Anti-human il-2 monoclonal antibody and use thereof

Abstract

This invention belongs to the field of antibody engineering technology, and relates to an anti-human IL-2 monoclonal antibody and its application, specifically involving the preparation and application of an anti-human IL-2 monoclonal antibody and antibody complex. The anti-human IL-2 monoclonal antibody provided by this invention, after binding to IL2, retains the binding of IL2 to IL2Rβ / γ (CD122 / 132), while simultaneously blocking the binding of IL-2 to IL-2Rα (CD25), effectively inhibiting the growth of xenografts in mice. Furthermore, this invention introduces a mutation into the Fc region of the anti-human IL-2 monoclonal antibody. When the Fc-mutated monoclonal antibody forms a complex with IL-2, it not only retains the in vivo anti-tumor activity of the IL-2 / antibody complex but also significantly reduces side effects and improves drug safety.

Description

Technical Field

[0001] This invention belongs to the field of antibody engineering technology, and relates to an anti-human IL-2 monoclonal antibody and its application, specifically to an anti-human IL-2 monoclonal antibody, the preparation of antibody complexes and their application. Background Technology

[0002] Interleukin-2 (IL-2) is a potent immunomodulatory cytokine that plays a role in T cell activation and growth, and is involved in anti-tumor effects and transplant rejection. Many drugs target IL-2, such as aldesleukin, and have a long history of clinical use in treating malignant tumors. However, numerous toxic side effects have been observed in the clinical application of IL-2, particularly liver and lung toxicity, limiting its use. Furthermore, the use of IL-2 has been found to stimulate Treg proliferation and induce activation-induced death (AICD) in immune cells, further impacting its efficacy.

[0003] IL-2 exerts its effect by binding to the IL-2 receptor, which consists of three subunits: α, β, and γ. α is a high-affinity receptor, while β and γ, although low-affinity receptors, mediate the signal transduction following IL-2 binding. The antitumor activity of IL-2 is mainly achieved by activating CD8-positive T cells and NK cells; however, its simultaneous expansion of CD4-positive Treg cells weakens or even completely eliminates its antitumor effect.

[0004] CD4-positive Treg cells highly express IL-2Rα (CD25). Since CD25 is a high-affinity receptor for IL-2, low concentrations of IL-2 preferentially bind to Treg cells, exerting an anti-tumor immunosuppressive effect by binding to CD25. CD8-positive T cells and NK cells, because they do not express or express low levels of α receptors but highly express β and γ receptors, are affected by high concentrations of IL-2. Therefore, reducing the binding of IL-2 to α receptors or enhancing the binding of IL-2 to β / γ receptors, thus causing IL-2 to preferentially act on CD8-positive T cells and NK cells, is an effective way to achieve the anti-tumor effect of IL-2.

[0005] However, current research has found that IL-2 monoclonal antibodies, IL-2-Fc fusion proteins, and antibody-IL-2 bifunctional molecules all have certain toxic side effects, including liver toxicity, when used to treat malignant tumors. Therefore, researching and developing an IL-2 drug with low toxicity and high anti-tumor activity is an urgent problem to be solved. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for preparing and applying anti-human IL-2 monoclonal antibodies and antibody complexes. Using recombinant human IL-2 protein as an immunogen, this invention prepares a murine anti-human IL-2 monoclonal antibody m22F8 via hybridoma technology. The binding activity and affinity of the murine antibody to IL-2 are analyzed, and its ability to block IL-2 binding to CD25 is investigated. A chimeric anti-human IL-2 antibody is prepared by determining the mouse hybridoma antibody gene. Based on the performance analysis of the murine antibody and chimeric antibody, a humanized anti-human IL-2 monoclonal antibody is constructed using CDR transplantation technology and CDR region mutation design. The affinity of the humanized antibody to IL-2 is measured, and its ability to block IL-2 binding to CD25 is analyzed. The anti-tumor activity of the anti-human IL-2 monoclonal antibody m22F8 and the antibody complex, as well as the synergistic anti-tumor activity of the antibody complex and PD1 monoclonal antibody, are studied, demonstrating promising applications in anti-tumor drugs.

[0007] This invention provides an anti-human IL-2 monoclonal antibody, which has the following characteristics:

[0008] The heavy chain variable region has the amino acid sequence of SEQ ID NO:4, and the light chain variable region has the amino acid sequence of SEQ ID NO:9.

[0009] Furthermore, the nucleotide encoding the aforementioned anti-human IL-2 monoclonal antibody has the following characteristics:

[0010] Nucleotide sequences of the heavy chain variable region as shown in SEQ ID NO:3; nucleotide sequences of the light chain variable region as shown in SEQ ID NO:8.

[0011] Furthermore, the antibody is derived from a mouse antibody.

[0012] This invention provides an anti-human IL-2 chimeric antibody, which has the following characteristics:

[0013] The amino acid sequence is the heavy chain sequence of SEQ ID NO:15 and the amino acid sequence is the light chain sequence of SEQ ID NO:16.

[0014] The present invention also provides an Fc-mutated chimeric anti-human IL-2 antibody having: a heavy chain sequence with the amino acid sequence SEQ ID NO:18 and a light chain sequence with the amino acid sequence SEQ ID NO:16.

[0015] The preparation method is as follows: using site-directed mutagenesis or gene synthesis technology, the H at position 310 of the human IgG4 constant region is converted to A and the H at position 435 is converted to Q to form the mutated human IgG4 constant region, with the amino acid sequence shown in SEQ ID NO:17; the heavy chain variable region of the aforementioned 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, with the amino acid sequence shown in SEQ ID NO:18; the light chain variable region sequence of the aforementioned 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, with the amino acid sequence shown in SEQ ID NO:16; then, the antibody is constructed into the pcDNA3.4 expression vector, transfected into Expi-293F cells, and purified by Protein G to obtain the Fc-mutated anti-human IL-2 chimeric antibody.

[0016] Furthermore, the present invention also provides a humanized anti-human IL-2 monoclonal antibody, which is a humanized anti-human IL-2 monoclonal antibody constructed by CDR transplantation technology and CDR region mutation design based on the aforementioned anti-human IL-2 monoclonal antibody.

[0017] Furthermore, the humanized anti-human IL-2 monoclonal antibody has: a heavy chain variable region with the amino acid sequence SEQ ID NO:24, and a light chain variable region with the amino acid sequence SEQ ID NO:25.

[0018] The present invention also provides a humanized anti-human IL-2 antibody, which has the following characteristics:

[0019] The amino acid sequence is the heavy chain sequence of SEQ ID NO:27 and the amino acid sequence is the light chain sequence of SEQ ID NO:28.

[0020] This invention also provides an Fc-mutated humanized anti-human IL-2 antibody, which has the following characteristics:

[0021] The amino acid sequence is the heavy chain sequence of SEQ ID NO:26 and the amino acid sequence is the light chain sequence of SEQ ID NO:28.

[0022] The preparation method is as follows: the variable region of the heavy chain of the humanized anti-human IL-2 monoclonal antibody is recombined with the constant region of the mutant IgG4 as shown in SEQ ID NO:17 to obtain the heavy chain of the Fc-mutant humanized anti-human IL-2 antibody, with the amino acid sequence shown in SEQ ID NO:26; the variable region sequence of the light chain of the anti-human IL-2 monoclonal antibody is recombined with the constant region of the human kappa chain as shown in SEQ ID NO:14 to obtain the light chain of the humanized anti-human IL-2 chimeric antibody, with the amino acid sequence shown in SEQ ID NO:28; then, it is constructed into the pcDNA3.4 expression vector, transfected into Expi-293F cells, and purified by Protein G to obtain the Fc-mutant humanized anti-human IL-2 antibody.

[0023] The present invention also provides an antibody complex prepared by mixing IL-2 (interleukin-2) with the anti-human IL-2 chimeric antibody obtained in the present invention or the Fc-mutated anti-human IL-2 chimeric antibody or the humanized anti-human IL-2 antibody or the Fc-mutated humanized anti-human IL-2 antibody at a mass ratio of 1:7.

[0024] Furthermore, the present invention also provides the use of the antibody complex in the preparation of therapeutic antitumor drugs.

[0025] Compared with existing technologies, the anti-human IL-2 monoclonal antibody prepared in this invention retains its binding to IL2Rβ / γ (CD122 / 132) while simultaneously blocking the binding of IL2 to IL2Rα (CD25), effectively inhibiting the growth of xenografts in mice. Furthermore, this invention introduces a mutation into the Fc region of the IL-2 monoclonal antibody. When the Fc-mutated monoclonal antibody forms a complex with IL-2, it not only retains the in vivo antitumor activity of the IL-2 / antibody complex but also significantly reduces side effects and improves drug safety.

[0026] To better understand this invention, some terms are first defined. Other definitions are listed throughout the detailed description section.

[0027] The term "IL-2" stands for interleukin-2, a cytokine in the chemokine family. IL-2 has a molecular weight of 15 kDa and is a glycoprotein containing 113 amino acid residues. In humans, it is encoded by a gene on chromosome 4.

[0028] The term "antibody" as used herein is intended to include full-length antibodies and any antigen-binding fragments (i.e., antigen-binding portions) or single chains. A full-length antibody is a glycoprotein consisting of at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain comprises a light chain variable region (VL) and a light chain constant region (CL).

[0029] The term "monoclonal antibody" or "monoclonal antibody" refers to an antibody molecule that consists of a single molecule. Monoclonal antibodies exhibit specific binding specificity and affinity for a particular epitope.

[0030] The term "EC50," also known as the half-maximal effect concentration, refers to the concentration that produces 50% of the maximum effect.

[0031] The term "IC50," also known as half-inhibition concentration, refers to the concentration of a drug or inhibitor required to inhibit a specified biological process or a component of that process (such as an enzyme, receptor, or cell) by half. Attached image description:

[0032] Figure 1 Affinity map of murine antibody m22F8 against target antigen IL-2;

[0033] Figure 2 The image shows the CD25-ECD pattern of murine antibody m22F8 blocking human IL-2 binding;

[0034] Figure 3 This is a diagram showing the antitumor activity of the IL-2 / m22F8 complex;

[0035] Figure 4 Serum ALT levels in surviving mice were measured after two doses of IL-2 / ch22F8mu complex at a dose of 3 mg / kg (calculated as IL-2).

[0036] Figure 5 Serum ALT levels in surviving mice were measured after 6 administrations of the IL-2 / ch22F8mu complex at a dose of 1 mg / kg (based on IL-2).

[0037] Figure 6 This is a graph showing the in vivo antitumor activity of the IL-2 / ch22F8mu complex.

[0038] Figure 7 Map of ch22F8mu and hu22F8mu binding to human IL-2;

[0039] Figure 8The diagram shows the blocking effect of hu22F8mu and ch22F8mu on IL-2 binding to CD25.

[0040] Figure 9 Image showing the proliferation of CTLL2 cells stimulated by the IL-2 / hu22F8mu complex;

[0041] Figure 10 Graphs showing the effects of the IL-2 / hu22F8mu complex and the binding of IL-2 / hu22F8 to human FcRn / β2M;

[0042] Figure 11 The graph shows the binding effect of the IL-2 / hu22F8mu complex and IL-2 / hu22F8 on mouse FcRn / β2M.

[0043] Figure 12 This is a graph showing the in vivo antitumor activity of the IL-2 / hu22F8mu complex.

[0044] Figure 13 The diagram shows the antitumor effect of SPGD01 (IL-2 / hu22F8mu);

[0045] Figure 14 The effect of SPGD01 (IL-2 / hu22F8mu) on serum ALT in experimental mice is shown in the figure.

[0046] Figure 15 The effect of SPGD01 (IL-2 / hu22F8mu) on the composition of CD4+ / CD8+ lymphocytes in experimental animals is shown in the figure.

[0047] Figure 16 The effect of SPGD01 (IL-2 / hu22F8mu) on the composition of CD4+ / CD25+ lymphocytes in experimental animals is shown in the figure.

[0048] Figure 17 Stability plots for SPGD01(IL-2 / hu22F8mu) and hu22F8mu;

[0049] Figure 18 Stability plots for SPGD01(IL-2 / hu22F8mu) and hu22F8mu

[0050] Figure 19 Stability plots for SPGD01(IL-2 / hu22F8mu) and hu22F8mu

[0051] Figure 20 Stability plots for SPGD01(IL-2 / hu22F8mu) and hu22F8mu. Detailed Implementation

[0052] The present invention will be further described below through specific embodiments, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not depart from the basic idea of ​​the present invention, they are all within the scope of the present invention.

[0053] Example 1: Antigen Immunization of Mice and Preparation and Screening of Hybridomas

[0054] Step 1: Balb / c mice (purchased from Shanghai Lingchang Biotechnology Co., Ltd.) were routinely immunized with human IL-2 protein expressed in prokaryotic Escherichia coli (purchased from Sino Biological, catalog number GMP-11848-HNAE, amino acid sequence as shown in SEQ ID NO:1). On day 1, IL-2 protein was emulsified with Freund's complete adjuvant and injected subcutaneously into Balb / c mice at multiple sites (human IL-2 protein, 50 μg / mouse / 0.5 ml). On day 21, human IL-2 protein was emulsified with Freund's incomplete adjuvant and injected subcutaneously into Balb / c mice (human IL-2 protein, 50 μg / mouse / 0.5 ml). On day 41, human IL-2 protein, 50 μg / mouse / 0.2 ml, was injected intraperitoneally to stimulate fusion. 3-4 days later, the spleens of the mice were harvested for fusion experiments.

[0055] Step 2: 3-4 days after the final immunization of mice, mouse spleen cells and mouse myeloma cells SP2 / 0 were electrofused using a standard hybridoma technique via an electrofusion apparatus (purchased from BTX). The fused cells were then resuspended in complete culture medium (RPMI 1640 and DMEM F12 medium mixed 1:1 with the addition of 1% Glutamine, 1% Sodium pyruvate, 1% MEM-NEAA (minimum basal medium - non-essential amino acid solution), 1% Penicillin-streptomycin, 50 μM β-mercaptoethanol, and 20% FBS (fetal bovine serum); all products were purchased from Gibco). The cells were then injected at a rate of 10... 5Cells were cultured at 100 μl / well in 25 wells of 96-well plates overnight. The next day, 100 μl of complete culture medium containing 2×HAT was added to each well, bringing the culture medium in each well to 200 μl (containing 1×HAT). After 7–12 days, the supernatant was harvested, and hybridoma wells showing positive human IL-2 binding activity were screened using an indirect enzyme-linked immunosorbent assay (ELISA), yielding 442 positive wells. Further screening was conducted to determine the effect of the hybridoma wells on blocking IL-2 binding to CD25, resulting in 25 positive wells. These hybridoma wells, which were positive for both human IL-2 binding and blocking IL-2 binding to CD25, were subjected to first and second rounds of subcloning using limiting dilution to obtain a hybridoma cell line named SPGD01-22F8.

[0056] The method for screening hybridoma wells with positive human IL-2 binding activity using indirect enzyme-linked immunosorbent assay (ELISA) is as follows: Recombinant human IL-2 protein was diluted to 1 μg / ml with coating buffer (50 mM carbonate coating buffer, pH 9.6), and 100 μl / well was added to the ELISA plate. The plate was coated overnight at 4°C. The plate was washed three times with PBST, and 200 μl / well of blocking buffer (2% BSA-PBST) was added. The plate was incubated at 37°C for 1 hour, followed by one wash with PBST. The collected hybridoma supernatant was then added sequentially to the blocked ELISA plate, 100 μl / well, and incubated at 37°C for 1 hour. Wash the plate three times with PBST, add HRP-labeled goat anti-mouse IgG secondary antibody (purchased from Millipore, catalog number AP181P), and incubate at 37°C for 30 min. After washing the plate five times with PBST, pat dry any remaining droplets on absorbent paper, add 100 μl of TMB (purchased from BD, catalog number 555214) to each well, and incubate at room temperature (20±5°C) in the dark for 5 min. Add 50 μl of 2M H2SO4 stop solution to each well to terminate the substrate reaction, and read the OD value at 450 nm using a microplate reader to analyze the binding ability of the test antibody to the target antigen IL-2.

[0057] The method for detecting the blocking of IL-2 binding to CD25 in hybridoma wells is as follows: Recombinant human CD25-ECD (purchased from Beijing Sino Biological Co., Ltd., catalog number 50292-M02H) was diluted to 1 μg / ml with coating buffer (50 mM carbonate coating buffer, pH 9.6), and 100 μl / well was added to the microplate and coated overnight at 4°C. The plate was washed 3 times with PBST, and 200 μl / well of blocking buffer (2% BSA-PBST) was added. After incubation at 37°C for 1 h, the plate was washed once with PBST. 60 μl of the collected supernatant from each hybridoma was added to 80 μl of biotin-labeled IL-2 (bio-IL-2, purchased from Sino Biological Co., Ltd., catalog number 11848-HNAE-B) diluted to 10 ng / ml and incubated at 37°C for 30 min. Then, 100 μl / well was added to the blocked microplate and incubated at 37°C for 1 h. Wash the plate three times with PBST, add HRP-labeled SA (SA-HRP, Pierce), and incubate at 37°C for 30 min. After washing the plate five times with PBST, pat dry any remaining droplets on absorbent paper, add 100 μl of TMB (purchased from BD, catalog number 555214) to each well, and incubate at room temperature (20±5°C) in the dark for 5 min. Add 50 μl of 2M H2SO4 stop solution to each well to terminate the substrate reaction, and read the OD value at 450 nm using a microplate reader to analyze the effect of the antibody in blocking IL-2 binding to CD25.

[0058] Example 2: Preparation of mouse-derived anti-human IL-2 monoclonal antibody m22F8

[0059] The hybridoma cell lines obtained through screening were amplified in complete culture medium (as described in Example 1), centrifuged, and transferred to serum-free SFM medium (purchased from Life Technologies, catalog number 12045-076) to achieve a cell density of 1–2 × 10⁶ cells / year. 7 The culture was cultured at 5% CO2 and 37°C for 1 week. The culture supernatant was obtained by centrifugation and purified by Protein G affinity chromatography to obtain the mouse-derived anti-human IL-2 monoclonal antibody m22F8.

[0060] Example 3: Determination of the affinity of mouse-derived anti-human IL-2 monoclonal antibody m22F8 for the target antigen IL2.

[0061] The affinity of mouse anti-human IL-2 monoclonal antibody m22F8 for recombinant human IL-2 protein was determined by ELISA. The experimental method is as follows:

[0062] Recombinant human IL-2 protein was diluted to 1 μg / ml with coating buffer (50 mM carbonate coating buffer, pH 9.6), and 100 μl / well was added to the microplate. The plate was incubated overnight at 4°C. The plate was washed three times with PBST, and 200 μl / well of blocking buffer (2% BSA-PBST) was added. After incubation at 37°C for 1 hour, the plate was washed once with PBST and set aside. Mouse anti-human IL-2 monoclonal antibody m22F8 was diluted with diluent (1% BSA-PBST) to 5000 / 1000 / 200 / 40 / 8 / 1.6 / 0.32 / 0 ng / ml, and 100 μl / well was added sequentially to the blocked microplate. The plate was incubated at 37°C for 1 hour. Wash the plate three times with PBST, add HRP-labeled goat anti-mouse IgG secondary antibody (purchased from Millipore, catalog number AP181P), and incubate at 37°C for 30 min. After washing the plate five times with PBST, pat dry any remaining droplets on absorbent paper, add 100 μl of TMB (purchased from BD, catalog number 555214) to each well, and incubate at room temperature (20±5°C) in the dark for 5 min. Add 50 μl of 2M H2SO4 stop solution to each well to terminate the substrate reaction, and read the OD value at 450 nm using a microplate reader to analyze the binding ability of the test antibody to the target antigen IL-2.

[0063] The results are as follows Figure 1 As shown, the EC50 of the mouse-derived anti-human IL-2 monoclonal antibody m22F8 binding to human IL-2 is 6.90 ng / ml, or 0.05 nM, indicating good affinity.

[0064] Example 4: Mouse-derived anti-human IL-2 monoclonal antibody m22F8 blocks human IL2 binding to CD25-ECD

[0065] Recombinant hCD25 (amino acid sequence as shown in SEQ ID NO:2) was diluted to 1 μg / ml with coating buffer (50 mM carbonate coating buffer, pH 9.6), and 100 μl / well was added to the microplate. The plate was incubated overnight at 4°C. After washing three times with PBST, 200 μl / well of blocking buffer (2% BSA-PBST) was added, and the plate was incubated at 37°C for 1 h. The plate was then washed once with PBST and set aside. Mouse anti-human IL-2 monoclonal antibody m22F8 was serially diluted with diluent (1% BSA-PBST) to 10000 / 2000 / 400 / 80 / 16 / 3.2 / 0.64 / 0 ng / ml, and mixed with an equal volume of bio-IL2 diluted to 20 ng / ml. After incubation at 37°C for 30 min, 100 μl / well was added to the blocked microplate and incubated at 37°C for 1 h. Wash the plate three times with PBST, add HRP-labeled SA (SA-HRP, Pierce), and incubate at 37°C for 30 min. After washing the plate five times with PBST, pat dry any remaining droplets on absorbent paper, add 100 μl of TMB (purchased from BD, catalog number 555214) to each well, and incubate at room temperature (20±5°C) in the dark for 5 min. Add 50 μl of 2MH2SO4 stop solution to each well to terminate the substrate reaction, and read the OD value at 450 nm using a microplate reader to analyze the effect of the antibody in blocking IL-2 binding to CD25.

[0066] The results are as follows Figure 2 As shown, the murine 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.

[0067] Example 5: IL-2 / m22F8 complex inhibits the growth of MC38 cell xenografts in mice.

[0068] Mouse colon cancer MC38 cells cultured in vitro were collected, and the cell suspension concentration was adjusted to 1×10⁻⁶. 7 / ml. Hair was shaved from the right rib area of ​​C57BL / 6 mice. Under aseptic conditions, 100 μl of cell suspension was subcutaneously injected into the right rib area of ​​C57 mice. The diameter of the subcutaneous xenografts in mice was measured using calipers. Tumors were allowed to grow to an average volume of 100-200 mm. 3 The animals were then randomly divided into groups of 8. IL-2 and m22F8 were mixed at a mass ratio of 1:7 and incubated at room temperature for 15 minutes to prepare the IL-2 / m22F8 complex. This complex was administered at a dose of 1 mg / kg (based on IL-2), while the control group received an equal volume of PBS. The complex was administered intraperitoneally twice weekly for two consecutive weeks. Throughout the experiment, the diameter of the transplanted tumor was measured twice weekly, and the mice were weighed simultaneously. The formula for calculating tumor volume (TV) is:

[0069] TV = 1 / 2 × a × b²

[0070] Where a and b represent length and width, respectively. The relative tumor volume (RTV) is calculated based on the measurement results using the formula: RTV = Vt / V0. Here, V0 is the tumor volume measured at the time of grouped drug administration (i.e., d0), and Vt is the tumor volume at each measurement. The evaluation index for antitumor activity is TGI (tumor inhibition rate %) / T / C (%), calculated using the following formula:

[0071] TGI% = 100% - T / C (%)

[0072] Relative tumor proliferation rate T / C (%) = (TRTV / CRTV) × 100

[0073] TRTV: RTV in the treatment group; CRTV: RTV in the negative control group.

[0074] The results are as follows Figure 3 As shown, the IL-2 / m22F8 complex exhibited excellent antitumor activity, with tumor inhibition rates approaching 100% after two doses. However, during the experiment, animals commonly exhibited clinical symptoms including reduced activity, decreased food intake, ruffled fur, and decreased body temperature, with 2 / 8 of the animals dying. This indicates that while the IL-2 / m22F8 complex possesses good in vivo antitumor activity, it has safety concerns, including the appearance of obvious clinical symptoms and even death.

[0075] Example 6: Determination of mouse hybridoma antibody gene and preparation of chimeric antibodies

[0076] In this embodiment, the heavy chain variable region and light chain variable region of hybridoma m22F8 were obtained through relevant molecular biology methods, and were further used to construct chimeric antibodies.

[0077] RNA was extracted from hybridoma cells using Trizol, and cDNA was obtained through reverse transcription of mRNA. Then, using cDNA as a template, PCR was performed using degenerate primers for the heavy and light chains of the mouse antibody (Antibody Engineering, Volume 1, Edited by Roland Kontermann and Stefan Dübel; the sequence of the combined primers is from page 323). The obtained PCR products were sequenced and analyzed using the Kabat database to confirm that the obtained sequence was the variable region sequence of the mouse antibody.

[0078] The relevant sequence information is as follows:

[0079] The m22F8 heavy chain variable region gene sequence is 351 bp 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 318 bp 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.

[0080] The variable region sequences of each hybridoma heavy chain were recombined with the human IgG4 constant region (containing 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 variable region sequences of the light chain 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).

[0081] 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 the NARA1 monoclonal antibody were synthesized according to the literature (patent US 2017 / 0183403A1). The heavy chain variable region of the 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 the 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).

[0082] The heavy and light chain genes were constructed into the pcDNA3.4 expression vector, respectively, and transfected into Expi-293F cells. The chimeric antibodies ch22F8 and NARA1 were obtained by purification with Protein A. The molecular weight of each expressed antibody was determined to be around 150 kDa by SDS-PAGE electrophoresis and SEC-HPLC, with antibody purity >95%. The antibodies were quantified, aliquoted, and stored at -80℃ for later use.

[0083] Example 7: Preparation of anti-human IL-2 chimeric antibody

[0084] Using site-directed mutagenesis or gene synthesis, the H at position 310 of the human IgG4 constant region (amino acid sequence as shown in SEQ ID NO: 13) is converted to A and the H at position 435 is converted to Q, while the other amino acid sequences remain unchanged, forming a mutated human IgG4 constant region (amino acid sequence as shown in SEQ ID NO: 17). The m22F8 heavy chain variable region is then 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 is then recombined with the mutated human IgG4 constant region to form a mutated NARA1 (NARA1mu) heavy chain (amino acid sequence as shown in SEQ ID NO: 22).

[0085] The aforementioned mutant heavy chain genes were constructed into the pcDNA3.4 expression vector, and the respective light chains prepared in Example 6 were transfected into Expi-293F cells. The anti-human IL-2 chimeric antibody (ch22F8mu) and NARA1mu were obtained by purification with Protein G. The molecular weight of each expressed antibody was determined to be around 150kD by SDS-PAGE electrophoresis and SEC-HPLC, and the antibody purity was >95%. The antibodies were quantified, aliquoted, and frozen at -80℃ for later use.

[0086] Example 8: Lethality of the IL-2 / ch22F8mu complex in experimental mice

[0087] IL-2 was mixed with each of the test antibodies (ch22F8, ch22F8mu, NARA1, NARA1mu) at a mass ratio of 1:7 and incubated at room temperature for 15 minutes to form a complex. The complex was then injected intraperitoneally into C57BL / 6 mice (Vioton Life Sciences) twice on days 1 and 4, based on the dosage of IL-2: 1 mg / kg and / or 3 mg / kg and / or 6 mg / kg (see Table 1 for details). The mortality of the experimental mice was observed on day 7. The control group was given the same volume of PBS.

[0088] Table 1 Dosage of each antibody

[0089] Dosage (IL-2) ch22F8 ch22F8mu NARA1 NARA1mu 1mg / kg √ - - - 3mg / kg √ √ √ √ 6mg / kg - √ - -

[0090] Table 2 Survival rate of experimental animals in each antibody dosage group

[0091] Antibody 1mg / kg 3mg / kg 6mg / kg IL-2 / ch22F8 40％ 0％ - IL-2 / ch22F8mu - 100％ 100％ NARA1 - 0％ - NARA1mu - 100％ -

[0092] The results are shown in Table 2:

[0093] (1) On day 7 of the experiment, at a dose of 3 mg / kg, all experimental animals in the ch22F8 and NARA1 groups died (survival rate 0%), but all experimental animals in the ch22F8mu and NARA1mu groups survived (survival rate 100%), indicating that after the IL-2 monoclonal antibody was modified as described, the complex formed with IL-2 had a significantly reduced toxicity to experimental mice.

[0094] (2) In the ch22F8mu group, the survival rate of experimental animals remained 100% when the dose was increased to 6 mg / kg, while in the ch22F8 group, 60% of the animals still died when the dose was reduced to 1 mg / kg. This further demonstrates that the toxicity of the complex formed by ch22F8mu and IL-2 in experimental mice is significantly reduced after Fc mutation. Example 9: IL-2 / ch22F8mu reduced liver damage in experimental animals.

[0095] IL-2 was mixed with the test antibodies ch22F8mu and ch22F8 at a mass ratio of 1:7 and incubated at room temperature for 15 minutes to form a complex. C57BL / 6 mice (Violenta Pharmaceuticals) were intraperitoneally injected twice on days 1 and 4, based on IL-2 at a dose of 3 mg / kg. C57BL / 6 mice (Violenta Pharmaceuticals) were intraperitoneally injected twice a week, based on IL-2 at a dose of 1 mg / kg, for a total of 6 times. On the day after the last administration, blood was collected from surviving mice to obtain serum and measure ALT.

[0096] result Figure 4 and Figure 5 As shown:

[0097] (1) At the IL-2 1 mg / kg dose, 3 / 8 mice in the IL-2 / ch22F8 group died, and the average ALT of the 5 / 8 surviving mice was 425.2 U / L. No animals died in the IL-2 / ch22F8mu group, and the average ALT of all 8 mice was 64.0 U / L.

[0098] (2) At the IL-2 dose of 3 mg / kg, 3 / 8 mice in the IL-2 / ch22F8 group died, and the average ALT of the surviving mice was 303.2 U / L. No animals died in the IL-2 / ch22F8mu group, and the average ALT of all 8 mice was 62.3 U / L.

[0099] (3) The average ALT level in the PBS control group was 29.4 U / L, indicating that the liver toxicity of IL-2 / 22F8mu was significantly reduced compared to IL-2 / ch22F8.

[0100] Example 10: In vivo antitumor activity of the IL-2 / ch22F8mu complex

[0101] The experimental method was the same as in Example 5. IL-2 was mixed with each of the test antibodies (ch22F8, ch22F8mu) at a mass ratio of 1:7, and incubated at room temperature for 15 minutes to form a complex. The complexes were administered at doses of 0.3 mg / kg and 1 mg / kg, respectively, with IL-2 monotherapy at a dose of 1 mg / kg as a control. The drugs were administered via intraperitoneal injection three times a week for two consecutive weeks.

[0102] The results are as follows Figure 6 As shown, neither IL-2 monotherapy nor ch22F8mu monotherapy, serving as controls, exhibited antitumor activity. At a dose of IL-2 0.3 mg / kg, both the IL-2 / ch22F8 and IL-2 / ch22F8mu groups showed weak activity, with TGIs of 24.6% and 38.2%, respectively. However, at a dose of 1.0 mg / kg (based on IL-2), both the IL-2 / ch22F8 and IL-2 / ch22F8mu groups showed strong activity, with TGIs of 72.9% and 71.9%, respectively, showing no significant difference. This indicates that IL-2 / ch22F8mu retained good in vivo antitumor activity.

[0103] Example 11: Preparation of humanized anti-human IL-2 monoclonal antibody and humanized anti-human IL-2 chimeric antibody

[0104] The amino acid sequences of the light chain variable region and heavy chain variable region of the candidate murine antibody from Example 1 were analyzed, and the three antigen complementarity-determining regions (CDRs) and four frame regions (FRs) of the murine antibody were determined according to Kabat rules. The amino acid sequences of the 22F8 heavy chain complementarity-determining regions are HCDR1: GFNIKNTY (amino acid sequence as shown in SEQ ID NO:5), HCDR2: IDPANGNT (amino acid sequence as shown in SEQ ID NO:6), and HCDR3: GRSRGYAMDY (amino acid sequence as shown in SEQ ID NO:7). The amino acid sequences of the light chain complementarity-determining regions are 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).

[0105] Humanized templates that best match the non-FR regions of the aforementioned murine antibodies were selected from the Germline database. Then, the CDR region of the murine antibody was transplanted onto the selected humanized template, replacing the CDR region of the human template. The heavy chain variable region was then recombined with the human IgG4 constant region (containing the S228P mutation), and the light chain variable region was recombined with the human kappa chain constant region. Simultaneously, based on the three-dimensional structure of this antibody, reversion mutations were performed on the embedded residues, residues that directly interact with the CDR region, and residues that significantly affect the conformation of the VL and VH of each antibody. Finally, the heavy chain variable region of the humanized anti-human IL-2 monoclonal antibody (hu22F8) (amino acid sequence as shown in SEQ ID NO:24) was obtained. This region was then 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 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). The light chain variable region of the humanized anti-human IL-2 monoclonal antibody (amino acid sequence as shown in SEQ ID NO:27) was also obtained. The light chain of the humanized anti-human IL-2 antibody (amino acid sequence shown in SEQ ID NO: 28) was obtained by recombination with the constant region of the human kappa chain (as shown in SEQ ID NO: 25). The heavy and light chains of each humanized antibody were constructed into the pcDNA3.4 expression vector, transfected into Expi-293F cells, and purified by Protein G to obtain the humanized anti-human IL-2 monoclonal antibody (hu22F8) and the mutant humanized anti-human IL-2 antibody (hu22F8mu). The molecular weight and purity (>95%) of each antibody were confirmed by SDS-PAGE electrophoresis and SEC-HPLC.

[0106] Example 12: ch22F8mu and hu22F8mu binding to human IL-2

[0107] The binding of each monoclonal antibody to human IL-2 was detected using the ELISA method, the same as in Example 3.

[0108] The results are as follows Figure 7 As shown, the EC50 values ​​of hu22F8 and hu22F8mu binding to human IL-2 by ELISA were 6.76 ng / ml and 6.01 ng / ml, respectively (0.05 nM and 0.04 nM). The EC50 values ​​of ch22F8 and ch22F8mu binding to human IL-2 were 6.29 ng / ml and 7.24 ng / ml, respectively (0.04 nM and 0.05 nM).

[0109] Example 13: hu22F8mu and ch22F8mu block IL-2 binding to CD25

[0110] The experimental method is the same as in Example 4.

[0111] The results are as follows Figure 8 As shown, the IC50 values ​​of hu22F8 and hu22F8mu for blocking human IL-2 binding to CD25 were 218.6 ng / ml and 227.9 ng / ml, respectively (1.46 nM and 1.52 nM); the IC50 values ​​of ch22F8 and ch22F8mu for blocking human IL-2 binding to CD25 were 256.7 ng / ml and 236.6 ng / ml, respectively (1.71 nM and 1.58 nM). This indicates that humanized or chimeric antibodies against the above-mentioned Fc mutations do not affect their ability to block IL-2 binding to CD25.

[0112] Example 14: IL-2 / hu22F8mu complex (SPGD01) stimulates CTLL2 cell proliferation.

[0113] This embodiment uses a CTLL2 cell proliferation assay to demonstrate the in vitro biological activities of the complexes IL-2 / hu22F8mu (SPGD01), IL-2 / hu22F8, IL-2 / ch22F8, and IL-2 / ch22F8mu. The method is as follows:

[0114] CTLL2 cells were diluted to 1E5 / ml with 1640 medium containing 10% FBS, and 100 μL / well was added to cell culture plates. IL-2 was mixed with hu22F8mu, hu22F8, ch22F8, and ch22F8mu at a mass ratio of 1:7 and incubated at room temperature for 30 minutes to form complexes SPGD01(IL2 / hu22F8mu), IL-2 / hu22F8, IL-2 / ch22F8, and IL-2 / ch22F8mu. Based on IL-2, IL-2 and the above complexes were diluted to 50 ng / ml with 1640 medium containing 10% FBS. After eight serial dilutions (two-fold), the solutions were added to the above-mentioned cell culture plates containing CTLL2 cells and incubated at 37°C in a 5% CO2 cell incubator for 72 h. After dilution, the relative cell count in each well was determined using a CCK8 assay, and EC50 was calculated to determine the viability of the samples.

[0115] The results are as follows Figure 9As shown, 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, while the EC50s of SPGD01 and IL-2 / hu22F8 are 1.01 ng / ml and 1.09 ng / ml, respectively. The EC50s of IL-2 / ch22F8 and IL-2 / ch22F8mu are 0.98 ng / ml and 1.04 ng / ml, respectively. This indicates that SPGD01 and IL-2 / hu22F8 have consistent biological activities, and IL-2 / ch22F8mu and IL-2 / ch22F8 also have similar biological activities. This further demonstrates that Fc mutations do not lead to the biological activity of the IL-2 / antibody complex.

[0116] Example 15: The ability of SPGD01 to bind to FcRn is significantly weakened.

[0117] The binding of SPGD01 (IL-2 / hu22F8mu) and IL-2 / hu22F8 to human FcRn / β2M (purchased from Beijing Yiqiao Shenzhou Co., Ltd., catalog number CT009-H08H) and mouse FcRn / β2M (purchased from Beijing Yiqiao Shenzhou Co., Ltd., catalog number CT029-M08H) was measured using ELISA, following the method described in Example 3. Mouse FcRn / β2M and human FcRn / β2M were coated onto the microplate at 1 μg / well and blocked. SPGD01 or IL-2 / hu22F8 was then diluted to 10 μg / ml (based on antibody mass) with PBS at pH 6.0, and added to the microplate after a 5-fold serial dilution. The entire experimental process used a buffer system at pH 6.0, and other procedures were the same as in Example 3.

[0118] The results are as follows Figure 10 As shown, the binding of SPGD01 (IL2 / hu22F8mu) to human FcRn / β2M is significantly weaker than that of IL2 / hu22F8. Overall, the binding of SPGD01 and IL2 / hu22F8 to human FcRn / β2M is relatively weak.

[0119] The results are as follows Figure 11 As shown, the binding ratio of SPGD01 (IL2 / hu22F8mu) to mouse FcRn / β2M is significantly weaker than that of IL2 / hu22F8. Overall, the binding ratios of SPGD01 and IL2 / hu22F8 to mouse FcRn / β2M are both relatively weak.

[0120] Example 16: SPGD01 (IL-2 / hu22F8mu) reduces lethality in experimental mice.

[0121] The experimental method is the same as in Example 8.

[0122] The results are shown in Table 3. At a dose of 3 mg / kg, 100% (10 / 10) of the experimental animals in the IL-2 / hu22F8 group died, while no experimental animals died (0 / 10) in the SPGD01 (IL-2 / hu22F8mu) group. At a dose of 1 mg / kg, 50% of the experimental animals in the IL-2 / hu22F8 group survived. In contrast, at high doses up to 6 mg / kg, 100% of the experimental animals in the SPGD01 group survived. This indicates that after the above mutation, the complex formed by the antibody Fc and IL-2 has a significantly reduced toxicity to experimental mice. This result is consistent with that of IL2 / ch22F8mu.

[0123] Table 3 Survival rate of experimental animals in each antibody dosage group

[0124] Antibody 1mg / kg 3mg / kg 6mg / kg IL-2 / hu22F8 50％ 0％ - IL-2 / SPGD01 - 100％ 100％

[0125] Example 17: In vivo antitumor activity of SPGD01 (IL-2 / hu22F8mu)

[0126] The experimental method is the same as in Example 5.

[0127] The results are as follows Figure 12 As shown, the IL-2 / hu22F8 complex exhibited good antitumor activity, with TGIs of 95.4% and 85.6% in the 0.5 mg / kg and 0.25 mg / kg groups, respectively. SPGD01 (IL-2 / hu22F8mu) also demonstrated excellent antitumor activity, with TGIs of 95.8%, 84.5%, and 75.4% in the 2 mg / kg, 1 mg / kg, and 0.5 mg / kg groups, respectively. Statistical analysis showed no significant difference in antitumor activity between the two (p>0.05).

[0128] During the experiment, animals in the 1 mg / kg dose group of IL2 / hu22F8 died after two doses, while no animals died in the other groups after six doses. This indicates that the IL2 / hu22F8 complex has strong toxicity, and the safety of SPGD01 (IL-2 / hu22F8mu) is significantly improved after Fc mutation.

[0129] Example 18: Synergistic antitumor activity of SPGD01 (IL-2 / hu22F8mu) and PD1 monoclonal antibody

[0130] The synergistic antitumor effect of SPGD01 (IL-2 / hu22F8mu) and anti-PD1 monoclonal antibody was evaluated using an MC38 xenograft model, with the experimental methods identical to those in Example 5. The SPGD01 dose was 4 mg / kg (based on IL-2), and the rat anti-mouse PD1 monoclonal antibody (purchased from BioXCell, catalog number BP0146) dose was 5 mg / kg, administered intraperitoneally three times per week for a total of six times. Three days after the last administration, mice were sacrificed, and their serum and spleen were collected. Serum alanine aminotransferase (ALT) levels were measured using an enzyme activity method, and CD4-positive, CD8-positive, and CD4 / CD25-positive cells in the spleen were analyzed using conventional flow cytometry (FACS). Other details are as per Example 5.

[0131] The results are as follows Figure 13 As shown, both SPGD01 (IL2 / hu22F8mu) monotherapy and PD1 monoclonal antibody (anti-mPD1) monotherapy exhibited moderate antitumor effects, with TGIs of 61.7% and 41.3%, respectively. The TGI of combination therapy reached 87.8%, demonstrating a good synergistic effect.

[0132] The results are as follows Figure 14 As shown, there was no significant difference in ALT values ​​between the drug administration groups and the PBS control group (p>0.05).

[0133] The results are as follows Figure 15 As shown, the ratio of CD4-positive cells to CD8-positive cells (CD4 / CD8) in the anti-mPD1 group was not significantly different from that in the PBS group (p>0.05), but the ratio in the SPGD01 group and the combined drug group (SPGD01+anti-mPD1) was significantly lower than that in the PBS group (p<0.01, p<0.001).

[0134] The results are as follows Figure 16 As shown, the ratio of CD4+CD25+ to CD3 positive cells (CD4+CD25+ / CD3+) in the anti-mPD1 group was not significantly different from that in the PBS group (p>0.05), but the ratio in the SPGD01 group and the combined drug group (SPGD01+anti-mPD1) was significantly lower than that in the PBS group (p<0.05).

[0135] Example 19, SPGD01(IL-2 / hu22F8mu) and hu22F8mu have good stability

[0136] IL-2 and hu22F8mu were mixed at a mass ratio of 1:7 to form SPGD01 (IL-2 / hu22F8mu). After being incubated with hu22F8mu at 4℃ for a corresponding time, the changes in purity of SPGD01 and hu22F8mu after different incubation times were detected by molecular sieve high-performance liquid chromatography (SEC-HPLC) to investigate the stability of SPGD01 and hu22F8mu. The method is as follows:

[0137] Chromatography was performed using a TSKgel G3000SWXL column (TSK) on an HPLC Ultimate 3000 (Thermo) system. The mobile phase was PBS (pH 7.4), the flow rate was constant at 0.8 mL / min, and the sample loading volume was 100 μg / 100 μL. Purity was expressed as the percentage of the target protein in the total protein calculated using the 280 nM absorption peak integration method.

[0138] The results are as follows Figure 17 , Figure 18 , Figure 19 Hehe Figure 20 As shown, SPGD01 (IL-2 / hu22F8mu) in PBS solution had a purity of 95% at week 0 and a purity of 94% after 24 weeks at 4°C, with a change rate of <2%. The monoclonal antibody hu22F8mu in PBS solution had a purity of 93% at week 0 and a purity of 95% after 6 weeks at 4°C, with a change rate of <3%. This indicates that the IL-2 / antibody complex SPGD01 and the related monoclonal antibody hu22F8mu remain stable in PBS buffer at 4°C.

[0139] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A monoclonal antibody against human IL-2, characterized in that, It has the following characteristics: The heavy chain variable region has the amino acid sequence of SEQ ID NO:4, and the light chain variable region has the amino acid sequence of SEQ ID NO:

9.

2. The nucleic acid encoding the anti-human IL-2 monoclonal antibody as described in claim 1, characterized in that, It has the following characteristics: Nucleotide sequences of the heavy chain variable region as shown in SEQ ID NO:3; nucleotide sequences of the light chain variable region as shown in SEQ ID NO:

8.

3. A chimeric antibody against human IL-2, characterized in that, It has the following characteristics: The amino acid sequence is the heavy chain sequence of SEQ ID NO:15 and the amino acid sequence is the light chain sequence of SEQ ID NO:

16.

4. An Fc-mutated chimeric anti-human IL-2 antibody, characterized in that, It has the following characteristics: The amino acid sequence is the heavy chain sequence of SEQ ID NO:18 and the amino acid sequence is the light chain sequence of SEQ ID NO:

16.

5. A humanized anti-human IL-2 monoclonal antibody, characterized in that, It is a humanized anti-human IL-2 monoclonal antibody constructed by CDR transplantation technology and CDR region mutation design based on the anti-human IL-2 monoclonal antibody described in claim 1; the humanized anti-human IL-2 monoclonal antibody has: a heavy chain variable region with the amino acid sequence SEQ ID NO:24, and a light chain variable region with the amino acid sequence SEQ ID NO:

25.

6. A humanized anti-human IL-2 antibody, characterized in that, It has the following characteristics: The amino acid sequence is the heavy chain sequence of SEQ ID NO:27 and the amino acid sequence is the light chain sequence of SEQ ID NO:

28.

7. A humanized anti-human IL-2 antibody with an Fc mutation, characterized in that, It has the following characteristics: The amino acid sequence is the heavy chain sequence of SEQ ID NO:26 and the amino acid sequence is the light chain sequence of SEQ ID NO:

28.

8. An antibody complex, characterized in that, It is prepared by mixing IL-2 with the anti-human IL-2 chimeric antibody of claim 3, the Fc-mutated anti-human IL-2 chimeric antibody of claim 4, the humanized anti-human IL-2 antibody of claim 6, or the Fc-mutated humanized anti-human IL-2 antibody of claim 7 at a mass ratio of 1:

7.

9. Use of the antibody complex as described in claim 8 in the preparation of antitumor drugs.

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