Humanized anti-human CD132 monoclonal antibody and its application

Abstract

The present invention belongs to the field of biomedicine, and in particular to a humanized anti-human CD132 monoclonal antibody and its application. Based on the monoclonal chimeras 2D4 and 5H10 previously obtained in this laboratory, the present invention screened and obtained high-affinity monoclonal humanized antibodies h2D4H4K12 and / or h5H10H6K4 that can target human CD132 by humanizing the target antibody. On the basis of not weakening the antibody activity, the immunogenicity of the maternal chimera was reduced, and the risk that the patient using the drug may produce an immune response to the antibody was further reduced. It has good application prospects in the treatment of autoimmune diseases (such as rheumatoid arthritis, systemic lupus erythematosus, etc.) associated with IL-4, IL-7, IL-9, IL-15 and / or IL-21 cytokines.

Description

[0001] The present invention is a divisional application of 2024100816460 humanized anti-human CD132 monoclonal antibody and its application filed on January 19, 2024. Technical Field

[0002] The present invention belongs to the field of biomedicine, and specifically relates to a humanized anti-human CD132 monoclonal antibody and its application. Background Art

[0003] CD132 (IL2Rγ) is the γ subunit of the interleukin-2 (IL-2) receptor. This subunit also serves as the γ subunit of the receptor complexes for six cytokines: IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Therefore, it is also known as γc (common γ chain). Systemic lupus erythematosus (SLE) is a chronic autoimmune disease affecting multiple organs and systems. Its etiology and pathogenesis are complex and are associated with the breakdown of autoimmune tolerance, but the exact cause and specific pathogenesis remain unclear. B cell dysfunction and the resulting accumulation of autoantibodies, which form immune complexes in patients, are key factors in the development and progression of SLE. Targeting B cells for the treatment of SLE is a research priority both domestically and internationally. These include the Blys-targeting monoclonal antibody belimumab and the fusion protein telitacicept, which simultaneously targets Blys and April, both of which are already approved for the treatment of SLE. However, in addition to B cell disorder, T cell overproliferation and activation are also core factors in SLE pathology. Therefore, targeting B cells alone does not meet the needs of SLE clinical treatment. + Follicular helper T cells (Tfh) and peripheral T helper cell populations (Th1, Th2, Th17, and Treg) mediate B cell activation and autoantibody production by providing co-stimulatory signals and cytokines. Therefore, some T cell-related cytokines have attracted much attention due to their role in promoting inflammation and organ damage in SLE, becoming promising therapeutic targets.

[0004] Numerous studies have demonstrated that IL4, IL-7, IL9, IL-15, and IL-21, members of the γ-chain family of cytokines, are significantly upregulated in immune cells in SLE. These γ-chain cytokines play a key regulatory role in the survival, proliferation, and differentiation of various lymphocytes. Given that SLE is a complex disease involving multiple cells and factors, and that the key regulatory role of γ-chain cytokines in various lymphocytes is closely associated with the development and progression of SLE, intervening in the functional activity of CD132, a γ-chain cytokine, may be a potential therapeutic approach for SLE. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a humanized anti-human CD132 monoclonal antibody in response to the deficiencies of the existing technology.

[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0007] A humanized anti-human CD132 monoclonal antibody, wherein the monoclonal antibody is a humanized monoclonal antibody,

[0008] The monoclonal antibody comprises a light chain complementary determining region and a heavy chain complementary determining region, wherein the light chain complementary determining region includes LCDR1, LCDR2 and LCDR3, and the heavy chain complementary determining region includes HCDR1, HCDR2 and HCDR3;

[0009] Specifically, the light chain complementary determining region and the heavy chain complementary determining region are selected from any one of the following groups:

[0010] Group 1: The amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs. 18, 14, and 15, respectively; the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs. 3, 4, and 5, respectively;

[0011] or,

[0012] The second group: the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs. 26, 27 and 31, respectively; the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs. 8, 9 and 10, respectively.

[0013] The monoclonal antibody comprises a light chain variable region and a heavy chain variable region, and the light chain variable region and the heavy chain variable region are selected from any one of the following groups:

[0014] Group 1: The amino acid sequence of the light chain variable region is shown in SEQ ID NO. 22; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 1;

[0015] or,

[0016] Group 2: The amino acid sequence of the light chain variable region is shown in SEQ ID NO.29; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.6.

[0017] The monoclonal antibody further comprises a constant region, and the constant region is the kappa chain constant region Ckappa and the human IgG4 constant region CH1-CH3.

[0018] The present invention also provides a nucleic acid molecule, specifically, a nucleotide sequence encoding the above-mentioned monoclonal antibody.

[0019] The present invention also provides an expression vector, specifically comprising the above nucleic acid molecule.

[0020] The present invention also provides a host cell, specifically comprising the above expression vector.

[0021] Wherein, the host cell is HEK293 cell.

[0022] The present invention also provides a detection reagent or kit, characterized in that it comprises the monoclonal antibodies h2D4H4K12 and / or h5H10H6K4.

[0023] The use of the above monoclonal antibodies in the preparation of drugs for inhibiting or reducing the activity of human CD132 is also within the scope of protection of the present invention.

[0024] Specifically, in some embodiments, in an in vitro activity experiment to detect the inhibitory activity of humanized candidate antibodies on CD132, the humanized anti-human CD132 antibody h2D4H4K12 can inhibit the activity of IL-21 stimulating NK92 cells to release IFN-γ; the humanized anti-human CD132 antibody h5H10H6K4 can inhibit the activity of IL-4 stimulating Ramos cells to express CD23.

[0025] Specifically, in some embodiments, C57BL / 6 background mice (C57BL / 6 background mice genetically modified from the Biocytogen breeding colony to replace the endogenous CD132 full-length domain with the corresponding human sequence) were subcutaneously administered with antibodies h2D4H4K12 or h5H10H6K4 at a dose of 8 mg / kg or 16 mg / kg, once every 4 days for 3 consecutive doses, and the absolute numbers and ratios of total immune cells, B cells, T cells, NK cells, neutrophils, monocytes, red blood cells and platelets in the peripheral blood at different time points (once every 10 days) were analyzed by flow cytometry to evaluate the effects of h2D4H4K12 and h5H10H6K4 antibodies on the absolute numbers of these cell types. The results showed that both h2D4H4K12 and h5H10H6K4 antibodies had varying degrees of inhibitory effects on B cells, T cells and NK cells, but did not affect the number of neutrophils, monocytes, red blood cells and platelets.

[0026] The use of the above monoclonal antibodies in the preparation of therapeutic drugs for preventing, neutralizing or treating autoimmune diseases is also within the scope of protection of the present invention.

[0027] The autoimmune diseases are immune diseases related to IL-4, IL-7, IL-9, IL-15 and / or IL-21 cytokine receptors, including systemic lupus erythematosus, rheumatoid arthritis, etc.

[0028] Specifically, in some embodiments, pristane (MCE) 0.5 mL was injected into the peritoneal cavity of C57BL / 6 background mice (C57BL / 6 background mice genetically modified from the Biocytogen breeding colony to replace the endogenous CD132 full-length domain with the corresponding human sequence), and an equal volume of PBS buffer (pH 7.2) was injected into the control group. h2D4H4K12 or h5H10H6K4 or anti KLH isotype control (all of the above antibodies are produced by Conoya Biotechnology Co., Ltd.) was administered subcutaneously at a dose of 20 mg / kg 3 days before modeling, with a frequency of 2 times a week for 6 weeks, or no administration (Table 5). The levels of urine protein, urine creatinine, and plasma anti-dsDNA in mice were monitored. The results showed that the two antibodies, h2D4H4K12 and h5H10H6K4, could significantly reduce the urine protein and plasma anti-dsDNA levels in pristane-induced lupus mice and improve the survival rate of mice.

[0029] Beneficial effects:

[0030] The present invention humanized two antibodies (2D4 and 5H10) previously obtained in the laboratory, and after screening, obtained two high-affinity monoclonal humanized antibodies (h2D4H4K12 and h5H10H6K4) that can target human CD132. The inhibitory activity of these two antibodies against five γc cytokines is consistent with that of the parent chimeras 2D4 and 5H10, while also reducing the immunogenicity of the parent chimeras, further reducing the risk that patients may develop an immune response to the antibodies. Both antibodies can be used in therapeutic drugs for the prevention, neutralization or treatment of autoimmune diseases (such as rheumatoid arthritis, systemic lupus erythematosus, etc.) associated with IL-4, IL-7, IL-9, IL-15 and / or IL-21 cytokines. The present invention also further illustrates the prospects of these two antibodies in drugs for the treatment of systemic lupus erythematosus through an animal model of systemic erythema. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0032] Figure 1 Inhibition of IFN-γ secretion by NK92 cells under constant IL-21 stimulation by the parental chimeric anti-human CD132 antibody 2D4 and multiple humanized 2D4 antibodies.

[0033] Figure 2 The parent chimeric anti-human CD132 antibody 5H10 and multiple humanized 5H10 antibodies inhibited CD23 expression by Ramos cells under constant IL-4 stimulation.

[0034] Figure 3 h5H10H6K4 inhibits IL21-stimulated IFN-γ secretion from NK92 cells.

[0035] Figure 4 h2D4H4K12 inhibits IL4-stimulated CD23 expression in Ramos cells.

[0036] Figure 5 h5H10H6K4( Figure 5-1 ) and h2D4H4K12( Figure 5-2 ) inhibits IL21-stimulated NK92 cells from secreting IFN-γ.

[0037] Figure 6 h5H10H6K4( Figure 6-1 ) and h2D4H4K12( Figure 6-2 ) inhibits IL7 stimulation of CD4 + T cell downstream phosphorylation.

[0038] Figure 7h5H10H6K4( Figure 7-1 ) and h2D4H4K12( Figure 7-2 ) inhibited IL9-stimulated M07E cell proliferation.

[0039] Figure 8 Immunogenicity of h2D4H4K12 and h5H10H6K4.

[0040] Figure 9 h2D4H4K12 and h5H10H6K4 antibodies against CD132 hu / hu Effects of background mouse T cells (9A), B cells (9B), NK cells (9C), neutrophils (9D), red blood cells (9E), monocytes (9F), platelets (9G) and Treg cells (9H).

[0041] Figure 10 h2D4H4K12 and h5H10H6K4 antibodies to pristane-induced CD132 hu / hu Anti-dsDNA levels (10A) and urine protein / creatinine ratio (10B) in a mouse lupus erythematosus model. DETAILED DESCRIPTION

[0042] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0043] In previous research by our research group, we have obtained monoclonal chimeras 2D4 and 5H10 that can target human CD132 through phage screening (detailed screening, construction, and sequence information for the monoclonal chimeras 2D4 and 5H10 of CD132 have been disclosed in patent CN116970081A). However, antibodies with chimera configurations are highly immunogenic and can easily lead to the production of anti-antibodies in the body, thereby weakening the antibody's efficacy. Therefore, this patent further humanizes the target antibodies 2D4 and 5H10 to reduce the chimera's immunogenicity without weakening the antibody's activity. At the same time, the target antibodies (h2D4H4K12 and h5H10H6K4) are validated by in vivo pharmacodynamics to further demonstrate the effectiveness of the two antibodies.

[0044] All experiments were approved and performed in accordance with the guidelines of the Ethics Committee of the Dermatology Hospital, Chinese Academy of Medical Sciences (Institute of Dermatology, Chinese Academy of Medical Sciences).

[0045] Example 1: Humanization of monoclonal antibodies 2D4 and 5H10

[0046] The variable region sequences of monoclonal antibodies 2D4 and 5H10 were compared with human germline antibody sequences to identify sequences with high homology for CDR transplantation. Simultaneously, computer modeling was performed to analyze the CDR regions and surrounding framework amino acid sequences to examine their spatial binding patterns. By calculating electrostatic forces, van der Waals forces, hydrophilicity, and entropy, key amino acids within the gene sequences of each positive monoclonal antibody were identified for potential interaction with CD132 and maintenance of the spatial framework. Back-mutation sites were then designed based on these analyses. HLA-DR affinity was analyzed to select human germline framework sequences with low immunogenicity. All of these humanization modifications were designed and completed by Conoya Biotechnology Co., Ltd.

[0047] For 2D4, one heavy chain variable region derivative (h2D4-VH4) and four light chain variable region derivatives (h2D4-VL3, h2D4-VL9, h2D4-VL11, and h2D4-VL12) were designed. For 5H10, one heavy chain variable region derivative (h5H10-VH6) and two light chain variable region derivatives (h5H10-VL3 and h5H10-VL4) were designed. The sequences of all the above light and heavy chain variable region derivatives are shown in Tables 1 and 2. The light chain variable region derivatives were cloned into the pHCT2 vector (purchased from Addgene), which contains the human IgGκ light chain constant region and regulatory elements and is developed by Conoya, to express the complete IgGκ light chain in mammalian cells. The heavy chain variable region derivatives were cloned into the pHCT1s vector (purchased from Addgene), a Conoya-developed vector containing the human IgG4 subtype heavy chain constant region and regulatory elements, to express the complete IgG4 subtype heavy chain in mammalian cells. After plasmid pairing, the plasmids were transfected into HEK293 cells (purchased from ATCC) and expressed for 5-6 days. The supernatant was collected, filtered, and purified using a protein A column to construct candidate humanized antibodies h2D4H4K3, h2D4H4K9, h2D4H4K11, h2D4H4K12, h5H10H6K4, and h5H10H6K5.

[0048] Table 1 Heavy chain variable region derivative sequences of candidate CD132 humanized antibodies (SEQ ID NO.** omitted before the numbers)

[0049]

[0050] Table 2: Light chain variable region derivative sequences of candidate CD132 humanized antibodies (SEQ ID NO.** omitted before the numbers)

[0051]

[0052] Specifically, the humanized antibody heavy chain variable region derivative sequence is:

[0053] 1) The amino acid sequence of h2D4-VH4 is shown in SEQ ID NO. 1, the encoding nucleotide sequence is shown in SEQ ID NO. 2, and the CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NOs. 3, 4, and 5, respectively.

[0054] <------------FR1------------>CDR1<----FR2----->CDR2

[0055] QVQLVQSGAEVKKPGSSVKVSCKTSGFNI EDIYLH WVRQAPGQGLEWMG RID PANGK

[0056] <-------------FR3-------------->CDR3<---FR4-

[0057] SNYDPKFQG RVTITADTSTSTAYMELSSLRSEDTAVYYCAA LRFFGLDY WGQG TLVT

[0058] -->

[0059] VSS

[0060] Nucleotide sequence

[0061] CAGGTTCAGCTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCAGCAGCGTGAAGGTGAGCTGCAAGACAAGCGGCTTCCAACATCGAGGACATCTACCTGCACTGGGTCCGACAGGCTCCAGGACAGGGAC TTGAGTGGATGGGCAGAATCGACCCTGCCAACGGCAAGAGCAACTACGACCCCAAGTTCCAGGGCAGAGTCACCATCACAGCCGACACCAGCACCAGCACCGCCTACATGGAACTGAGCAGCCTGAGAAGCGAG GACACCGCCGTGTACTACTGTGCCGCTCTGAGATTCTTCGGCCTGGACTACTGGGGCCAGGGAACACTGGTTACCGTGTCTAGT

[0062] 2) The amino acid sequence of h5H10-VH6 is shown in SEQ ID NO. 6, the encoding nucleotide sequence is shown in SEQ ID NO. 7, and the CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NOs. 8, 9, and 10, respectively.

[0063] <------------FR1------------>CDR1<----FR2----->CDR2

[0064] QVQLVQSGAEVKKPGASVKVSCKASGYTF TNYWLG WVRQAPGQGLEWMG D IYPGGGY

[0065] <-------------FR3-------------->CDR3<--FR4

[0066] TNYNEKFQG RVTMTADTSTSTVYMELSSLRSEDTAVYFCAR GDYGSSWFPY W GQGTL

[0067] ---->

[0068] VTVSS

[0069] Nucleotide sequence

[0070] CAGGTTCAGCTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGGTGAGCTGCAAGGCTAGCGGCTACACCTTCACCAACTACTGGCTCGGCTGGGTCCGACAGGCTCCTGGACAGGGACTGGAATGGATGGGCGACATCTACCCTGGCGGCGGATACACAAACT ACAACGAGAAGTTCCAGGGCAGAGTCACAATGACCGCCGACACCTCTACCAGCACAGTCTACATGGAACTGAGCAGCCTGAGAAGCGAGGATACCGCCGTGTACTTCTGTGCCAGAGGCGACTACGGCTCTAGCTGGTTTCCTTACTGGGGCCAGGGAACCCTGGTCACCGTTTCTTCT

[0071] Specifically, the sequence of the humanized antibody light chain variable region derivative is:

[0072] 3) The amino acid sequence of h2D4-VL3 is shown in SEQ ID NO. 11, the encoding nucleotide sequence is shown in SEQ ID NO. 12, and the CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NOs. 13, 14, and 15, respectively.

[0073] <---------FR1--------->CDR1<-----FR2----->CDR2<-

[0074] DMQMTQSPSSLSASVGDRVTITC SATQSVSYMY WYQQKPGKAPELWIY RTSN LAS GV

[0075] -------------FR3-------------->CDR3<--FR4--->

[0076] PSRFSGSGSGTDYTFTISSLQPEDIATYYC QQWSSNPPT FGQGTKLEIK

[0077] Nucleotide sequence

[0078] GACATGCAGATGACACAGAGCCCTTCTAGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCATCACATGTAGCGCCACACAGAGCGTGTCCTACATGTACTGGTATCAGCAGAAGCCCGGCAAGGCCCTGAGCTTTGGATCTACAGAACAAGCAACCTG GCCAGCGGCGTGCCCTCTAGATTTTCTGGCTCTGGCAGCGGCACCGACTACACCTTCACAATCTCTAGCCTGCAGCCTGAGGATATCGCCACCTACTACTGCCAGCAGTGGTCTAGCAACCCTCCTACATTCGGCCAGGGCACCAAGCTGGAAATCAAG

[0079] 4) The amino acid sequence of h2D4-VL9 is shown in SEQ ID NO. 16, its encoding nucleotide sequence is shown in SEQ ID NO. 17, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 18, 14 and 15, respectively.

[0080] <---------FR1--------->CDR1<-----FR2----->CDR2<-

[0081] EIVLTQSPATLSLSPGERATLSC RATSSVSYMY WYQQKPGQAPRLLIY RTSNLAS GI

[0082] -------------FR3------------->CDR3<--FR4--->

[0083] PARFSGSGSGTDYTLTISSLEPEDFAVYYC QQWSSNPPT FGQGTKLEIK

[0084] Nucleotide sequence

[0085] GAGATCGTGCTGACACAGAGCCCTGCCACACTGTCACTGTCTCCAGGCGAGAGAGCCACACTGAGCTGTCGCGCCACCAGCAGCGTGTCCTACATGTACTGGTATCAGCAGAAGCCCGGCCAGGCTCCTCGGCTTCTGATCTACAGAACAAGCAACCTG GCCAGCGGCATCCCCGCTAGATTTTCTGGCTCTGGCAGCGGCACCGACTACACCCTGACAATCTCTAGCCTGGAACCTGAGGACTTCGCCGTGTACTACTGCCAGCAGTGGTCTAGCAACCCTCCTACATTCGGCCAGGGCACCAAGCTGGAAATCAAG

[0086] 5) The amino acid sequence of h2D4-VL11 is shown in SEQ ID NO. 19, the encoding nucleotide sequence is shown in SEQ ID NO. 20, and the CDR1, CDR2, and CDR3 thereof are shown in SEQ ID NOs. 21, 14, and 15, respectively.

[0087] <---------FR1--------->CDR1<-----FR2----->CDR2<-

[0088] DIQMTQSPSSLSASVGDRVTITC QATQSVSYMY WYQQKPGKAPELLIY RTSNL AS GV

[0089] -------------FR3------------->CDR3<--FR4--->

[0090] PSRFSGSGSGTDYTFTISSLQPEDIATYYC QQWSSNPPT FGQGTKLEIK

[0091] Nucleotide sequence

[0092] GACATCCAGATGACACAGAGCCCTTCTAGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCATCACATGTCAGGCCACACAGAGCGTGTCCTACATGTACTGGTATCAGCAGAAGCCCGGCAAGGCCCTGAGCTTCTGATCTACAGAACAAGCAACCTG GCCAGCGGCGTGCCCTCTAGATTTTCTGGCTCTGGCAGCGGCACCGACTACACCTTCACAATCTCTAGCCTGCAGCCTGAGGATATCGCCACCTACTACTGCCAGCAGTGGTCTAGCAACCCTCCTACATTCGGCCAGGGCACCAAGCTGGAAATCAAG

[0093] 6) The amino acid sequence of h2D4-VL12 is shown in SEQ ID NO. 22, the encoding nucleic acid thereof is shown in SEQ ID NO. 23, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 18, 14 and 15, respectively.

[0094] <---------FR1--------->CDR1<-----FR2----->CDR2<-

[0095] EIVLTQSPATLSLSPGERATLSC RATSSVSYMY WYQQKPGQAPELLIY RTSNLAS GI

[0096] -------------FR3------------->CDR3<--FR4--->

[0097] PARFSGSGSGTDYTLTISSLEPEDFAVYYC QQWSSNPPT FGQGTKLEIK

[0098] Nucleotide sequence

[0099] GAGATCGTGCTGACACAGAGCCCTGCCACACTGTCACTGTCTCCAGGCGAGAGAGCCACACTGAGCTGTCGCGCCACCAGCAGCGTGTCCTACATGTACTGGTATCAGCAGAAGCCCGGCCAGGCTCCTGAGCTTCTGATCTACAGAACAAGCAACCTG GCCAGCGGCATCCCCGCTAGATTTTCTGGCTCTGGCAGCGGCACCGACTACACCCTGACAATCTCTAGCCTGGAACCTGAGGACTTCGCCGTGTACTACTGCCAGCAGTGGTCTAGCAACCCTCCTACATTCGGCCAGGGCACCAAGCTGGAAATCAAG

[0100] 7) The amino acid sequence of h5H10-VL3 is shown in SEQ ID NO. 24, the encoding nucleotide sequence is shown in SEQ ID NO. 25, and the CDR1, CDR2 and CDR3 thereof are shown in SEQ ID NOs. 26, 27 and 28, respectively.

[0101] <---------FR1--------->CDR1<-----FR2----->CDR2<-

[0102] DIQMTQSPSSLSASVGDRVTITC RASQSISNNLH WYQQKPGKAPKLLIK YASQSI S G

[0103] -------------FR3------------->CDR3<--FR4--->

[0104] VPSRFSGSGSGTDFTLTISSLQPEDFATYFC QQSDSWLT FGQGTKVEIK

[0105] Nucleotide sequence

[0106] GACATCCAGATGACACAGAGCCCTTCTAGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCATCACATGTAGAGCCAGCCAGAGCATCAGCAACAACCTGCACTGGTATCAGCAGAAGCCCGGCAAGGCTCCCAAGCTGCTGATTAAGTACGCCAGCCAG TCCATCTCCGGCGTGCCATCTAGATTCAGCGGCTCTGGCTCTGGCACCGACTTCACCCTGACAATCTCTAGCCTGCAGCCTGAGGACTTCGCTACCTACTTCTGCCAGCAGAGCGACAGCTGGCTGACATTTGGCCAGGGCACCAAGGTGGAAATCAAG

[0107] 8) The amino acid sequence of h5H10-VL4 is shown in SEQ ID NO. 29, the encoding nucleotide sequence is shown in SEQ ID NO. 30, and the CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NOs. 26, 27, and 31, respectively.

[0108] <---------FR1--------->CDR1<-----FR2----->CDR2<-

[0109] DIQMTQSPSSLSASVGDRVTITC RASQSISNNLH WYQQKPGKAPKLLIK YASQSI S G

[0110] ---------------FR3------------->CDR3<--FR4--->

[0111] VPSRFSGSGSGTDFTLTISSLQPEDFATYFC QQSDSYLT FGQGTKVEIK

[0112] Nucleotide sequence

[0113] GACATCCAGATGACACAGAGCCCTTCTAGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCATCACATGTAGAGCCAGCCAGAGCATCAGCAACAACCTGCACTGGTATCAGCAGAAGCCCGGCAAGGCTCCCAAGCTGCTGATTAAGTACGCCAGCCAG TCCATCTCCGGCGTGCCATCTAGATTCAGCGGCTCTGGCTCTGGCACCGACTTCACCCTGACAATCTCTAGCCTGCAGCCTGAGGACTTCGCTACCTACTTCTGCCAGCAGAGCGACAGCTACCTGACATTTGGCCAGGGCACCAAGGTGGAAATCAAG

[0114] Example 2: In vitro activity assay to detect the inhibitory activity of candidate humanized antibodies against CD132

[0115] To further evaluate the activity gap between the candidate humanized antibodies and the parent chimera, the experimental method in patent CN116970081A was referred to. Because the parent chimera 2D4 has strong inhibitory activity against IL21, the method of IL21 stimulating the NK92 cell line to secrete IFN-γ was used to evaluate the 2D4 candidate humanized antibody. Because the activity gap between the 5H10 candidate humanized antibodies was more significant in the IL4 inhibition experimental system, the method of IL4 stimulating the Ramos cell line to upregulate CD23 was selected to further evaluate the 5H10 candidate humanized antibody.

[0116] 2.1 Verification of the 2D4 humanized anti-human CD132 candidate antibody's ability to inhibit IFN-γ secretion from NK92 cells

[0117] 2 × 10 cells were seeded in a 96-well plate using growth medium (prepared according to the instructions of Pronose, but without IL-2). 4 NK-92 cells (Punosai CL-0530) were incubated at 37°C in 5% CO2 and starved overnight. The next day, each 2D4 humanized anti-human CD132 candidate antibody was serially diluted in a 3-fold gradient from 400nM to 1.6nM in growth medium (without IL-2), added to NK-92 cells and incubated for 30 minutes. After incubation, 250pM IL-21 was added to NK-92 cells containing different 2D4 humanized anti-human CD132 candidate antibodies. After incubation for 72h at 37°C in 5% CO2, IFN-γ secretion was measured by ELISA.

[0118] Using a human IFN-γ ELISA kit (BD Catalog No. 555142), the capture antibody was coated at a 1:250 ratio on a 96-well microtiter plate and incubated overnight at 4°C. The next day, the plates were washed three times with PBS buffer (pH 7.2), blocked with 10% FBS for 1 hour at room temperature, and then washed three times with PBS buffer (pH 7.2). Then, 100 μl of sample (cell supernatant) and standard were added, and the plates were incubated for 2 hours at room temperature. The plates were then washed five times with PBS buffer (pH 7.2). 5AV-HRP was added at a 1:250 ratio in a dilution containing 0.4% detection antibody. Finally, the plates were incubated for 1 hour at room temperature, washed seven times with PBS buffer (pH 7.2), and 100 μl of color development solution (TMB solution, Sigma Catalog No. T2885) was added. The plates were incubated at 37°C for 10 minutes, and the reaction was terminated by adding 50 μl of 2M concentrated sulfuric acid solution. The plates were immediately placed in a microtiter plate and the OD was read. 450 The value of Figure 1 The results showed that the activity of humanized anti-human CD132 antibody h2D4H4K12 in inhibiting IL-21-stimulated IFN-γ release from NK92 cells was comparable to that of the parental 2D4 chimera.

[0119] 2.2 Verification of the 5H10 humanized anti-CD132 candidate antibody's ability to inhibit IL-4-stimulated CD23 expression in Ramos cells

[0120] IL-4 can stimulate Ramos cells to express CD23, so this property can be used to evaluate the inhibitory effect of the 5H10 humanized anti-CD132 candidate antibody on IL-4 signaling. Ramos cell line (ATCC) was cultured in RPMI 1640 complete medium containing 10% FBS at 37°C and 5% CO2, with a cell concentration of 2×10 cells per ml. 5 -2×10 6 Cells were plated in 96-well flat-bottom cell culture plates, with 100 μl per well. Serial dilutions of each 5H10 humanized anti-CD132 candidate antibody were prepared in RPMI 1640 complete medium, with a four-fold serial dilution from 200 nM to 0.003 nM. After incubation with the cells for 30 minutes, 50 μl of IL-4 was added for a final concentration of 2.67 nM, and the cells were cultured at 37°C, 5% CO2 for 48 hours.

[0121] The cultured cells were transferred to a U-shaped 96-well cell culture plate, centrifuged at 300 g for 3 minutes at 4°C, discarded the supernatant, and washed twice with flow cytometry buffer (PBS buffer containing 4% calf serum, pH 7.2), 200 μl per well. Take 50 μl of blocking solution (buffer containing 100 μg / ml hIgG) and add it to the cells, block it on ice for 10 minutes, centrifuge it at 300g for 3 minutes and discard the supernatant, then add 50 μl of anti-human CD23 FITC antibody diluent (BD EBVCS-5) to each well, block it on ice for 20 minutes; centrifuge it at 300g for 3 minutes and discard the supernatant, wash the cells twice with flow cytometry buffer, 200 μl per well; take PI staining solution and add it to the cells, 100 μl per well, ice bath in the dark for 5 minutes, centrifuge it at 300g for 3 minutes and discard the supernatant, wash the cells twice with 200 μl of flow cytometry buffer per well, and then resuspend the cells with 100 μl of PBS buffer (pH 7.2) per well. Read the mean fluorescence intensity by flow cytometer and record the measurement results. Figure 2 As shown, the activity of the humanized anti-human CD132 antibody h5H10H6K4 in inhibiting IL-4-stimulated CD23 expression in Ramos cells was comparable to that of the parental 5H10 chimera.

[0122] Example 3: Revalidation of humanized anti-human CD132 antibodies h5H10H6K4 and h2D4H4K12

[0123] In Example 2, the inhibitory activity of candidate humanized antibodies against CD132 was tested by in vitro activity experiments, and two humanized anti-human CD132 antibodies, h5H10H6K4 and h2D4H4K12, were screened. This example further verified whether the inhibitory activity of the two antibodies against five γc cytokines was consistent with that of the parent chimeras 2D4 and 5H10.

[0124] 3.1 Humanized anti-human CD132 antibody h5H10H6K4 inhibits IL21-stimulated NK92 cell secretion of IFN-γ

[0125] To further evaluate the ability of h5H10H6K4 to block IFN-γ secretion from NK92 cells stimulated by IL-21, the specific experimental steps are as detailed in Example 2.1, except that the antibody was replaced with h5H10H6K4. Figure 3 , Figure 3 The results showed that the anti-human CD132 antibody h5H10H6K4 could inhibit the release of IFN-γ from NK92 cells stimulated by IL-21, but Figure 1 Compared with the results of h2D4H4K12, its inhibitory activity was weaker than that of h2D4H4K12.

[0126] 3.2 Humanized anti-human CD132 antibody h2D4H4K12 inhibits IL4-stimulated CD23 expression in Ramos cells

[0127] To further evaluate the ability of h2D4H4K12 to block the secretion and expression of CD23 by Ramos cells stimulated by IL-4, the specific experimental steps are as detailed in Example 2.2, except that the antibody was replaced with h2D4H4K12. Figure 4 , Figure 4 The results showed that the anti-human CD132 antibody h2D4H4K12 could inhibit the expression of CD23 by Ramos cells stimulated by IL-4.

[0128] 3.3 Humanized anti-human CD132 antibodies h2D4H4K12 and h5H10H6K4 inhibit IL15-stimulated NK92 cell secretion of IFN-γ

[0129] To further evaluate the ability of h2D4H4K12 and h5H10H6K4 to block IL-15-stimulated NK92 cell secretion of IFN-γ, the specific experimental steps are as detailed in Example 2.1, using antibodies h5H10H6K4 and h2D4H4K12, respectively, and replacing 250pM IL-21 with 2.5nM IL-15. The experimental results are shown in Figure 2. Figure 5-1 and as shown in 5-2. Figure 5-1 and Figure 5-2 The results showed that both humanized anti-human CD132 antibodies h5H10H6K4 and h2D4H4K12 could inhibit the release of IFN-γ from NK92 cells stimulated by IL-15, but the inhibitory activity of h5H10H6K4 was weaker than that of h2D4H4K12.

[0130] 3.4 Flow cytometric analysis of STAT phosphorylation in human CD4+ T cells (human PBMC)

[0131] To further evaluate the in vitro properties of humanized anti-human CD132 antibodies h5H10H6K4 and h2D4H4K12, their ability to block IL-7-induced CD4 + The experimental process was the same as that of Example 8 in patent CN116970081A, except that the chimeric anti-human CD132 antibodies 5H10 and 2D4 were replaced with humanized anti-human CD132 antibodies h5H10H6K4 and h2D4H4K12. Figure 6-1 and 6-2. Figure 6-1 and Figure 6-2 The results showed that anti-human CD132 antibodies h5H10H6K4 and h2D4H4K12 could inhibit STAT5 phosphorylation downstream of IL-7-stimulated human peripheral blood CD4+ cells.

[0132] 3.5 Humanized anti-human CD132 antibodies h2D4H4K12 and h5H10H6K4 inhibit IL-9-stimulated M07E cell proliferation

[0133] The inhibitory activity of humanized anti-human CD132 antibodies h5H10H6K4 and h2D4H4K12 against IL-9 was further evaluated. The experimental process was the same as that of Example 10 in patent CN116970081A, except that the chimeric anti-human CD132 antibodies 5H10 and 2D4 were replaced by humanized anti-human CD132 antibodies h5H10H6K4 and h2D4H4K12. The results are shown in Figure 2. Figure 7 As shown, Figure 7-1 and Figure 7-2 The results showed that humanized anti-human CD132 antibodies h5H10H6K4 and h2D4H4K12 could inhibit the proliferation of M07E cells stimulated by IL-9.

[0134] Example 4: Evaluation of the immunogenicity of humanized anti-human CD132 antibodies h2D4H4K12 and h5H10H6K4

[0135] Sample preparation:

[0136] Under sterile conditions, h2D4H4K12, h5H10H6K4, and KLH (independently expressed by Conoya) were diluted to 200 μg / mL using X-vivo15 medium (Shanghai Peiyuan) and sterilized for use.

[0137] Determination method:

[0138] Peripheral venous blood was collected from 25 healthy volunteers and PBMCs were collected by Ficoll centrifugation (Healthcare). PBMCs were washed once with PBS, counted, and the cell density was adjusted to 2.5×10 5 / mL, 100 μL was added to a 96-well U-shaped plate (corning). 100 μL of the diluted sample was added to the cells and incubated at 37°C, 5% CO2 for 48 hours. KLH was used as a positive control, and wells without sample were used as negative controls. The same method was used for analysis. The 96-well plate was discarded, 200 μL / well of the supernatant was washed once with flow cytometry buffer (PBS + 10% FBS). 100 μg / mL hIgG (Jackson) was added, 50 μL per well, and incubated on ice for 30 minutes. 1 μL / well of FITC anti-human CD4, APC anti-human CD137 (OX40), and PE anti-human CD134 (4-1BB) fluorescent antibodies (all purchased from Biolegend) were added and incubated on ice for 45 minutes. 50 μL of 5 μg / mL PI was added for staining for 5 minutes. The cells were washed twice with flow cytometry buffer, resuspended in 100 μL of PBS, and analyzed by flow cytometry.

[0139] Data Analysis:

[0140] (1) Lymphocytes were gated based on cell size (FSC-A) and granularity (SSC-A);

[0141] (2) In the lymphocyte population, adherent cells were excluded based on FSC-A and FSC-H, and single cell populations were circled;

[0142] (3) Circle the living cell population in the single cell population based on FSC-A and PI light signals;

[0143] (4) Circle the CD4-positive T cells among the living cells and analyze the proportion of CD137 (OX40) and CD134 (4-1BB) double-positive cells in this group of cells.

[0144] The final result is as follows Figure 8 As shown, the double-positive cells of humanized anti-human CD132 antibodies h2D4H4K12 and h5H10H6K4 were comparable to those of the negative control, indicating that the above two antibodies have extremely low immunogenicity.

[0145] Example 5: In vivo immunosuppressive experiment evaluating the effects of anti-human CD132 antibodies h2D4H4K12 and h5H10H6K4 on immune cell populations in the blood

[0146] Experimental Procedure: C57BL / 6 background mice (C57BL / 6 background mice from the Biocytogen breeding colony that have been genetically modified to replace the endogenous full-length CD132 domain with the corresponding human sequence) were subcutaneously administered with antibodies h2D4H4K12 or h5H10H6K4 at a dose of 8 mg / kg or 16 mg / kg once every four days for three doses. No administration of antibodies h2D4H4K12 or h5H10H6K4 served as a control.

[0147] Table 3 Experimental drug administration and treatment schemes for mice in each group

[0148] Grouping Recipient strain Number of each group Antibody treatment Group A <![CDATA[CD132 hu / hu ]]> 6 h2D4H4K12, 8mg / kg Group B <![CDATA[CD132 hu / hu ]]> 6 h2D4H4K12, 16 mg / kg Group C <![CDATA[CD132 hu / hu ]]> 6 h5H10H6K4, 8mg / kg Group D <![CDATA[CD132 hu / hu ]]> 6 h5H10H6K4, 16mg / kg Group E <![CDATA[CD132 hu / hu ]]> 6 Not applied

[0149] Analysis of immune cell populations in the blood over a period of time by flow cytometry. The specific process is as follows: The number of total immune cells, B cells, T cells, NK cells, and neutrophils in peripheral blood at different time points (once every 10 days) was analyzed by flow cytometry to evaluate the effect of h2D4H4K12 and h5H10H6K4 antibodies on the absolute number of these cell types. Briefly, at each time point, blood samples were collected from each mouse, and 50-100 μL of each blood sample was incubated in red blood cell lysis buffer (Biyuntian) at room temperature for 10 minutes to lyse red blood cells. If lysis was not complete, a second round of lysis was performed. After washing twice in PBS buffer (Shanghai Peiyuan, pH 7.2), mouse IgG (Jackson Immunoresearch) was diluted to 200 μg / ml in flow buffer (4% FBS (ExCell Bio)) to prepare FC receptor blocking solution. 50 μL of blocking solution was added to each lysed blood sample and blocked at room temperature for 10 minutes. The cells were then stained for cell surface markers by adding a cocktail of fluorescently labeled antibodies (described in Table 4) diluted in flow buffer to identify CD45 + Cells, T cells, B cells, and NK cells (the absolute counts of neutrophils, monocytes, red blood cells, and platelets were all determined using a mouse blood routine analyzer). Finally, the samples were washed twice in flow cytometry buffer, resuspended in PBS buffer (pH 7.2), and sample data were acquired on a BDcelesta flow cytometer. Data analysis was performed using FlowJov10 software. + Immune cells were defined as singlet, viable cells, and within this group, T cells were further defined as CD3 + , Treg cells were further defined as CD4 + 、CD25 + , B cells were further defined as CD3 - 、CD19 + NK cells are further defined as CD3 - 、CD19-、NK1.1 + .

[0150] Table 4 Antibodies used in flow cytometry analysis

[0151] Antibody Fluorescent antibodies Manufacturer Final dilution CD45 PE BD 1:400 CD3 APC-Cy7 BD 1:200 CD4 FITC Biolegend 1:200 CD8a BV510 BD 1:200 CD19 APC Biolegend 1:200 CD25 PerCP-Cy5.5 BD 1:200 NK1.1 BV650 BD 1:200

[0152] The results are as follows Figure 9 As shown: Treatment of background mice with h2D4H4K12 or h5H10H6K4 (8 mg / kg and 16 mg / kg) resulted in a decrease in T cells in their blood ( Figure 9 A) and B cells ( Figure 9 B) and NK cells ( Figure 9C) were significantly decreased, while neutrophils ( Figure 9 D) red blood cells ( Figure 9 E), monocytes ( Figure 9 F), platelet count ( Figure 9 G) were not affected, among which h2D4H4K12 had no significant effect on the proportion of Treg cells ( Figure 9 H). After three doses, serum concentrations of h2D4H4K12 and h5H10H6K4 decreased over time. By the end of the study, all of these populations had returned to levels similar to those observed before treatment. These results suggest that both h2D4H4K12 and h5H10H6K4 antibodies have varying degrees of inhibitory effects on B cells, T cells, and NK cells, but do not affect the number of neutrophils, monocytes, red blood cells, or platelets. Furthermore, h2D4H4K12 has a weaker inhibitory effect on Treg cells than h5H10H6K4.

[0153] Example 6: Establishment of a pristane-induced mouse lupus erythematosus model for evaluating the blocking activity of anti-human CD132 antibodies h2D4H4K12 and h5H10H6K4.

[0154] Experimental procedure: C57BL / 6 background mice (C57BL / 6 background mice genetically modified from the Biocytogen breeding colony to replace the endogenous CD132 full-length domain with the corresponding human sequence) were injected with 0.5 mL of pristane (MCE) into the peritoneal cavity, and the control group was injected with an equal volume of PBS buffer (pH 7.2). h2D4H4K12 or h5H10H6K4 or anti-KLH isotype control (all antibodies are produced by Conoya Biotechnology Co., Ltd.) was administered subcutaneously at a dose of 20 mg / kg 3 days before modeling, twice a week for 6 weeks, or not (Table 5). The levels of urine protein, urine creatinine, and plasma anti-dsDNA in the mice were monitored. The mice were killed by cervical dislocation at week 12.

[0155] Specific monitoring experimental steps:

[0156] 1. Use the Mouse Urine Microalbumin ELISA Detection Kit (Elabscience) to detect mouse urine microalbumin: Collect 20μl of urine from each mouse and perform the test according to the operating procedures. Briefly, dilute the urine of each mouse 1000 times with the standard / sample diluent, add 100μl to each well of the 96-well ELISA plate that has been coated in the kit, incubate at 37°C for 90 minutes, dry, add 100μl of biotinylated antibody working solution to each well, and incubate at 37°C for 1 hour. Wash 4 times, add 100μl of HRP enzyme conjugate working solution to each well, incubate at 37°C for 30 minutes, wash 4 times, add 90μl of color development solution, place at 37°C for 10 minutes, add 50μl of stop solution, and immediately place in the microplate reader to read the OD 450 value.

[0157] 2. ELISA test for anti-dsDNA level in mouse plasma: Collect 50-100 μl of blood from each mouse, centrifuge at 4500 rpm, take the supernatant and freeze it at -80°C for subsequent testing. TM Salmon sperm DNA solution (Invitrogen) was incubated at 4°C overnight. The next day, the cells were washed three times with PBS buffer (pH 7.2) and blocked with 10% BSA (Sangon) at 37°C for 1 hour. The cells were then washed three times with PBS buffer (pH 7.2). A 50-fold diluted plasma sample was added to each well, 100 μl, and the cells were incubated at 37°C for 90 minutes. The cells were then washed five times with PBS buffer (pH 7.2) and HRP-labeled Fc-specific anti-mouse IgG1 (abclonal) was added at a ratio of 1:4000. The cells were incubated at room temperature for 1 hour, washed seven times with PBS buffer (pH 7.2), and 100 μl of color development solution (TMB solution, Sigma catalog number T2885) was added. The cells were incubated at 37°C for 10 minutes, and then 50 μl of 2M concentrated sulfuric acid solution was added to terminate the reaction. The cells were immediately placed in a microplate reader and the OD was read. 450 value.

[0158] Table 5 Experimental drug administration and treatment scheme for each group of mice

[0159] Grouping Recipient strain Number of each group Modeling situation mAb treatment Group A <![CDATA[CD132 hu / hu ]]> 5 PBS injection No mAb Group B <![CDATA[CD132 hu / hu ]]> 10 Injection of pristane anti KL (isotype) Group C <![CDATA[CD132 hu / hu ]]> 10 Injection of pristane h2D4H4K12 Group D <![CDATA[CD132 hu / hu ]]> 10 Injection of pristane h5H10H6K4

[0160] The results are as follows Figure 10 As shown: h2D4H4K12 or h5H10H6K4 antibodies can reduce the level of autoantibodies anti-dsDNA in mouse plasma ( Figure 10 A) Reduce the urine protein / creatinine ratio of mice ( Figure 10B) This indicates that h2D4H4K12 and h5H10H6K4 antibodies can significantly reduce urine protein and plasma anti-dsDNA levels in pristane-induced lupus mice and improve their survival rate.

[0161] In summary, the above in vitro and in vivo experiments have shown that the humanized anti-human CD132 antibodies h2D4H4K12 and h5H10H6K4 can inhibit the activity of IL4, IL-7, IL9, IL-15 and / or IL-21. Further experiments in lupus erythematosus model mice have found that both antibodies can significantly improve the phenotype of lupus model mice. Therefore, these two antibodies may have potential value in treating autoimmune diseases in which the secretion of these cytokines is an important pathophysiological change.

[0162] The present invention provides a humanized anti-human CD132 monoclonal antibody and its application ideas and methods. There are many methods and approaches to implement this technical solution. The above is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention. Such improvements and modifications should also be considered as the scope of protection of the present invention. All components not specified in this embodiment can be implemented using existing technologies.

Claims

1. A humanized anti-human CD132 monoclonal antibody, characterized in that The monoclonal antibody is a humanized monoclonal antibody, The monoclonal antibody comprises a light chain complementary determining region and a heavy chain complementary determining region, wherein the light chain complementary determining region includes LCDR1, LCDR2 and LCDR3, and the heavy chain complementary determining region includes HCDR1, HCDR2 and HCDR3; The light chain complementary determining region and heavy chain complementary determining region are as follows: The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs. 26, 27 and 31, respectively; the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs. 8, 9 and 10, respectively.

2. The monoclonal antibody according to claim 1, characterized in that The monoclonal antibody comprises a light chain variable region and a heavy chain variable region, and the light chain variable region and the heavy chain variable region are as follows: The amino acid sequence of the light chain variable region is shown in SEQ ID NO. 29; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

6.

3. The monoclonal antibody according to claim 1, characterized in that The monoclonal antibody further comprises a constant region, which is a kappa chain constant region Ckappa and a human IgG4 constant region CH1-CH3.

4. A nucleic acid molecule, characterized in that Encodes the monoclonal antibody according to any one of claims 1 to 3.

5. An expression vector, characterized in that Comprising the nucleic acid molecule of claim 4.

6. A host cell, characterized in that Comprising the expression vector according to claim 5.

7. The host cell according to claim 6, characterized in that The host cell is HEK293 cell.

8. A detection reagent or kit, characterized in that: The method comprises the monoclonal antibody according to any one of claims 1 to 3.

9. Use of the monoclonal antibody according to any one of claims 1 to 3 in the preparation of a medicament for treating autoimmune diseases; in, The autoimmune disease is systemic lupus erythematosus.

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