和ch22F8mu阻断IL-2结合CD25图；
图9为IL-2/hu22F8mu复合物刺激CTLL2细胞增殖图；
图10为IL-2/hu22F8mu复合物和IL-2/hu22F8结合人FcRn/β2M效果图；
图11为IL-2/hu22F8mu复合物和IL-2/hu22F8结合鼠FcRn/β2M效果图；
图12为IL-2/hu22F8mu复合物的体内抗肿瘤活性图；
图13为SPGD01(IL-2/hu22F8mu)的抗肿瘤作用图；
图14为SPGD01(IL-2/hu22F8mu)对实验小鼠血清ALT的影响图；
图15为SPGD01(IL-2/hu22F8mu)对实验动物CD4+/CD8+淋巴细胞组成的影响图；
图16为SPGD01(IL-2/hu22F8mu)对实验动物CD4+/CD25+淋巴细胞组成的影响图；
图17为SPGD01(IL-2/hu22F8mu)和hu22F8mu的稳定性图；
图18为SPGD01(IL-2/hu22F8mu)和hu22F8mu的稳定性图
图19为SPGD01(IL-2/hu22F8mu)和hu22F8mu的稳定性图
图20为SPGD01(IL-2/hu22F8mu)和hu22F8mu的稳定性图。
具体实施方式
以下通过具体实施方式的描述对本发明作进一步说明，但这并非是对本发明的限制，本领域技术人员根据本发明的基本思想，可以做出各种修改或改进，但是只要不脱离本发明的基本思想，均在本发明的范围之内。
实施例1、抗原免疫小鼠以及杂交瘤的制备和筛选
步骤一：用原核细胞大肠杆菌表达的人IL-2蛋白(购自sino biological公司，货号GMP-11848-HNAE，氨基酸序列如SEQ ID NO:1所示)常规免疫Balb/c小鼠(购自上海灵畅生物科技有限公司)；第1天，IL-2蛋白与弗氏完全佐剂乳化后，对Balb/c小鼠进行皮下多点注射(人IL-2蛋白，50μg/鼠/0.5ml)，第21天，人IL-2蛋白与弗氏不完全佐剂乳化后，对Balb/c小鼠进行皮下注射(人IL-2蛋白，50μg/鼠/0.5ml)，在第41天，人IL-2蛋白，50μg/小鼠/0.2ml，腹腔内注射激发，3～4天后，取小鼠脾脏进行融合实验；
步骤二：在小鼠末次免疫后3～4天，使用常规的杂交瘤技术方案，将小鼠脾细胞与小鼠骨髓瘤细胞SP2/0通过电融合仪(购自BTX公司)进行电融合，融合后的细胞在完全培养基(即将RPMI1640和DMEM F12培养基1：1混匀后加 入1％的Glutamine(谷氨酰胺)，1％Sodium pyruvate(丙酮酸钠)，1％MEM-NEAA(最小基本培养基-非必需氨基酸溶液)，1％Penicillin-streptomycin(青霉素-链霉素)，50μM的β-巯基乙醇及20％FBS(胎牛血清)；所有产品均购自Gibco公司)中悬浮均匀，按10 5个细胞/100μl/孔，分入共25块96孔培养板中培养过夜，次日，每孔加入100μl孔含有2×HAT的完全培养基，使96孔板内培养液为200μl/孔(含1×HAT)；在7～12天后，收获上清液，通过间接酶联免疫吸附测定法(ELISA)筛选人IL-2结合活性阳性的杂交瘤孔，共获得442个阳性孔。通过检测杂交瘤孔对阻断IL-2结合CD25的作用，进一步筛选获得25个阳性孔。将人IL-2结合阳性且阻断IL-2结合CD25阳性的杂交瘤孔通过有限稀释法进行第一、第二轮亚克隆，获得杂交瘤细胞株，命名为SPGD01-22F8。
其中，间接酶联免疫吸附测定法筛选人IL-2结合活性阳性的杂交瘤孔的方法如下：将重组人IL-2蛋白以包被液(50mM的碳酸盐包被缓冲液，pH 9.6)稀释至1μg/ml，100μl/孔加入酶标板，4℃包被过夜。PBST洗板3次，加入200μl/孔封闭液(2％BSA-PBST)，37℃放置1h后PBST洗板1次待用。将收取的杂交瘤上清液依次加入封闭后的酶标板，100μl/孔，37℃放置1h。PBST洗板3次，加入HRP标记的羊抗小鼠IgG二抗(购自Millipore,货号AP181P)，37℃放置30min；PBST洗板5次后，在吸水纸上尽量拍干残留液滴，每孔加入100μl的TMB(购自BD公司，货号555214)，室温(20±5℃)避光放置5min；每孔加入50μl 2M H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，分析待测抗体与靶抗原IL-2结合能力。
其中，检测杂交瘤孔阻断IL-2结合CD25的方法如下：重组人CD25-ECD(购自北京义翘神州公司，货号50292-M02H)以包被液(50mM的碳酸盐包被缓冲液，pH 9.6)稀释至1μg/ml，100μl/孔加入酶标板，4℃包被过夜。PBST洗板3次，加入200μl/孔封闭液(2％BSA-PBST)，37℃放置1h后PBST洗板1次待用。将收取的各杂交瘤上清液60ul与稀释至10ng/ml的80ul生物素标记的IL-2(bio-IL-2，购自sino biological公司，货号11848-HNAE-B)37℃孵育30min后加入封闭后的酶标板，100μl/孔，37℃放置1h。PBST洗板3次，加入HRP标记的SA(SA-HRP，Pierce公司)，37℃放置30min；PBST洗板5次后，在吸水纸上尽量拍干残留液滴，每孔加入100μl的TMB(购自BD公司，货号555214)， 室温(20±5℃)避光放置5min；每孔加入50μl 2M H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，分析待测抗体阻断IL-2结合CD25的作用。
实施例2、鼠源抗人IL-2单克隆抗体m22F8的制备
在完全培养基(如实施例1所述)中扩增筛选获得的杂交瘤细胞株，离心换液至无血清培养液SFM培养基(购自life technologies公司，货号12045-076)，使细胞密度为1～2×10 7/ml，在5％CO 2，37℃条件下培养1周，离心获取培养上清，通过Protein G亲和层析进行纯化，获得鼠源抗人IL-2单克隆抗体m22F8。
实施例3、鼠源抗人IL-2单克隆抗体m22F8对靶抗原IL2的亲和力的测定
通过ELISA的方法测定鼠源抗人IL-2单克隆抗体m22F8对重组人IL-2蛋白的亲和力，实验方法如下：
将重组人IL-2蛋白以包被液(50mM的碳酸盐包被缓冲液，pH 9.6)稀释至1μg/ml，100μl/孔加入酶标板，4℃包被过夜。PBST洗板3次，加入200μl/孔封闭液(2％BSA-PBST)，37℃放置1h后PBST洗板1次待用。鼠源抗人IL-2单克隆抗体m22F8以稀释液(1％BSA-PBST)稀释至5000/1000/200/40/8/1.6/0.32/0ng/ml，依次加入封闭后的酶标板，100μl/孔，37℃放置1h。PBST洗板3次，加入HRP标记的羊抗小鼠IgG二抗(购自Millipore,货号AP181P)，37℃放置30min；PBST洗板5次后，在吸水纸上尽量拍干残留液滴，每孔加入100μl的TMB(购自BD公司，货号555214)，室温(20±5℃)避光放置5min；每孔加入50μl 2M H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，分析待测抗体与靶抗原IL-2结合能力。
结果如图1所示，鼠源抗人IL-2单克隆抗体m22F8结合人IL-2的EC50为6.90ng/ml，即0.05nM，显示良好的亲和力。
实施例4、鼠源抗人IL-2单克隆抗体m22F8阻断人IL2结合CD25-ECD
重组hCD25(氨基酸序列如SEQ ID NO:2所示)以包被液(50mM的碳酸盐包被缓冲液，pH 9.6)稀释至1μg/ml，100μl/孔加入酶标板，4℃包被过夜。PBST洗板3次，加入200μl/孔封闭液(2％BSA-PBST)，37℃放置1h后PBST洗板1 次待用。鼠源抗人IL-2单克隆抗体m22F8以稀释液(1％BSA-PBST)梯度稀释至10000/2000/400/80/16/3.2/0.64/0ng/ml后，与相同体积稀释至20ng/ml的bio-IL2混合，37℃孵育30min后，加入至封闭后的酶标板，100μl/孔，37℃放置1h。PBST洗板3次，加入HRP标记的SA(SA-HRP，Pierce公司)，37℃放置30min；PBST洗板5次后，在吸水纸上尽量拍干残留液滴，每孔加入100μl的TMB(购自BD公司，货号555214)，室温(20±5℃)避光放置5min；每孔加入50μl 2M H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，分析待测抗体阻断IL-2结合CD25的作用。
结果如图2所示，鼠源抗人IL-2单克隆抗体m22F8能够有效抑制IL-2结合CD25，IC50为253.1ng/ml，即1.69nM。
实施例5、IL-2/m22F8复合物抑制MC38细胞移植瘤在小鼠体内生长
收集体外培养的小鼠结肠癌MC38细胞，将细胞悬液浓度调整为1×10 7/ml。C57BL/6小鼠右侧肋部剃毛。在无菌条件下，接种100μl细胞悬液于C57小鼠右侧肋部皮下。小鼠皮下移植瘤用游标卡尺测量移植瘤直径，待平均肿瘤体积生长至100-200mm 3后将动物随机分组，8只/组。IL-2与m22F8按质量比1:7混合，室温孵育15分钟后制得IL-2/m22F8复合物，以IL-2计，1mg/kg剂量给药，对照组给等量PBS，每周腹腔注射给药2次，连续给药2次。整个实验过程中，每周2次测量移植瘤直径，同时称小鼠体重。肿瘤体积(tumor volume,TV)的计算公式为：
TV＝1/2×a×b2
其中a、b分别表示长、宽。根据测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为：RTV＝Vt/V0。其中V0为分组给药时(即d0)测量所得肿瘤体积，Vt为每一次测量时的肿瘤体积。抗肿瘤活性的评价指标
为TGI(肿瘤抑制率％)T/C(％)，计算公式如下：
TGI％＝100％-T/C(％)
相对肿瘤增殖率T/C(％)＝(TRTV/CRTV)×100
TRTV：治疗组RTV；CRTV：阴性对照组RTV。
结果如图3所示，IL-2/m22F8复合物体现了极好的抗肿瘤活性，给药2次，抑瘤率均接近100％。但实验过程中，动物普遍出现包括活动减少、进食减少、毛发松散和体温下降等临床症状，出现2/8动物死亡。说明IL-2/m22F8复合物虽然具有良好的体内抗肿瘤作用，但存在安全性问题，包括出现明显的临床症状，甚至是死亡。
实施例6、小鼠杂交瘤抗体基因的测定和嵌合抗体制备
本实施例通过分子生物学的相关方法获取杂交瘤m22F8的重链可变区和轻链可变区，并进一步用于构建嵌合抗体。
通过Trizol提取杂交瘤细胞的RNA并进行mRNA反转录获取cDNA，随后以cDNA为模板，分别用鼠源抗体的重链和轻链简并引物(《Antibody Engineering》Volume 1，Edited by Roland Kontermann and Stefan Dübel，组合引物的序列来自第323页)进行PCR，对所获得的PCR产物进行测序并通过kabat数据库分析，确定所获得的序列为鼠源抗体的可变区序列。
相关序列信息如下：
m22F8重链可变区基因序列，全长为351bp，编码117个氨基酸残基，核苷酸序列如SEQ ID NO:3所示，氨基酸如SEQ ID NO:4所示；m22F8单抗轻链可变区基因序列全长318bp，编码106个氨基酸残基，核苷酸序列如SEQ ID NO:8所示，氨基酸序列如SEQ ID NO:9所示。
对所得的各杂交瘤重链可变区序列分别与人的IgG4恒定区(包含S228P突变)(氨基酸序列如SEQ ID NO:13所示)重组，形成嵌合ch22F8单抗重链(氨基酸序列如SEQ ID NO:15所示)；轻链可变区序列与人的kappa链恒定区(氨基酸序列如SEQ ID NO:14所示)重组，形成嵌合ch22F8单抗轻链(氨基酸序列如SEQ ID NO:16所示)。
按文献(专利US 2017/0183403 Al)合成NARA1单抗的重链可变区(氨基酸序列如SEQ ID NO:19所示)和轻链可变区(氨基酸序列如SEQ ID NO:20所示)。NARA1单抗的重链可变区与人IgG4恒定区重组形成NARA1单抗重链(氨基酸序列如SEQ ID NO:21所示)，NARA1单抗轻链可变区与人的kappa链恒定区重组形成NARA1单抗轻链(氨基酸序列如SEQ ID NO:23所示)。
上述重链和轻链基因分别构建至pcDNA3.4表达载体，配对转染Expi-293F细胞，通过Protein A纯化获得嵌合抗体ch22F8和NARA1，通过SDS-PAGE电泳及SEC-HPLC确定所表达各抗体分子量在150kD左右，抗体纯度>95％，定量，分装，冻存于-80℃备用。
实施例7、抗人IL-2嵌合抗体的制备
采用基因定点突变技术或基因合成技术，将人IgG4恒定区(氨基酸序列如SEQ ID NO:13所示)第310位H转换成A和第435位氨基酸H转换成Q，其他氨基酸序列保持不变，形成突变的人IgG4恒定区(氨基酸序列如SEQ ID NO:17所示)，将m22F8重链可变区与突变的人IgG4恒定区重组，形成抗人IL-2嵌合抗体重链(氨基酸序列如SEQ ID NO:18所示)；NARA1单抗的重链可变区与突变的人IgG4恒定区重组形成突变NARA1(NARA1mu)重链(氨基酸序列如SEQ ID NO:22所示)。
上述突变重链基因分别构建至pcDNA3.4表达载体，配对实施例6制得的各自轻链转染Expi-293F细胞，通过Protein G纯化获得抗人IL-2嵌合抗体(ch22F8mu)和NARA1mu，通过SDS-PAGE电泳及SEC-HPLC确定所表达各抗体分子量在150kD左右，抗体纯度>95％，定量，分装，冻存于-80℃备用。
实施例8、IL-2/ch22F8mu复合物对实验小鼠的致死性
IL-2分别与各受试抗体(ch22F8，ch22F8mu，NARA1，NARA1mu)按质量比1：7混合，室温孵育15分钟后形成复合物，按IL-2计，1mg/kg和/或3mg/kg和/或6mg/kg剂量(具体见表1)，于第1天和第4天，腹腔注射C57BL/6小鼠(维通利华公司)共2次，于第7天观察实验小鼠的死亡情况，对照组给药同体积的PBS。
表1各抗体给药剂量
剂量(IL-2) ch22F8 ch22F8mu NARA1 NARA1mu
1mg/kg √ - - -
3mg/kg √ √ √ √
6mg/kg - √ - -
表2各抗体剂量组实验动物存活率
抗体 1mg/kg 3mg/kg 6mg/kg
IL-2/ch22F8 40％ 0％ -
IL-2/ch22F8mu - 100％ 100％
NARA1 - 0％ -
NARA1mu - 100％ -
结果如表2所示：
(1)在实验第7天，在3mg/kg剂量下，ch22F8，NARA1组所有实验动物死亡(存活率0％)，但是ch22F8mu，NARA1mu组所有实验动物均存活(存活率100％)，说明IL-2单抗采用所述的突变后，与IL-2形成的复合物，对实验小鼠的毒性明显下降。
(2)ch22F8mu组在剂量升高至6mg/kg时，实验动物存活率仍然为100％，而ch22F8在剂量降低至1mg/kg时，仍然有60％动物死亡，进一步说明，ch22F8mu在Fc突变后，与IL-2形成的复合物对实验小鼠的毒性明显下降。
实施例9、IL-2/ch22F8mu降低了对实验动物的肝脏损害
IL-2分别与受试抗体ch22F8mu和ch22F8按质量比1:7混合，室温孵育15分钟形成复合物，按IL-2计，3mg/kg剂量，于第1天和第4天，腹腔注射C57BL/6小鼠(维通利华公司)共2次；按IL2计1mg/kg剂量，2次/周，腹腔注射C57BL/6小鼠(维通利华公司)共6次，于末次给药后次日，存活小鼠取血获取血清，测定ALT。
结果图4和图5所示：
(1)在IL-2 1mg/kg剂量下，IL-2/ch22F8组3/8小鼠死亡，存活的5/8小鼠ALT平均值为425.2U/L，IL-2/ch22F8mu组无动物死亡，所有8只小鼠的ALT平均值为64.0U/L。
(2)在IL-2 3mg/kg剂量下，IL-2/ch22F8组3/8小鼠死亡，存活小鼠ALT平均值为303.2U/L，IL-2/ch22F8mu组无动物死亡，所有8只小鼠的ALT平均值为62.3U/L。
(3)PBS对照组ALT平均值则为29.4U/L，说明IL-2/22F8mu的肝脏毒性较IL-2/ch22F8明显减小。
实施例10、IL-2/ch22F8mu复合物的体内抗肿瘤活性
实验方法同实施例5，IL-2分别与各受试抗体(ch22F8，ch22F8mu)按质量比1:7混合，室温孵育15分钟后形成复合物，按IL-2计，0.3mg/kg和1mg/kg剂量，IL-2单药按1mg/kg剂量作为对照。每周腹腔注射给药3次，连续给药2周。
结果如图6所示，作为对照的IL-2单药和ch22F8mu单药均未显示抗肿瘤作用，在按IL-2 0.3mg/kg剂量下，IL-2/ch22F8和IL-2/ch22F8mu组均体现弱活性，TGI分别为24.6％和38.2％；而在按IL-2计1.0mg/kg剂量下，IL-2/ch22F8和IL-2/ch22F8mu组均体现强活性，TGI分别为72.9％和71.9％，无显著差别，说明IL-2/ch22F8mu保留了良好的体内抗肿瘤活性。
实施例11、人源化抗人IL-2单克隆抗体和人源化抗人IL-2嵌合抗体的制备
对实施例1的候选鼠源抗体轻链可变区和重链可变区的氨基酸序列进行分析，依据Kabat规则确定鼠源抗体的3个抗原互补决定区(CDR)和4个框架区(FR)。22F8重链互补决定区的氨基酸序列为HCDR1：GFNIKNTY(氨基酸序列如SEQ ID NO:5所述)、HCDR2：IDPANGNT(氨基酸序列如SEQ ID NO:6所示)、HCDR3：GRSRGYAMDY(氨基酸序列如SEQ ID NO:7所示)，轻链互补决定区的氨基酸序列为LCDR1：DHINNW(氨基酸序列如SEQ ID NO:10所示)、LCDR2：GATSLET(氨基酸序列如SEQ ID NO:11所示)和LCDR3：QQYWSTPT(氨基酸序列如SEQ ID NO:12所示)。
在Germline数据库中选取与上述各鼠源抗体非FR区匹配最好的人源化模板。然后将鼠源抗体的CDR区移植到所选择的人源化模板上，替换人源模板的CDR区，重链可变区再与人IgG4恒定区(包含S228P突变)重组，轻链可变区与人的kappa链恒定区重组，同时以该抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基，以及对各抗体的VL和VH的构象有重要影响的残基进行回复突变，最终获得人源化抗人IL-2单克隆抗体(hu22F8)重链可变区(氨基酸序列如SEQ ID NO:24所示)，与人IgG4恒定区重组获得重组人源化抗人IL-2单克隆抗体重链(氨基酸序列如SEQ ID NO:27所示)，与突变的人IgG4恒定区重组获得人源化抗人IL-2抗体(hu22F8mu)重链(氨基酸序列如SEQ ID NO:26所示)，人源化抗人IL-2单克隆抗体轻链可变区(氨基酸序列如SEQ ID  NO:25所示)，与人的kappa链恒定区重组获得人源化抗人IL-2抗体的轻链(氨基酸序列如SEQ ID NO:28所示)。分别构建各人源化抗体的重链和轻链至pcDNA3.4表达载体，转染Expi-293F细胞，通过Protein G纯化获得人源化抗人IL-2单克隆抗体(hu22F8)和突变的人源化抗人IL-2抗体(hu22F8mu)，并通过SDS-PAGE电泳及SEC-HPLC确定各抗体分子量大小正确及纯度>95％。
实施例12、ch22F8mu和hu22F8mu结合人IL-2
采用ELISA方法检测各单抗结合人IL-2，方法同实施例3。
结果如图7所示，ELISA法检测hu22F8和hu22F8mu结合人IL-2的EC50分别为：6.76ng/ml和6.01ng/ml，即0.05nM和0.04nM，ch22F8和ch22F8mu结合人IL-2的EC50分别为：6.29ng/ml和7.24ng/ml，即0.04nM和0.05nM。
实施例13、hu22F8mu和ch22F8mu阻断IL-2结合CD25
实验方法同实施例4。
结果如图8所示，hu22F8和hu22F8mu阻断人IL-2结合CD25的IC50分别为：218.6ng/ml和227.9ng/ml，即1.46nM和1.52nM；ch22F8和ch22F8mu阻断人IL-2结合CD25的IC50分别为：256.7ng/ml和236.6ng/ml，即1.71nM和1.58nM。表明Fc的上述突变的人源化抗体或嵌合抗体不影响其阻断IL-2结合CD25的作用。
实施例14、IL-2/hu22F8mu复合物(SPGD01)刺激CTLL2细胞增殖
本实施例以CTLL2细胞增殖实验说明复合物IL-2/hu22F8mu(SPGD01)、IL-2/hu22F8、IL-2/ch22F8和IL-2/ch22F8mu的体外生物学活性。方法如下：
CTLL2细胞以含10％FBS的1640培养液稀释至1E5/ml，100ul/孔加入细胞培养板。IL-2分别与hu22F8mu、hu22F8、ch22F8和ch22F8mu按质量比1:7混合，室温放置30分钟，形成复合物SPGD01(IL2/hu22F8mu)、IL-2/hu22F8、IL-2/ch22F8和IL-2/ch22F8mu，按IL-2计，将IL-2、上述各复合物以含10％FBS的1640培养液稀释至50ng/ml，后2倍比稀释共8个梯度后分别加入上述含 CTLL2细胞的培养板，37℃、5％CO 2细胞培养箱培养72h，稀释后以CCK8测定各孔的相对细胞数，并计算EC50，判定样品的活性。
结果如图9所示，IL-2，SPGD01(IL-2/hu22F8mu)、IL-2/hu22F8、IL-2/ch22F8和IL-2/ch22F8mu均可以刺激CTLL2细胞的增殖，IL-2的EC50为0.74ng/ml，SPGD01、IL-2/hu22F8的EC50分别为1.01ng/ml和1.09ng/ml，IL-2/ch22F8、IL-2/ch22F8mu的EC50分别为0.98ng/ml和1.04ng/ml，说明SPGD01与IL-2/hu22F8具有一致的生物学活性，IL-2/ch22F8mu和IL-2/ch22F8也具有一种的生物学活性，进一步说明，Fc的突变，不会导致IL-2/抗体复合物的生物学活性。
实施例15、SPGD01结合FcRn的能力明显减弱
采用ELISA方法测定SPGD01(IL-2/hu22F8mu)和IL-2/hu22F8结合人FcRn/β2M(购自北京义翘神州公司，货号CT009-H08H)和小鼠FcRn/β2M(购自北京义翘神州公司，货号CT029-M08H)，方法参考实施例3。小鼠FcRn/β2M和人FcRn/β2M以1ug/孔包被酶标板并封闭后，以pH为6.0的PBS稀释SPGD01或IL2/hu22F8至10ug/ml(按抗体质量计)，5倍比梯度稀释后加入酶标板，整个实验过程均采用pH为6.0的缓冲体系，其他同实施例3。
结果如图10所示，SPGD01(IL2/hu22F8mu)结合人FcRn/β2M较IL2/hu22F8明显减弱，总体上，SPGD01和IL2/hu22F8结合人FcRn/β2M均较微弱。
结果如图11所示，SPGD01(IL2/hu22F8mu)结合鼠FcRn/β2M较IL2/hu22F8明显减弱，总体上，SPGD01和IL2/hu22F8结合鼠FcRn/β2M均较微弱。
实施例16、SPGD01(IL-2/hu22F8mu)降低对实验小鼠的致死性
实验方法同实施例8。
结果如表3所示，在3mg/kg剂量下，IL-2/hu22F8组100％(10/10)实验动物死亡，SPGD01(IL-2/hu22F8mu)组则无实验动物死亡(0/10)；IL-2/hu22F8组在1mg/kg剂量下，50％实验动物存活；而SPGD01组在高剂量至6mg/kg下，100％实验动物存活，说明，抗体Fc在上述突变后，与IL-2形成的复合物对实验小鼠的毒性显著下降，这个结果与IL2/ch22F8mu一致。
表3各抗体剂量组实验动物存活率
抗体 1mg/kg 3mg/kg 6mg/kg
IL-2/hu22F8 50％ 0％ -
IL-2/SPGD01 - 100％ 100％
实施例17、SPGD01(IL-2/hu22F8mu)的体内抗肿瘤活性
实验方法同实施例5。
结果如图12所示，IL-2/hu22F8复合物体现良好的抗肿瘤活性，0.5mg/kg和0.25mg/kg组的TGI分别达到95.4％和85.6％；SPGD01(IL-2/hu22F8mu)同样体现极好的抗肿瘤活性，2mg/kg，1mg/kg和0.5mg/kg各组的TGI分别达到95.8％、84.5％和75.4％。统计分析表明，两者的抗肿瘤作用无显著差别(p>0.05)。
实验过程中，IL2/hu22F8的1mg/kg剂量组给药2次后出现动物死亡，其他各组则在完成6次给药均无动物死亡，说明IL2/hu22F8复合物具有较强的毒性，SPGD01(IL-2/hu22F8mu)在Fc突变后安全性明显提高。
实施例18、SPGD01(IL-2/hu22F8mu)与PD1单抗体内协同抗肿瘤活性
采用MC38移植瘤模型评价SPGD01(IL-2/hu22F8mu)与抗PD1单抗体内协同抗肿瘤作用，实验方法同实施例5。SPGD01剂量按IL-2计为4mg/kg，大鼠抗小鼠PD1单抗(购自Bio X Cell公司，货号BP0146)的剂量为5mg/kg，腹腔注射给药，3次/周，共6次。末次给药后3天，处死小鼠，获取小鼠血清和小鼠脾脏，采用酶活性方法测定小鼠血清中谷丙转氨酶(ALT)含量，采用常规流式方法(FACS)分析脾脏细胞CD4阳性细胞、CD8阳性细胞和CD4、CD25阳性细胞。其他参考实施例5。
结果如图13所示，SPGD01(IL2/hu22F8mu)单药和PD1单抗(anti-mPD1)单药均体现中等的抗肿瘤作用，TGI分别为61.7％和41.3％，联合用药的TGI则达到87.8％，体现良好的协同作用。
结果如图14所示，各给药组与PBS对照组相比，ALT值均无显著差异(p>0.05)。
结果如图15所示，anti-mPD1组CD4阳性细胞与CD8阳性细胞比值(CD4/CD8)与PBS组无显著差异(p>0.05)，但SPGD01组和联合用药组(SPGD01+anti-mPD1)组明显低于PBS组，相差非常显著(p<0.01,p<0.001)。
结果如图16所示，anti-mPD1组CD4+CD25+与CD3阳性细胞比值(CD4+CD25+/CD3+)与PBS组无显著差异(p>0.05)，但SPGD01组和联合用药组(SPGD01+anti-mPD1)组明显低于PBS组，相差显著(p<0.05)。
实施例19、SPGD01(IL-2/hu22F8mu)和hu22F8mu具有良好的稳定性
将IL-2和hu22F8mu按质量比1:7混匀，即形成SPGD01(IL-2/hu22F8mu)，与hu22F8mu置4℃保持相应时间后，采用分子筛高效液相色谱(SEC-HPLC)检测SPGD01和hu22F8mu放置不同时间后纯度的变化，考察SPGD01和hu22F8mu的稳定性。方法如下：
采用TSKgel G3000SWXL色谱柱(TSK公司)，在HPLC Ultimate 3000(Thermo公司)色谱仪上进行。检测流动相为PBS(pH 7.4)，恒定流速0.8ml/min，上样量为100ug/100ul。采用280nM吸收峰积分法计算目标蛋白在总蛋白中的含量(％)来表示纯度。
结果如图17、图18、图19和和图20所示，SPGD01(IL-2/hu22F8mu)在PBS溶液中，0周是纯度为95％，4℃放置24周后，纯度为94％，变化率<2％；单抗hu22F8mu在PBS溶液中，0周时纯度为93％，在4℃放置6周后，纯度为95％，变化率<3％，表明IL-2/抗体复合物SPGD01和相关单抗hu22F8mu在PBS缓冲液中，4℃条件下保持稳定。
上述实施例仅示例性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。

Claims (10)

  1. 一种抗人IL-2单克隆抗体，其特征在于，其具有：
    氨基酸序列为SEQ ID NO:4的重链可变区，和氨基酸序列为SEQ ID NO:9的轻链可变区。
  2. 编码如权利要求1所述的抗人IL-2单克隆抗体的核苷酸，其特征在于，其具有：
    如SEQ ID NO:3所示的重链可变区的核苷酸序列；如SEQ ID NO:8所示的轻链可变区的核苷酸序列。
  3. 一种抗人IL-2嵌合抗体，其特征在于，其具有：
    氨基酸序列为SEQ ID NO:15的重链序列，和氨基酸序列为SEQ ID NO:16的轻链序列。
  4. 一种Fc突变的抗人IL-2嵌合抗体，其特征在于，其具有：
    氨基酸序列为SEQ ID NO:18的重链序列，和氨基酸序列为SEQ ID NO:16的轻链序列。
  5. 一种人源化抗人IL-2单克隆抗体，其特征在于，其是在权利要求1所述的抗人IL-2单克隆抗体的基础上，采用CDRs移植技术、CDR区突变设计构建的人源化抗人IL-2单克隆抗体。
  6. 如权利要求5所述的人源化抗人IL-2单克隆抗体，其特征在于，其具有：氨基酸序列为SEQ ID NO:24的重链可变区，和氨基酸序列为SEQ ID NO:25的轻链可变区。
  7. 一种人源化抗人IL-2抗体，其特征在于，其具有：
    氨基酸序列为SEQ ID NO:27的重链序列，和氨基酸序列为SEQ ID NO:28的轻链序列。
  8. 一种Fc突变的人源化抗人IL-2抗体，其特征在于，其具有：
    氨基酸序列为SEQ ID NO:26的重链序列，和氨基酸序列为SEQ ID NO:28的轻链序列。
  9. 一种抗体复合物，其特征在于，其由IL-2与权利要求3所述的抗人IL-2嵌合抗体或权利要求4所述的Fc突变的抗人IL2嵌合抗体或权利要求7所述的人源化抗人IL-2抗体或权利要求8所述的Fc突变的人源化抗人IL-2抗体按质量比1:7混合制得。
  10. 如权利要求9所述的抗体复合物在制备治疗抗肿瘤药物中的用途。
PCT/CN2022/141438 2022-10-13 2022-12-23 一种抗人il-2单克隆抗体及其应用 WO2024077773A1 (zh)

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Publication number Priority date Publication date Assignee Title
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CN109071648A (zh) * 2015-10-23 2018-12-21 辉瑞有限公司 抗il-2抗体及其组合物和用途
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WO2021161287A2 (en) * 2020-02-16 2021-08-19 Aulos Bioscience, Inc Engineered anti-il-2 antibodies
WO2021164722A1 (zh) * 2020-02-21 2021-08-26 江苏恒瑞医药股份有限公司 抗il-2抗体、其抗原结合片段及其医药用途

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CN101189265A (zh) * 2005-06-01 2008-05-28 米克罗麦特股份公司 抗il2抗体
CN109071648A (zh) * 2015-10-23 2018-12-21 辉瑞有限公司 抗il-2抗体及其组合物和用途
CN110087681A (zh) * 2016-09-28 2019-08-02 佐马美国有限公司 结合白细胞介素-2的抗体和其用途
WO2021161287A2 (en) * 2020-02-16 2021-08-19 Aulos Bioscience, Inc Engineered anti-il-2 antibodies
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