CN117886940A - Humanized anti-human CD132 monoclonal antibody and application thereof - Google Patents
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Abstract
The invention belongs to the field of biological medicine, and in particular relates to a humanized anti-human CD132 monoclonal antibody and application thereof. The invention screens and obtains the high-affinity monoclonal humanized antibody H2D4H4K12 and/or H5H10H6K4 which can target human CD132 by humanized reconstruction of the target antibody on the basis of monoclonal chimeric 2D4 and 5H10 obtained in the laboratory, reduces the immunogenicity of the female parent chimeric on the basis of not weakening the activity of the antibody, and further reduces the risk that a patient taking the drug possibly generates immune response to the antibody. Has good application prospect in the treatment medicaments for preventing, neutralizing or treating autoimmune diseases (such as rheumatoid arthritis, systemic lupus erythematosus and the like) related to IL-4, IL-7, IL-9, IL-15 and/or IL-21 cytokines.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a humanized anti-human CD132 monoclonal antibody and application thereof.
Background
CD132 (IL 2 Rgamma), the gamma subunit of the interleukin 2 (Interleukin-2, IL-2) receptor, which is simultaneously the gamma subunit of the following 6 cytokine receptor complexes: IL2, IL-4, IL-7, IL-9, IL-15, IL-21. And is therefore also referred to as yc (common gamma chain). Systemic lupus erythematosus (Systemic lupus erythematosus, SLE) is a chronic autoimmune disease involving multiple organs, multiple systems. The etiology and pathogenesis are complex and are associated with the destruction of autoimmune tolerance, but the exact etiology and specific pathogenesis are not yet clear. Among them, B cell dysfunction, deposition of the produced autoantibodies in patients to form immune complexes are key causes of SLE development. Targeting B cell therapy SLE is a focus of research at home and abroad, including Belimumab mab targeting blys that has been approved for treatment of lupus erythematosus and fusion protein telicaccept that can target blys and april simultaneously. However, in addition to B cell disorders, T cell hyperproliferation and activation are also central factors in SLE pathology. Thus, targeting B cells alone does not meet the need for clinical treatment of SLE. CD4 in SLE patients with impaired number and function + Follicular helper T cells (Tfh) and peripheral helper T cell populations (Th 1, th2, th17, treg) mediate B cell activation and autoantibody production by providing costimulatory signals and cytokines. Thus, some T cell-associated cytokines have been of great interest for their promoting role in SLE inflammation and organ damage, and have become promising therapeutic targets.
Several documents demonstrate that IL4, IL-7, IL9, IL-15 and IL-21 among gamma-chain family cytokines are significantly upregulated in immune cells of SLE, and these gamma-chain cytokines play a key role in the survival, proliferation and differentiation of various lymphocytes. Given that SLE is a complex disease with multiple cells and multiple factors involved together and that gamma-chain cytokines play a key role in various lymphocytes, and is closely related to the occurrence and development of SLE, the intervention of the functional activity of CD132 in gamma-chain cytokines may become a potential treatment means for SLE.
Disclosure of Invention
The invention aims to solve the technical problem of providing a humanized anti-human CD132 monoclonal antibody aiming at the defects of the prior art.
In order to solve the technical problems, the invention adopts the following technical scheme:
a humanized anti-human CD132 monoclonal antibody, the monoclonal antibody is a humanized monoclonal antibody,
wherein the monoclonal antibody comprises a light chain complementarity determining region and a heavy chain complementarity determining region, wherein the light chain complementarity determining region comprises LCDR1, LCDR2 and LCDR3, and the heavy chain complementarity determining region comprises HCDR1, HCDR2 and HCDR3;
specifically, the light chain complementarity determining region and the heavy chain complementarity determining region select any one of the following groups:
a first group: the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.18, 14 and 15; the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.3, 4 and 5 in sequence;
or,
second group: the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.26, 27 and 28; the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.8, 9 and 10 in sequence.
Wherein 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:
a first group: the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 22; the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 1;
or,
second group: the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 29; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 6.
Wherein the monoclonal antibody further comprises constant regions, and the constant regions are kappa chain constant regions Ckappa and human IgG4 constant regions CH1-CH3.
The invention also provides a nucleic acid molecule, in particular a nucleotide sequence for encoding the monoclonal antibody.
The invention also provides an expression vector, in particular, the expression vector comprises the nucleic acid molecule.
The invention also provides a host cell, in particular to a host cell containing the expression vector.
Wherein the host cell is HEK293 cell.
The invention also provides a detection reagent or a kit, which is characterized by comprising the monoclonal antibodies H2D4H4K12 and/or H5H10H6K4.
The use of the monoclonal antibodies described above for the preparation of a medicament for inhibiting or reducing the activity of human CD132 is also within the scope of the invention.
Specifically, in some embodiments, in detecting the inhibitory activity of a humanized candidate antibody on CD132 in an in vitro activity assay, the humanized anti-human CD132 antibody H2D4H4K12 may inhibit the activity of IL-21 to stimulate release of IFN- γ by NK92 cells; humanized anti-human CD132 antibody H5H10H6K4 can inhibit the activity of IL-4 in stimulating expression of CD23 by Ramos cells.
Specifically, in some embodiments, C57BL/6 background mice (genetically modified to replace endogenous CD132 full length domain with corresponding human sequences from the bazedox breeding cluster C57BL/6 background mice) were given antibody H2D4H4K12 or H5H10H6K4 subcutaneously at a dose of 8mg/kg or 16mg/kg, with a frequency of 4 days for 3 doses, and the effect of H2D4H4K12 and H5H10H6K4 antibodies on the absolute numbers of total immune cells, B cells, T cells, NK cells, neutrophils, monocytes, erythrocytes and platelets in peripheral blood at different time points (1 per 10 days) was analyzed by flow cytometry to assess the absolute numbers of these cell types. As a result, it was found that the two antibodies of H2D4H4K12 and H5H10H6K4 had different degrees of inhibition on B cells, T cells and NK cells, but did not affect the numbers of neutrophils, monocytes, erythrocytes and platelets.
The use of the monoclonal antibodies described above for the preparation of a therapeutic agent for the prevention, neutralization or treatment of autoimmune diseases is also within the scope of the present invention.
Wherein the autoimmune disease is an immune disease associated with IL-4, IL-7, IL-9, IL-15 and/or IL-21 cytokine receptors, comprising: systemic lupus erythematosus, rheumatoid arthritis, and the like.
Specifically, in some embodiments, 0.5mL of prine (MCE) is injected into the abdominal cavity of C57BL/6 background mice (C57 BL/6 background mice genetically modified from the baisai pattern breeding clusters to replace the endogenous CD132 full-length domain with the corresponding human sequence), and an equal volume of PBS buffer (pH 7.2) is injected into the control group. H2D4H4K12 or H5H10H6K4 or anti-KLH isotype control (antibodies above were all manufactured by Kannoa Biotechnology Co., ltd.) were subcutaneously administered at a dose of 20mg/kg 3 days prior to molding, 2 times per week for 6 weeks or no administration (Table 5). Mouse urine protein, urinary creatinine, and plasma anti-dsDNA levels were monitored. As a result, the two antibodies H2D4H4K12 and H5H10H6K4 can obviously reduce the urine protein and plasma anti-dsDNA level of the prine-induced lupus mice, and improve the survival rate of the mice.
The beneficial effects are that:
the invention obtains two monoclonal humanized antibodies (H2D 4H4K12 and H5H10H6K 4) with high affinity, which can target human CD132, through humanized modification of two antibodies (2D 4 and 5H 10) obtained in the prior laboratory and screening. The inhibitory activity of the two antibodies on 5 gamma c cytokines is consistent with that of the female parent chimeras 2D4 and 5H10, and meanwhile, the immunogenicity of the female parent chimeras is reduced, and the risk that a patient taking the drug possibly generates immune response on the antibodies is further reduced. Both antibodies can be used in therapeutic agents for preventing, neutralizing or treating autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosus, etc.) associated with IL-4, IL-7, IL-9, IL-15 and/or IL-21 cytokines. The invention further illustrates the prospect of the two antibodies applied to the medicines for treating the systemic lupus erythematosus through a systemic lupus erythematosus animal model.
Drawings
The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.
FIG. 1 inhibition of NK92 cell secretion IFN-gamma by maternal chimeric anti-human CD132 antibody 2D4 and multiple humanized 2D4 antibodies under constant IL-21 stimulation.
FIG. 2 maternal chimeric anti-human CD132 antibody 5H10 and multiple humanized 5H10 antibodies inhibited Ramos cell expression CD23 under constant IL-4 stimulation.
FIG. 3H5H10H6K4 inhibits secretion of IFN-gamma by IL 21-stimulated NK92 cells.
FIG. 4H2D4H4K12 inhibits IL4 from stimulating expression of CD23 by Ramos cells.
FIGS. 5H5H10H6K4 (FIG. 5-1) and H2D4H4K12 (FIG. 5-2) inhibit IL21 from stimulating secretion of IFN- γ by NK92 cells.
FIG. 6H5H10H6K4 (FIG. 6-1) and H2D4H4K12 (FIG. 6-2) inhibit IL7 from stimulating CD4 + Downstream phosphorylation of T cells.
FIGS. 7H5H10H6K4 (FIG. 7-1) and H2D4H4K12 (FIG. 7-2) inhibit IL9 from stimulating M07E cell proliferation.
FIG. 8H2D4H4K12 and H5H10H6K4 immunogenicity.
FIG. 9H2D4H4K12 and H5H10H6K4 antibody pair CD132 hu/hu Background effects of T cells (9A), B cells (9B), NK cells (9C), neutrophils (9D), erythrocytes (9E), monocytes (9F), platelets (9G) and Treg cells (9H) in mice.
FIG. 10H2D4H4K12 and H5H10H6K4 antibodies to prine-induced CD132 hu/hu Anti dsDNA amount (10A) and urine protein/creatinine ratio (10B) of the mouse lupus erythematosus model.
Detailed Description
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, are commercially available.
In previous studies of the subject group, monoclonal chimeras 2D4 and 5H10 that can target human CD132 have been obtained by phage selection (detailed screening, construction information and sequence information of monoclonal chimeras 2D4 and 5H10 of CD132 have been disclosed in patent CN116970081 a), but antibodies in the chimeric configuration are highly immunogenic, easily causing antibodies to be produced by the body, thus impairing antibody efficacy. Therefore, the patent further discloses humanized modification of the target antibodies 2D4 and 5H10, reduces the immunogenicity of the chimeric on the basis of not weakening the activity of the antibodies, and simultaneously performs in vivo pharmacodynamics verification on the target antibodies (H2D 4H4K12 and H5H10H6K 4) so as to further prove the effectiveness of the two antibodies.
All experiments were approved and conducted as directed by the ethical committee of the dermatology hospital of the national academy of medical science (the dermatology institute of the national academy of medical science).
Example 1: humanized engineering of monoclonal antibodies 2D4 and 5H10
Comparing the variable region sequences of the monoclonal antibodies 2D4 and 5H10 with the human germline antibody sequences, finding out sequences with high homology, and carrying out CDR transplantation; and then, simultaneously carrying out homologous modeling by using a computer, analyzing the CDR region and the frame amino acid sequences around the CDR region, and examining the spatial stereo combination mode. The key amino acid individuals possibly acting with CD132 and maintaining space frames in the gene sequences of each positive monoclonal antibody are analyzed by calculating electrostatic force, van der Waals force, hydrophilicity and hydrophobicity and entropy values, and the back mutation sites are designed on the basis. HLA-DR affinity was examined by analysis, and a human germline framework sequence with low immunogenicity was selected. The above humanized modifications were all designed and completed by Kannoa biotechnology Co.
A total of 1 heavy chain variable region derivative (h 2D4-VH 4) and 4 light chain variable region derivatives (h 2D4-VL3, h2D4-VL9, h2D4-VL11, h2D4-VL 12) were designed for 2D 4; a total of 1 heavy chain variable region derivative (H5H 10-VH 6) and 2 light chain variable region derivatives (H5H 10-VL3, H5H10-VL 4) were designed for 5H 10. All the light and heavy chain variable region derivative sequences described above are shown in tables 1 and 2. The light chain variable region derivatives were cloned into pHCT2 vectors (available from Addgene) containing human IgG kappa light chain constant regions and regulatory elements, developed independently by Kang Nuoya company, to express intact IgG kappa light chains in mammalian cells. The heavy chain variable region derivatives were cloned into pHCT1s vectors (available from Addgene) containing human IgG4 subtype heavy chain constant regions and regulatory elements, developed independently by Kang Nuoya company, to express the complete IgG4 subtype heavy chain in mammalian cells. The plasmids were paired and transfected into HEK293 cells (purchased from ATCC) for 5-6 days, and the supernatants were collected and filtered and purified by using a protein A column to construct candidate humanized antibodies H2D4H4K3, H2D4H4K9, H2D4H4K11, H2D4H4K12, H5H10H6K4 and H5H10H6K5.
Table 1 heavy chain variable region derivative sequences of candidate CD132 humanized antibodies (numbers previously omitted SEQ ID No.: x)
Table 2 light chain variable region derivative sequences of candidate CD132 humanized antibodies (numbers preceded by the omission of SEQ ID No..: x)
Specifically, humanized antibody heavy chain variable region derivative sequences:
1) The amino acid sequence of h2D4-VH4 is shown as SEQ ID NO.1, the encoding nucleotide is shown as SEQ ID NO.2, and CDR1, CDR2 and CDR3 are shown as SEQ ID NO.3, 4 and 5 respectively.
<------------FR1------------>CDR1<----FR2----->CDR2
QVQLVQSGAEVKKPGSSVKVSCKTSGFNIEDIYLHWVRQAPGQGLEWMGRIDPANGK
<-------------FR3-------------->CDR3<---FR4-
SNYDPKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCAALRFFGLDYWGQGTLVT
-->
VSS
Nucleotide sequence
CAGGTTCAGCTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCAGCAGCGTGAAGGTGAGCTGCAAGACAAGCGGCTTCAACATCGAGGACATCTACCTGCACTGGGTCCGACAGGCTCCAGGACAGGGACTTGAGTGGATGGGCAGAATCGACCCTGCCAACGGCAAGAGCAACTACGACCCCAAGTTCCAGGGCAGAGTCACCATCACAGCCGACACCAGCACCAGCACCGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTACTGTGCCGCTCTGAGATTCTTCGGCCTGGACTACTGGGGCCAGGGAACACTGGTTACCGTGTCTAGT
2) The amino acid sequence of H5H10-VH6 is shown as SEQ ID NO.6, the coding nucleotide is shown as SEQ ID NO.7, and CDR1, CDR2 and CDR3 are shown as SEQ ID NO.8, 9 and 10 respectively.
<------------FR1------------>CDR1<----FR2----->CDR2
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWVRQAPGQGLEWMGDIYPGGGY
<-------------FR3-------------->CDR3<--FR4
TNYNEKFQGRVTMTADTSTSTVYMELSSLRSEDTAVYFCARGDYGSSWFPYWGQGTL
---->
VTVSS
Nucleotide sequence
CAGGTTCAGCTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGGTGAGCTGCAAGGCTAGCGGCTACACCTTCACCAACTACTGGCTCGGCTGGGTCCGACAGGCTCCTGGACAGGGACTGGAATGGATGGGCGACATCTACCCTGGCGGCGGATACACAAACTACAACGAGAAGTTCCAGGGCAGAGTCACAATGACCGCCGACACCTCTACCAGCACAGTCTACATGGAACTGAGCAGCCTGAGAAGCGAGGATACCGCCGTGTACTTCTGTGCCAGAGGCGACTACGGCTCTAGCTGGTTTCCTTACTGGGGCCAGGGAACCCTGGTCACCGTTTCTTCT
Specifically, humanized antibody light chain variable region derivative sequences:
3) The amino acid sequence of h2D4-VL3 is shown as SEQ ID NO.11, the encoding nucleotide is shown as SEQ ID NO.12, and CDR1, CDR2 and CDR3 are shown as SEQ ID NO.13, 14 and 15 respectively.
<---------FR1--------->CDR1<-----FR2----->CDR2<-
DMQMTQSPSSLSASVGDRVTITCSATQSVSYMYWYQQKPGKAPELWIYRTSNLASGV
-------------FR3-------------->CDR3<--FR4--->
PSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPPTFGQGTKLEIK
Nucleotide sequence
GACATGCAGATGACACAGAGCCCTTCTAGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCATCACATGTAGCGCCACACAGAGCGTGTCCTACATGTACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTGAGCTTTGGATCTACAGAACAAGCAACCTGGCCAGCGGCGTGCCCTCTAGATTTTCTGGCTCTGGCAGCGGCACCGACTACACCTTCACAATCTCTAGCCTGCAGCCTGAGGATATCGCCACCTACTACTGCCAGCAGTGGTCTAGCAACCCTCCTACATTCGGCCAGGGCACCAAGCTGGAAATCAAG
4) The amino acid sequence of h2D4-VL9 is shown as SEQ ID NO.16, the encoding nucleotide is shown as SEQ ID NO.17, and CDR1, CDR2 and CDR3 are shown as SEQ ID NO.18, 14 and 15 respectively.
<---------FR1--------->CDR1<-----FR2----->CDR2<-
EIVLTQSPATLSLSPGERATLSCRATSSVSYMYWYQQKPGQAPRLLIYRTSNLASGI
-------------FR3------------->CDR3<--FR4--->
PARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPTFGQGTKLEIK
Nucleotide sequence
GAGATCGTGCTGACACAGAGCCCTGCCACACTGTCACTGTCTCCAGGCGAGAGAGCCACACTGAGCTGTCGCGCCACCAGCAGCGTGTCCTACATGTACTGGTATCAGCAGAAGCCCGGCCAGGCTCCTCGGCTTCTGATCTACAGAACAAGCAACCTGGCCAGCGGCATCCCCGCTAGATTTTCTGGCTCTGGCAGCGGCACCGACTACACCCTGACAATCTCTAGCCTGGAACCTGAGGACTTCGCCGTGTACTACTGCCAGCAGTGGTCTAGCAACCCTCCTACATTCGGCCAGGGCACCAAGCTGGAAATCAAG
5) The amino acid sequence of h2D4-VL11 is shown as SEQ ID NO.19, the encoding nucleotide is shown as SEQ ID NO.20, and CDR1, CDR2 and CDR3 are shown as SEQ ID NO.21, 14 and 15 respectively.
<---------FR1--------->CDR1<-----FR2----->CDR2<-
DIQMTQSPSSLSASVGDRVTITCQATQSVSYMYWYQQKPGKAPELLIYRTSNLASGV
-------------FR3------------->CDR3<--FR4--->
PSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPPTFGQGTKLEIK
Nucleotide sequence
GACATCCAGATGACACAGAGCCCTTCTAGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCATCACATGTCAGGCCACACAGAGCGTGTCCTACATGTACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTGAGCTTCTGATCTACAGAACAAGCAACCTGGCCAGCGGCGTGCCCTCTAGATTTTCTGGCTCTGGCAGCGGCACCGACTACACCTTCACAATCTCTAGCCTGCAGCCTGAGGATATCGCCACCTACTACTGCCAGCAGTGGTCTAGCAACCCTCCTACATTCGGCCAGGGCACCAAGCTGGAAATCAAG
6) The amino acid sequence of h2D4-VL12 is shown as SEQ ID NO.22, the encoding nucleic acid is shown as SEQ ID NO.23, and CDR1, CDR2 and CDR3 are shown as SEQ ID NO.18, 14 and 15 respectively.
<---------FR1--------->CDR1<-----FR2----->CDR2<-
EIVLTQSPATLSLSPGERATLSCRATSSVSYMYWYQQKPGQAPELLIYRTSNLASGI
-------------FR3------------->CDR3<--FR4--->
PARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPPTFGQGTKLEIK
Nucleotide sequence
GAGATCGTGCTGACACAGAGCCCTGCCACACTGTCACTGTCTCCAGGCGAGAGAGCCACACTGAGCTGTCGCGCCACCAGCAGCGTGTCCTACATGTACTGGTATCAGCAGAAGCCCGGCCAGGCTCCTGAGCTTCTGATCTACAGAACAAGCAACCTGGCCAGCGGCATCCCCGCTAGATTTTCTGGCTCTGGCAGCGGCACCGACTACACCCTGACAATCTCTAGCCTGGAACCTGAGGACTTCGCCGTGTACTACTGCCAGCAGTGGTCTAGCAACCCTCCTACATTCGGCCAGGGCACCAAGCTGGAAATCAAG
7) The amino acid sequence of H5H10-VL3 is shown as SEQ ID NO.24, the encoding nucleotide is shown as SEQ ID NO.25, and CDR1, CDR2 and CDR3 are shown as SEQ ID NO.26, 27 and 28 respectively.
<---------FR1--------->CDR1<-----FR2----->CDR2<-
DIQMTQSPSSLSASVGDRVTITCRASQSISNNLHWYQQKPGKAPKLLIKYASQSISG
-------------FR3------------->CDR3<--FR4--->
VPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQSDSWLTFGQGTKVEIK
Nucleotide sequence
GACATCCAGATGACACAGAGCCCTTCTAGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCATCACATGTAGAGCCAGCCAGAGCATCAGCAACAACCTGCACTGGTATCAGCAGAAGCCCGGCAAGGCTCCCAAGCTGCTGATTAAGTACGCCAGCCAGTCCATCTCCGGCGTGCCATCTAGATTCAGCGGCTCTGGCTCTGGCACCGACTTCACCCTGACAATCTCTAGCCTGCAGCCTGAGGACTTCGCTACCTACTTCTGCCAGCAGAGCGACAGCTGGCTGACATTTGGCCAGGGCACCAAGGTGGAAATCAAG
8) The amino acid sequence of H5H10-VL4 is shown as SEQ ID NO.29, the encoding nucleotide is shown as SEQ ID NO.30, and CDR1, CDR2 and CDR3 are shown as SEQ ID NO.26, 27 and 28 respectively.
<---------FR1--------->CDR1<-----FR2----->CDR2<-
DIQMTQSPSSLSASVGDRVTITCRASQSISNNLHWYQQKPGKAPKLLIKYASQSISG
--------------FR3------------->CDR3<--FR4--->
VPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQSDSYLTFGQGTKVEIK
Nucleotide sequence
GACATCCAGATGACACAGAGCCCTTCTAGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCATCACATGTAGAGCCAGCCAGAGCATCAGCAACAACCTGCACTGGTATCAGCAGAAGCCCGGCAAGGCTCCCAAGCTGCTGATTAAGTACGCCAGCCAGTCCATCTCCGGCGTGCCATCTAGATTCAGCGGCTCTGGCTCTGGCACCGACTTCACCCTGACAATCTCTAGCCTGCAGCCTGAGGACTTCGCTACCTACTTCTGCCAGCAGAGCGACAGCTACCTGACATTTGGCCAGGGCACCAAGGTGGAAATCAAG
Example 2: in vitro Activity assay to test the inhibitory Activity of candidate humanized antibodies against CD132
To further evaluate the activity gap between the candidate humanized antibody and the maternal chimera, refer to the experimental method in patent CN116970081a, since maternal chimera 2D4 has strong inhibitory activity on IL21, the 2D4 candidate humanized antibody was evaluated by a method in which IL21 stimulates NK92 cell line to secrete IFN- γ; because the activity gap between 5H10 candidate humanized antibodies was more pronounced in the inhibition assay system for IL4, the method of selecting IL4 to stimulate Ramos cell lines to up-regulate CD23 was further evaluated for 5H10 candidate humanized antibodies.
2.1 Verification that 2D4 humanized anti-human CD132 candidate antibody inhibits NK92 cell secretion IFN-gamma
Inoculation of 2X 10 in 96-well plates with growth medium (prepared according to the instructions for Pronoxel, but without IL-2) 4 NK-92 cells (Prunorace CL-0530) and at 37℃in 5% CO 2 Starvation was performed overnight. The following 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 min, respectively. After incubation, 250pMIL-21 was added separately to NK-92 cells containing different 2D4 humanized anti-human CD132 candidate antibodies. At 37℃in 5% CO 2 After 72h of incubation, IFN-. Gamma.secretion was measured by ELISA.
The capture antibodies (capture antibodies) were coated at 1:250 on 96-well ELISA plates using human IFN- γ ELISA kit (BD cat No. 555142) and incubated overnight at 4 ℃. After 3 washes with PBS buffer (pH 7.2) the next day, the cells were blocked with 10% FBS at room temperature for 1 hour, PBS buffer (pH 7.2) followed by 3 washes, then 100. Mu.l of the sample (cell supernatant) and the standard were added, respectively, incubated at room temperature for 2 hours, and then washed with PBS buffer (pH 7.2) 5 times, 5AV-HRP was added to a dilution containing 0.4% of the detection antibody (Detection Antibody) at a ratio of 1:250. Finally, the incubation was carried out for 1 hour at room temperature, the mixture was washed 7 times with PBS buffer (pH 7.2), 100. Mu.l of a color-developing solution (TMB solution, sigma stock number T2885) was added, the reaction was stopped by adding 50. Mu.l of a 2M concentrated sulfuric acid solution after 10 minutes at 37℃and the OD was read immediately in an ELISA reader 450 Figure 1 shows that the humanized anti-human CD132 antibody H2D4H4K12 inhibits IL-21 to stimulate NK92 cells to release IFN- γ with activity comparable to that of the parental 2D4 chimera.
2.2 Verification that 5H10 humanized anti-CD 132 candidate antibody inhibits IL-4 from stimulating Ramos cells to express CD23
IL-4 stimulates Ramos cells to express CD23, and this feature can be used to evaluate the inhibition of IL-4 signaling by 5H10 humanized anti-CD 132 candidate antibodies. Ramos cell line (ATCC) was completely cultured with RPMI 1640 containing 10% FBS at 37℃on the basis of 5% CO 2 Culturing under conditions to control cell concentration to 2×10 per 1ml 5 -2×10 6 Individual cells, which were seeded into 96-well flat bottom cell culture plates at 100 μl per well. Preparation of consecutive 5H10 humanized anti-CD 132 candidate antibodies in RPMI 1640 complete MediumDilutions were performed in 4-fold gradient of antibody from 200nM to 0.003nM, and after incubation with cells for 30 min 50. Mu.l IL-4 was added at a final concentration of 2.67nM, 5% CO at 37 ℃ 2 Culturing for 48 hours under the condition.
The cultured cells were transferred to a U-type 96-well cell culture plate, centrifuged at 300g for 3 minutes at 4℃and the supernatant was discarded, and the cells were washed twice with a flow buffer (PBS buffer containing 4% calf serum, pH 7.2) and 200. Mu.l per well. 50 μl of blocking solution (buffer containing 100 μg/ml hIgG) was added to the cells, blocking was performed in an ice bath for 10 min, centrifugation was performed for 3 min at 300g, and the supernatant was discarded, then 50 μl of anti-human CD23 FITC antibody diluent (BD EBVCS-5) was added per well, and blocking was performed in an ice bath for 20 min; after centrifugation at 300g for 3 min, the supernatant was discarded, the cells were washed twice with flow buffer, 200 μl per well; the PI staining solution was added to the cells, 100. Mu.l of each well was kept away from light for 5 minutes in an ice bath, the supernatant was discarded by centrifugation for 3 minutes at 300g, and after washing the cells twice with 200. Mu.l of each well of flow buffer, the cells were resuspended with 100. Mu.l of PBS buffer (pH 7.2) per well, and the average fluorescence intensity was read by flow cytometry, and the measurement results were recorded. As shown in FIG. 2, the humanized anti-human CD132 antibody H5H10H6K4 inhibited IL-4 from stimulating Ramos cells to express CD23 with activity comparable to that of the parental 5H10 chimera.
Example 3: humanized anti-human CD132 antibodies H5H10H6K4 and H2D4H4K12 were re-validated
The inhibitory activity of the candidate humanized antibodies against CD132 was tested by in vitro activity assay in example 2, which screened for two humanized anti-human CD132 antibodies H5H10H6K4 and H2D4H4K12, and this example further re-validated whether the two antibodies were identical to the maternal chimeras 2D4 and 5H10 for the inhibitory activity of the two antibodies against the 5 yc cytokines.
3.1 humanized anti-human CD132 antibody H5H10H6K4 inhibiting IL21 stimulates NK92 cells to secrete IFN-gamma
To further assess the ability of H5H10H6K4 to block secretion of IFN-gamma by IL-21 stimulated NK92 cells, specific experimental procedures are detailed in example 2.1, substituting only the antibody for H5H10H6K4. The results are shown in FIG. 3, and FIG. 3 shows that the anti-human CD132 antibody H5H10H6K4 can inhibit IL-21 from stimulating NK92 cells to release IFN-gamma, but has weaker inhibitory activity than H2D4H4K12 compared with the results of H2D4H4K12 in FIG. 1.
3.2 humanized anti-human CD132 antibody H2D4H4K12 inhibition of IL4 stimulation of Ramos cells to express CD23
To further assess the ability of H2D4H4K12 to block secretion of CD23 expressing Ramos cells stimulated by IL-4, specific experimental procedures are detailed in example 2.2, substituting only the antibody for H2D4H4K12. The results are shown in FIG. 4, and FIG. 4 shows that the anti-human CD132 antibody H2D4H4K12 can inhibit IL-4 from stimulating Ramos cells to express CD23.
3.3 humanized anti-human CD132 antibodies H2D4H4K12 and H5H10H6K4 inhibiting IL15 stimulating NK92 cells to secrete IFN-gamma
To further evaluate the ability of H2D4H4K12 and H5H10H6K4 to block secretion of IFN-gamma by IL-15 stimulated NK92 cells, specific experimental procedures are detailed in example 2.1 using antibodies H5H10H6K4 and H2D4H4K12, respectively, and 250pM IL-21 was replaced with 2.5nM IL-15. The experimental results are shown in FIGS. 5-1 and 5-2. FIGS. 5-1 and 5-2 show that both humanized anti-human CD132 antibodies H5H10H6K4 and H2D4H4K12 can inhibit IL-15 stimulated NK92 cells from releasing IFN-. Gamma.but that H5H10H6K4 has a weaker inhibitory activity than H2D4H4K12, respectively.
3.4 flow cytometry analysis of STAT phosphorylation in human cd4+ T cells (human PBMC)
To further evaluate the in vitro properties of humanized anti-human CD132 antibodies H5H10H6K4 and H2D4H4K12, they were further assayed by protein phosphorylation flow cytometry to block IL-7 induced CD4 + T cell activation ability. The experimental procedure 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. The experimental results are shown in FIGS. 6-1 and 6-2. FIGS. 6-1 and 6-2 show that anti-human CD132 antibodies H5H10H6K4 and H2D4H4K12, respectively, can inhibit IL-7 from stimulating downstream STAT5 phosphorylation in human peripheral blood CD4+ cells.
3.5 humanized anti-human CD132 antibodies H2D4H4K12 and H5H10H6K4 inhibit IL-9 from stimulating M07E cell proliferation
Further evaluation of the inhibitory activity of the humanized anti-human CD132 antibodies H5H10H6K4 and H2D4H4K12 on IL-9, the procedure was the same as that of example 10 in patent CN116970081a, except that the chimeric anti-human CD132 antibodies 5H10 and 2D4 were replaced with the humanized anti-human CD132 antibodies H5H10H6K4 and H2D4H4K12. The results are shown in FIG. 7, where FIGS. 7-1 and 7-2 show that humanized anti-human CD132 antibodies H5H10H6K4 and H2D4H4K12 inhibit IL-9 from stimulating M07E cell proliferation, respectively.
Example 4: evaluation of immunogenicity of humanized anti-human CD132 antibodies H2D4H4K12 and H5H10H6K4
Sample preparation:
under aseptic conditions, H2D4H4K12, H5H10H6K4 and KLH (autonomously expressed by Kang Nuoya company) were diluted to 200. Mu.g/mL with X-vivo15 (Shanghai Pegyo) medium and sterilized for use.
The measuring method comprises the following steps:
peripheral venous blood was collected from 25 healthy volunteers and PBMCs were collected by centrifugation through Ficoll (Healthcare). PBS was washed once, counted, and the cell density was adjusted to 2.5X10 with X-vivol5 medium 5 mu.L of 100/mL was added to 96-well U-shaped plates (burning). 100. Mu.L of diluted sample was added to the cells at 37℃with 5% CO 2 Culturing for 48 hours under the same method, wherein KLH is used as a positive control, and a sample hole is not added as a negative control. The 96-well plates were discarded at 200. Mu.L/well, washed once with streaming buffer (PBS+10% FBS), added with hIgG (Jackson) at 100. Mu.L/well, incubated on ice for 30 min, 1. Mu.L/well added with FITC anti-human CD4, APC anti-human CD137 (OX 40), PE anti-human CD134 (4-1 BB) fluorescent antibodies (all available from Biolegend), incubated on ice for 45 min, stained with 5. Mu.g/mL PI at 50. Mu.L for 5 min, washed with streaming buffer for 2 times, resuspended with 100. Mu.L PBS, and analyzed by flow cytometry.
Data analysis:
(1) Lymphocyte loop gate was performed according to cell size (FSC-A) and particle size (SSC-A);
(2) Depletion of adherent cells from the lymphocyte population according to FSC-A and FSC-H, single cell population in the circle;
(3) Circling se:Sub>A living cell population according to FSC-A and PI optical signals in the single cell population;
(4) CD4 positive T cells were circled in the living cells and the proportion of the double positive cell population of CD137 (OX 40) and CD134 (4-1 BB) in this population was analyzed.
The end result is shown in fig. 8, where humanized anti-human CD132 antibodies H2D4H4K12 and H5H10H6K4 double positive cells were comparable to negative controls, indicating that both antibodies had very low immunogenicity.
Example 5: in vivo immunosuppression experiments to evaluate the effects of anti-human CD132 antibodies H2D4H4K12 and H5H10H6K4 on immune cell populations in blood
Experimental procedure: the C57BL/6 background mice (C57 BL/6 background mice genetically modified from the baisai-graph breeding cluster to replace the endogenous CD132 full-length domain with the corresponding human sequences) were given the antibodies H2D4H4K12 or H5H10H6K4 subcutaneously at a dose of 8mg/kg or 16mg/kg, once a frequency of 4 days, for 3 doses, with no administration of the antibodies H2D4H4K12 or H5H10H6K4 as controls.
Table 3 experimental dosing and treatment protocols for mice of each group
| Grouping | Recipient strain | Number of groups | Antibody treatment |
| Group A | CD132 hu/hu | 6 | h2D4H4K12，8mg/kg |
| Group B | CD132 hu/hu | 6 | h2D4H4K12，16mg/kg |
| Group C | CD132 hu/hu | 6 | h5H10H6K4，8mg/kg |
| Group D | CD132 hu/hu | 6 | h5H10H6K4，16mg/kg |
| Group E | CD132 hu/hu | 6 | Not applied |
Analysis of immune cell populations in blood over a period of time by flow cytometry. The specific process is as follows: total immune, B, T, NK and neutrophil numbers in peripheral blood at different time points (1 per 10 days) were analyzed by flow cytometry to assess the effect of H2D4H4K12 and H5H10H6K4 antibodies on the absolute numbers of these cell types. Briefly, at each time point, blood samples from each mouse were collected and 50-100 μl of each blood sample was incubated in red blood cell lysis buffer (bi-cloud) for 10 minutes at room temperature to lyse red blood cells. If the cleavage is not in place, a second round of cleavage is performed. Mice IgG (Jackson Immunoresearch) were washed twice in PBS buffer (Shanghai culture source, pH 7.2) and then diluted to 200 μg/ml with streaming buffer (4% FBS (Excell Bio)) to make up FC receptor blocking solution. To each of the lysed blood samples, 50. Mu.l of a blocking solution was added, and the mixture was blocked at room temperature for 10 minutes. Subsequently, cells were stained for cell surface markers by adding diluted fluorescent-labeled antibody mixtures (described in table 4) in streaming buffer to identify CD45 + The absolute numbers of cells, T cells, B cells and NK cells (neutrophils, monocytes, erythrocytes and platelets) were all obtained from mouse bloodGauge analyzer). Finally, the samples were washed twice in flow buffer, resuspended in PBS buffer (pH 7.2) and the sample data was obtained on BD celesta flow cytometer. Data analysis was performed using FlowJov10 software. CD45 + Immune cells are defined as singlet, living cells, and in this population T cells are further defined as CD3 + Treg cells are further defined as CD4 + 、CD25 + B cells are further defined as CD3 - 、CD19 + NK cells are further defined as CD3 - 、CD19 - 、NK1.1 + 。
TABLE 4 antibodies used in flow cytometric analysis
| Antibodies to | Fluorescent antibodies | Manufacturer (S) | 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 |
The results are shown in FIG. 9: treatment of background mice with H2D4H4K12 or H5H10H6K4 (8 mg/kg and 16 mg/kg) resulted in a significant reduction in the number of T cells (fig. 9A) and B cells (fig. 9B) and NK cells (fig. 9C) in the blood, whereas neutrophils (fig. 9D), erythrocytes (fig. 9E), monocytes (fig. 9F), platelet counts (fig. 9G) were unaffected, with H2D4H4K12 having no significant effect on Treg cell proportion (fig. 9H). After 3 doses were completed, serum concentrations of H2D4H4K12 and H5H10H6K4 decreased over time. By the end of the study, all of these groups recovered to similar levels as observed prior to treatment. This result shows that both antibodies H2D4H4K12 and H5H10H6K4 have different degrees of inhibition on B cells, T cells and NK cells, but do not affect the numbers of neutrophils, monocytes, erythrocytes and platelets, and that H2D4H4K12 has a weaker inhibition on Treg cells than H5H10H6K4.
Example 6: modeling of a pristand-induced mouse lupus erythematosus model for evaluating anti-human CD132 antibodies H2D4H4K12 and H5H10H6K4 blocking activity.
Experimental procedure: the abdominal cavity of a C57BL/6 background mouse (C57 BL/6 background mouse genetically modified from the baisai's reproductive cluster to replace the endogenous CD132 full-length domain with the corresponding human sequence) was injected with prine (MCE) 0.5mL, and the control group was injected with an equal volume of PBS buffer (pH 7.2). H2D4H4K12 or H5H10H6K4 or anti-KLH isotype control (antibodies above were all manufactured by Kannoa Biotechnology Co., ltd.) were subcutaneously administered at a dose of 20mg/kg 3 days prior to molding, 2 times per week for 6 weeks or no administration (Table 5). Mouse urine protein, urinary creatinine, and plasma anti-dsDNA levels were monitored. And mice were sacrificed at week 12 cervical breaks.
The specific monitoring experiment steps are as follows:
1. mouse urine microalbumin was detected using a mouse urine microalbumin ELISA detection kit (Elabscience): mu.l of urine from each mouse was collected and tested according to the procedure. Briefly, each mouse urine was diluted 1000-fold with standard/sample dilutions, 100 μl per well was added to a 96-well ELISA plate well-wrapped in the kit, incubated for 90 min at 37 ℃, and 100 μl of biotinylated antibody working solution per well after spin-drying was added, and incubated for 1 hr at 37 ℃. Washing for 4 times, adding 100 μl of HRP enzyme conjugate working solution into each well, incubating at 37deg.C for 30 min, washing for 4 times, adding 90 μl of chromogenic solution, standing at 37deg.C for 10 min, adding 50 μl of stop solution, immediately placing in an ELISA reader for reading OD 450 Is a value of (2).
2. ELISA detection of anti-dsDNA levels in mouse plasma: 50-100 mu l of blood of each mouse is collected, centrifuged at 4500rpm, and the supernatant is frozen at-80 ℃ to facilitate subsequent detection. Coating UltraPure on 96 well ELISA plates at 100. Mu.g/ml TM Salmon sperm DNA solution (Invitrogen), incubated overnight at 4 ℃. After 3 washes with PBS buffer (pH 7.2) the next day, blocking with 10% BSA (Bio-industry) at 37℃for 1 hour, washing with PBS buffer (pH 7.2) then 3 times, then adding 50-fold diluted plasma samples, 100. Mu.l each well, incubating at 37℃for 90 minutes, then washing with PBS buffer (pH 7.2) 5 times, adding HRP-labeled Fc-specific anti-room in a ratio of 1:4000mouse IgG1 (abclon). Incubation for 1 hour at room temperature, washing with PBS buffer (pH 7.2) for 7 times, adding 100 μl of chromogenic solution (TMB solution, sigma stock number T2885), standing at 37deg.C for 10 min, adding 50 μl of 2M concentrated sulfuric acid solution to terminate reaction, immediately placing in a microplate reader, and reading OD 450 Is a value of (2).
Table 5 experimental dosing and treatment regimens for groups of mice
| Grouping | Recipient strain | Number of groups | Molding conditions | mAb treatment |
| Group A | CD132 hu/hu | 5 | PBS injection | mAb-free |
| Group B | CD132 hu/hu | 10 | Injecting pristand | anti KL (same type) |
| Group C | CD132 hu/hu | 10 | Injection ofpristane | h2D4H4K12 |
| Group D | CD132 hu/hu | 10 | Injecting pristand | h5H10H6K4 |
The results are shown in FIG. 10: the use of H2D4H4K12 or H5H10H6K4 antibodies reduced the autoantibody anti-dsDNA content in mouse plasma (FIG. 10A) and reduced the mouse urine protein/creatinine ratio (FIG. 10B). This indicates that the two antibodies, H2D4H4K12 and H5H10H6K4, significantly reduced urine protein and plasma anti-dsDNA levels in prine-induced lupus mice, improving survival in mice.
In summary, the above in vitro and in vivo experiments show 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, and further through lupus erythematosus model mice experiments, both antibodies can significantly improve the phenotype of lupus erythematosus model mice. Thus, both antibodies may be of potential value for the treatment of autoimmune diseases where the above cytokine secretion in large amounts is an important pathophysiological change.
The invention provides a humanized anti-human CD132 monoclonal antibody, and a thought and a method for application thereof, and a method and a way for realizing the technical scheme are numerous, the above is only a preferred embodiment of the invention, and it should be pointed out that a plurality of improvements and modifications can be made to those skilled in the art without departing from the principle of the invention, and the improvements and modifications are also considered as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.
Claims (10)
1. A humanized anti-human CD132 monoclonal antibody, characterized in that the monoclonal antibody is a humanized monoclonal antibody,
wherein the monoclonal antibody comprises a light chain complementarity determining region and a heavy chain complementarity determining region, wherein the light chain complementarity determining region comprises LCDR1, LCDR2 and LCDR3, and the heavy chain complementarity determining region comprises HCDR1, HCDR2 and HCDR3;
the light chain complementarity determining region and the heavy chain complementarity determining region select any one of the following groups:
a first group: the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.18, 14 and 15; the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.3, 4 and 5 in sequence; or,
second group: the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.26, 27 and 28; the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.8, 9 and 10 in sequence.
2. The monoclonal antibody of claim 1, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region and the heavy chain variable region selected from any one of the group consisting of:
a first group: the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 22; the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 1;
or,
second group: the amino acid sequence of the light chain variable region is shown as 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 of claim 1, further comprising constant regions, wherein the constant regions are kappa chain constant region Ckappa and human IgG4 constant regions CH1-CH3.
4. A nucleic acid molecule encoding the monoclonal antibody of any one of claims 1-3.
5. An expression vector comprising the nucleic acid molecule of claim 4.
6. A host cell comprising the expression vector of claim 5.
7. The host cell of claim 6, wherein the host cell is a HEK293 cell.
8. A detection reagent or kit comprising the monoclonal antibody of any one of claims 1-3.
9. Use of a monoclonal antibody according to any one of claims 1-3 for the manufacture of a medicament for inhibiting or reducing the activity of human CD 132.
10. Use of a monoclonal antibody according to any one of claims 1-3 in the manufacture of a medicament for the prophylaxis, neutralization or treatment of autoimmune diseases.
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| CN202410081646.0A CN117886940A (en) | 2024-01-19 | 2024-01-19 | Humanized anti-human CD132 monoclonal antibody and application thereof |
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| CN202410081646.0A CN117886940A (en) | 2024-01-19 | 2024-01-19 | Humanized anti-human CD132 monoclonal antibody and application thereof |
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