阅读说明：本技术 抗cd127的抗体、分泌该抗体的细胞株及其制备方法和应用 (anti-CD127 antibody, cell strain secreting antibody, preparation method and application thereof ) 是由 余迪 田文静 张宇 张�浩 于 2019-12-05 设计创作，主要内容包括：本发明提供一种抗CD127的抗体、分泌该抗体的细胞株及其制备方法和应用,属于生物医药和免疫化学技术领域。所述抗体或抗体的抗原结合片段,其特异性结合CD127并且特异性识别CD127的表位序列。本发明制备得到的抗hCD127单克隆抗体及分泌该单克隆抗体的单克隆抗体杂交瘤细胞株CD127-7E7能够特异性识别人CD127,且具有良好的亲和力,其不仅可用于CD127蛋白的定性或定量检测；同时还可对CD127蛋白表位或功能域进行判定,因此具有良好的实际应用之价值。(The invention provides an anti-CD127 antibody, a cell strain secreting the antibody, and a preparation method and application thereof, and belongs to the technical field of biomedicine and immunochemistry. The antibody or antigen-binding fragment of an antibody that specifically binds to CD127 and specifically recognizes an epitope sequence of CD 127. The anti-hCD 127 monoclonal antibody and the monoclonal antibody hybridoma cell strain CD127-7E7 secreting the monoclonal antibody prepared by the invention can specifically recognize human CD127, have good affinity, and can be used for qualitative or quantitative detection of CD127 protein; meanwhile, the epitope or the functional domain of the CD127 protein can be judged, so that the method has good practical application value.)

1. An antibody or antigen-binding fragment of said antibody, characterized in that it specifically binds to CD127 and specifically recognizes the epitope sequence of CD 127;

the epitope sequence of CD127 is selected from: a sequence comprising or consisting of SEQ ID No. 1.

2. The antibody or antigen-binding fragment of said antibody of claim 1, wherein said antibody is a monoclonal antibody against human CD127 or wherein said antibody subtype is IgG2 a.

3. A nucleic acid molecule encoding the antibody or antigen-binding fragment of the antibody of claim 1 or 2.

4. The monoclonal antibody hybridoma cell strain CD127-7E7, which has been preserved in China general microbiological culture Collection center (CGMCC) at 11/19 in 2019, and the biological preservation number is CGMCC No. 18897.

5. The method for producing an antibody according to claim 1 or 2, which comprises immunizing a non-human animal with an antigen comprising an epitope sequence of CD 127.

6. The method of claim 5, comprising:

initial CD4⁺Immunizing a mouse by using the T cells, and then immunizing the mouse by using the synthetic OVA coupled polypeptide as an antigen to obtain immune spleen cells; fusing the spleen cells with myeloma cells to obtain hybridoma cells; culturing the hybridoma cells, collecting cell culture supernatant, and purifying to obtain the hybridoma cells;

preferably, the synthetic OVA-conjugated polypeptide is an OVA carrier protein with CD127 epitope; the epitope sequence of the CD127 antigen is SEQ ID NO. 1;

preferably, the initial CD4⁺The T cell obtaining method comprises the following steps: sterile sorting of CD4 from spleen cells of OT-II mice⁺A T cell;

preferably, the initial CD4⁺The T cell is CD44^lowCD62L⁺CD25^-CD4⁺CD8^-B220^-CD3⁺Initial CD4⁺T cells.

7. The method of claim 6, wherein the method specifically comprises:

initial CD4⁺After a T cell immunizes a mouse, the synthesized OVA coupled polypeptide is mixed with Freund adjuvant to immunize a BALB/c mouse; freund's complete adjuvant for primary immunizationAn agent, Freund's incomplete adjuvant for multiple booster immunizations; finally, performing sprint immunization by using the synthetic OVA coupled polypeptide, fusing immune spleen cells of mice with myeloma cells SP2/0, performing indirect competitive enzyme-linked immune screening and subcloning to obtain a cell strain CD127-7E7, culturing the cell strain CD127-7E7, and collecting and purifying to obtain the recombinant vector;

the monoclonal antibody hybridoma cell strain CD127-7E7 obtained by the culture is preserved in the China general microbiological culture Collection center on 11/19 of 2019, and the biological preservation number is CGMCC No. 18897.

8. Use of the antibody or antigen-binding fragment of an antibody according to claim 1 or 2, the nucleic acid molecule according to claim 3 and/or the cell line CD127-7E7 according to claim 4 in any one or more of the following 1) -2):

1) a pharmaceutical composition for treating immune-related diseases or a pharmaceutical composition for preparing the pharmaceutical composition for treating the immune-related diseases;

2) a detection kit or a preparation detection kit.

9. A pharmaceutical composition for treating an immune-related disorder, comprising the antibody or antigen-binding fragment of an antibody of claim 1 or 2, the nucleic acid molecule of claim 3, and/or a secretion of the cell line CD127-7E7 or the cell line CD127-7E7 of claim 4;

preferably, the pharmaceutical composition further comprises a carrier, an excipient and a diluent.

10. A test kit comprising the antibody or antigen-binding fragment of an antibody according to claim 1 or 2, the nucleic acid molecule according to claim 3 and/or the secretion of the cell line CD127-7E7 or cell line CD127-7E7 according to claim 4;

preferably, the antibody also comprises a label combined with the antibody of claim 1 or 2, wherein the label is any one or more of a fluorescent label, a radioactive label and an enzyme-labeled label;

preferably, the kit has any one or more of the following applications:

a) qualitative or quantitative detection of CD127 protein;

b) and (3) judging the CD127 protein epitope or functional domain.

Technical Field

The invention belongs to the technical field of biomedicine and immunochemistry, and particularly relates to an anti-CD127 antibody, a cell strain secreting the antibody, and a preparation method and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.

CD127 is an interleukin-7 (IL-7) receptor α chain (IL-7R α) that plays a role in different stages of the T lymphocyte life cycle, (1) it plays an important role in the development of T lymphocytes in the thymus, differentiation of thymocytes depends on the expression of IL-7, and lack of expression of CD127 in only two stages throughout the T lymphocyte life cycle, (2) it is an important regulator of peripheral mature T cell homeostasis, under T cell deficiency conditions, CD127 signaling can stimulate T cell proliferation independently of TCR, promoting recovery of T cell numbers, and CD127 can also regulate memory CD8 independently of TCR interaction with its major histocompatibility complex antigens⁺T cell environmental homeostasis; in addition, effector CD4 in vivo⁺Survival and memory of T cells CD4⁺CD127 also plays an important role during T cell transformation. (3) CD127 also plays a very important role in the immune response following viral infection and in the generation and maintenance of virus-specific CD4 and CD8 memory T cell pools. CD127 as CD8⁺Surface marker for T cell differentiation, in virus-clearing specific CD8⁺High expression on the surface of T cells, e.g., CD127 begins at virus-specific CD8 after acute Hepatitis B Virus (HBV) infection is effectively controlled⁺T cell surface expression, and CD127⁺HBV specific effector CD8⁺T cell proliferation is significantly enhanced and secretes numerous antiviral cytokines, most of which are specific CD8 when the virus is cleared⁺The T cell is CD127⁺Subtype, CD127 is therefore the effector CD8 after viral infection⁺T cell memorySex CD8⁺Important markers for T cell transformation. (4) The surface of the regulatory functional Treg cell is low to express CD127, which can be used as a Treg cell surface marker through CD25⁺CD127^loThe Treg cells were purified. CD127, as a receptor for IL-7, plays an important role in T lymphocyte development, maintenance of T cell homeostasis, and differentiation and survival of memory T cells, and is also a useful marker for recognition of memory and effector T cells. Based on the multiple actions of CD127, the development of better anti-CD127 monoclonal antibodies becomes especially valuable in the market and in the application prospect. However, the inventors found that the immunogen of the current anti-CD127 monoclonal antibody is generally recombinant protein or recombinant extracellular protein, so that the epitope or functional domain of the specific site of CD127 is difficult to be determined by the prepared monoclonal antibody.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides an anti-CD127 antibody, a cell strain secreting the antibody, a preparation method and application thereof, the invention prepares a brand-new anti-CD127 monoclonal antibody and a monoclonal antibody hybridoma cell strain CD127-7E7 secreting the monoclonal antibody, the monoclonal antibody can specifically identify human CD127, the monoclonal antibody has stronger affinity when being combined with the human CD127, and meanwhile, the monoclonal antibody is suitable for judging the epitope or functional domain of the specific site of the CD127, so the monoclonal antibody has good practical application value.

In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:

in a first aspect of the invention, there is provided an antibody, or an antigen-binding fragment of said antibody, which specifically binds to CD127 and specifically recognizes the epitope sequence of CD 127;

the epitope sequence of CD127 is selected from: a sequence comprising or consisting of SEQ ID No. 1.

Further, the antibody may be a chimeric antibody or a humanized antibody or a deimmunized antibody.

Further, the antibody is a monoclonal antibody of anti-human CD127(hCD127), and the antibody subtype is IgG2 a.

In a second aspect of the invention, there is provided a nucleic acid molecule encoding the above antibody or antigen-binding fragment of an antibody.

In the third aspect of the invention, a monoclonal antibody hybridoma cell strain CD127-7E7 is provided, which has been preserved in the general microbiological center of China Committee for culture Collection of microorganisms (address: No.3 of Xilu No.1 of Beijing Kogyo, Chaoyang, China) 11.19.2019, and the biological preservation number is CGMCC No. 18897. The cell line can secrete the antibody.

In a fourth aspect of the invention, there is provided an antigen consisting of an epitope sequence of CD127, said antigen comprising or consisting of the sequence of SEQ ID No. 1.

In a fifth aspect of the present invention, there is provided a method for producing the above antibody or an antigen-binding fragment of the antibody, which comprises immunizing a non-human animal against the above antigen. Such non-human animals include, but are not limited to, mice, rats, guinea pigs, rabbits, and monkeys.

In a sixth aspect of the present invention, there is provided a use of the above antibody or an antigen-binding fragment thereof, the above nucleic acid molecule and/or the above cell line 7E7 in any one or more of the following 1) -2):

1) a pharmaceutical composition for treating immune-related diseases or a pharmaceutical composition for preparing the pharmaceutical composition for treating the immune-related diseases;

2) a detection kit or a preparation detection kit.

In a seventh aspect of the present invention, there is provided a pharmaceutical composition comprising the above antibody or an antigen-binding fragment thereof, the above cell line CD127-7E7 and/or a secretion of the above cell line CD127-7E 7; may also contain pharmaceutically acceptable carriers, excipients and diluents.

In an eighth aspect of the present invention, there is provided a detection kit, comprising the antibody or an antigen-binding fragment thereof, the cell line CD127-7E7, and/or the secretion of the cell line CD127-7E7, and a label bound to the monoclonal antibody, wherein the label is one or more of a fluorescent label, a radioactive label, and an enzyme-labeled label. The kit has any one or more of the following applications:

a) for qualitative or quantitative detection of CD127 protein;

b) and (3) judging the CD127 protein epitope or functional domain.

The invention has the beneficial technical effects that:

the invention provides an anti-hCD 127 monoclonal antibody and a hybridoma cell strain CD127-7E7 secreting the monoclonal antibody, which can specifically recognize human CD127 and have good affinity through experimental verification, and can be used for qualitative or quantitative detection of CD127 protein; meanwhile, the epitope or the functional domain of the CD127 protein can be judged, so that the method has good practical application value.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a representation of the initial CD4 of OT-II mice in example 1 of the present invention⁺Constructing a T enhanced immune response model;

FIG. 2 is a graph showing the correlation between the ratio of GC-B cells to B cells in the CD45.2 mouse immune model in example 1 of the present invention;

FIG. 3 is a diagram showing the affinity of mAb 7E7 for the antigen CD127-225-239-OVA in example 1 of the present invention;

FIG. 4 is a drawing showing the amino acid sequence of CD127 protein in 293T, Hela, K562 cells by mAb 7E7 in example 2 of the present invention;

FIG. 5 is the in-situ recognition map of monoclonal antibody 7E7 on over-expressed CD127 protein in 293T cells in example 2 of the present invention.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. It is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.

As mentioned above, the immunogen of the current anti-CD127 monoclonal antibody is generally recombinant protein or recombinant extracellular protein, so that the epitope or functional domain of the specific site of CD127 is difficult to be determined by the prepared monoclonal antibody.

In view of the above, in one embodiment of the present invention, there is provided an antibody or an antigen-binding fragment thereof that specifically binds to CD127 and specifically recognizes the epitope sequence of CD 127;

the epitope sequence of CD127 is selected from: a sequence comprising or consisting of SEQ ID No. 1.

An "antigen-binding fragment" of an antibody of the invention is a portion of an antibody, i.e. a molecule corresponding to a portion of the structure of an antibody of the invention, which exhibits antigen-binding ability to the IL-7 receptor of human IL-7, possibly in its native form; such fragments in particular exhibit the same or substantially the same antigen binding specificity for said antigen as compared to the antigen binding specificity of the corresponding four-chain antibody. Advantageously, the antigen binding fragment has a similar binding affinity as the corresponding four-chain antibody. However, antigen-binding fragments having reduced antigen-binding affinity relative to the corresponding four-chain antibody are also encompassed within the invention. The antigen binding capacity can be determined by measuring the affinity of the antibody and the fragment under consideration. These antigen binding fragments may also be referred to as functional fragments of antibodies.

An antigen-binding fragment of an antibody is a fragment comprising its hypervariable region which contains the antigen recognition site, i.e., the IL-7 receptor CDR (complementarity determining region) of human IL-7, or a portion thereof, thereby defining the antigen recognition specificity.

In one embodiment of the invention, the antibody may be a chimeric antibody or a humanized antibody or a deimmunized antibody.

In one embodiment of the invention, the antibodies are modified, and thus are chimeric antibodies, comprising domains or chains of amino acid residues of different antibodies bound together in a functional antibody, in particular antibodies obtained from different animal species.

In a particular embodiment of the invention, humanization may be performed by surface reconstruction according to known techniques or by CDRR (complementarity determining region) grafting. Resurfacing is achieved, inter alia, by substituting rodent residues for human amino acid residues. Substitutions are made in a manner that preserves the framework structure and CDR presentation of the original antibody, thereby allowing the framework and CDR interactions in the resurfaced antibody to maintain the native conformation of the surface in contact with the antigen such that it retains antigen binding affinity.

In one embodiment of the invention, the murine antibody is humanized to reduce its immunogenicity in humans, and deimmunization is a useful alternative. Deimmunization can reduce the immunogenicity of antibodies in humans by removing epitopes from the variable regions of rodent antibodies that may be immunogenic in humans. Such as removal of potentially immunogenic B and T cell epitopes or by chemical replacement of the constant region of murine antibodies with human antibody constant regions.

In one embodiment of the invention, the antibody is a monoclonal antibody against human CD127(hCD127), and thus the composition of these antibodies is homogeneous or identical with respect to antigen binding specificity and with respect to variable region composition. Thus, even if the antibody is obtained by an alternative technique to the hybridoma technique, the antibody may be referred to as a monoclonal antibody. The antibody subtype is IgG2 a.

In yet another embodiment of the present invention, there is provided a nucleic acid molecule encoding the above antibody or antigen-binding fragment of an antibody.

In another embodiment of the present invention, a monoclonal antibody hybridoma cell strain CD127-7E7 is provided, which has been deposited in the general microbiological culture Collection center of China Committee for culture Collection of microorganisms at 11/19/2019 (address: No.3, Xilu 1. Beichen, the sunward, Beijing, China), and has a biological preservation number of CGMCC No. 18897. The cell lines can be used to secrete the anti-hCD 127 monoclonal antibodies described above.

In yet another embodiment of the present invention, there is provided an antigen consisting of the epitope sequence of CD127, said antigen comprising or consisting of the sequence of SEQ ID No. 1.

In another embodiment of the present invention, there is provided a method for producing the above antibody, which comprises immunizing a non-human animal against the above antigen. Such non-human animals include, but are not limited to, mice, rats, guinea pigs, rabbits, and monkeys.

In another embodiment of the present invention, the preparation method comprises:

initial CD4⁺T(naive CD4⁺T) immunizing a mouse by using the cells, and then immunizing the mouse by using the synthetic OVA coupled polypeptide as an antigen to obtain immune spleen cells; fusing the spleen cells with myeloma cells to obtain hybridoma cells; culturing the hybridoma cells (in vitro culture or in vivo culture), and collecting and purifying to obtain the hybridoma.

In yet another embodiment of the present invention, the initial CD4⁺The T cell obtaining method comprises the following steps: sterile sorting of primary CD4 from spleen cells of OT-II mice⁺T cells. Further, the initial CD4⁺The T cell is CD44^lowCD62L⁺CD25^-CD4⁺CD8^-B220^-CD3⁺Initial CD4⁺T cells.

In the present invention, the original CD4⁺T cells can specifically recognize the polypeptide of OVA protein 323-339, so that more polypeptide-OVA antigen is presented. Thereby more can be obtainedThe GC B cell of (1). And further differentiated into effector B cells capable of producing antibodies, thereby producing a large amount of antibodies.

In yet another embodiment of the invention, the mouse is a female C57BL/B6 mouse.

The synthetic OVA coupling polypeptide is an OVA carrier protein with CD127 epitope; the epitope sequence of the CD127 antigen is SEQ ID NO. 1;

in another embodiment of the present invention, the preparation method specifically comprises:

initial CD4⁺After a T cell immunizes a mouse, the synthesized OVA coupled polypeptide is mixed with Freund adjuvant to immunize a BALB/c mouse; freund's complete adjuvant (CFA) for the first immunization, Freund's incomplete adjuvant (IFA) for the multiple booster immunizations; finally, the immune of the mouse immune spleen cell and myeloma cell SP2/0 are fused by using the synthetic OVA coupled polypeptide, and the cell strain CD127-7E7 is obtained by indirect competitive enzyme-linked immune screening and subcloning, and the antibody secreted by the cell strain 7E7 is obtained by culturing the cell strain (in vitro culture or in vivo culture) and purifying.

In the invention, the CD127 monoclonal antibody cell strain CD127-7E7 obtained by the culture is preserved in the common microorganism center of China Committee for culture Collection of microorganisms (address: No.3 of the Xilu No.1 of the North West Chen of the Chaoyang district, Beijing, China) in 11 months and 19 days in 2019, and the biological preservation number is CGMCC No. 18897.

In still another embodiment of the present invention, there is provided a use of the above antibody or an antigen-binding fragment thereof, the above nucleic acid molecule and/or the above cell line CD127-7E7 in any one or more of the following 1) -2):

1) a pharmaceutical composition for treating immune-related diseases or a pharmaceutical composition for preparing the pharmaceutical composition for treating the immune-related diseases;

2) a detection kit or a preparation detection kit.

The immune-related diseases include, but are not limited to, rheumatoid arthritis, multiple sclerosis, type I diabetes, autoimmune thyroiditis or lupus, or inflammatory diseases such as Inflammatory Bowel Disease (IBD) and encephalomyelitis, or allergic diseases, or cancer, or respiratory diseases, or diseases associated with transplantation.

In yet another embodiment of the present invention, there is provided a pharmaceutical composition for treating an immune-related disease, comprising the antibody or antigen-binding fragment of the antibody or secretion of cell line 7E7 or cell line 7E7 as described above.

The pharmaceutical composition may further comprise a suitable amount of carriers, excipients and diluents commonly used. Further, the composition can be prepared into oral preparations such as powder, granule, tablet, capsule, suspension, emulsion, syrup, and spray, external preparations, suppositories, and sterile injectable solutions according to a conventional method.

In yet another embodiment of the present invention, the non-pharmaceutically active ingredients such as carriers, excipients and diluents which may be included are well known in the art and can be determined by one of ordinary skill in the art to meet clinical criteria.

In still another embodiment of the present invention, the carrier, excipient and diluent include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

In yet another embodiment of the present invention, the medicament of the present invention may be administered into the body by known means. For example, by intravenous systemic delivery or local injection into the tissue of interest. Optionally via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration may be via a single dose or multiple doses. It will be understood by those skilled in the art that the actual dosage to be administered in the present invention may vary greatly depending on a variety of factors, such as the target cell, the type of organism or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.

In another embodiment of the present invention, the present invention provides a detection kit, which comprises the above antibody or antigen binding fragment of the antibody or secretion of cell strain CD127-7E7 or cell strain CD127-7E7, and a label bound to the above antibody, wherein the label is any one or more of a fluorescent label, a radioactive label and an enzyme-labeled label. The reagent kit has any one or more of the following applications:

a) for qualitative or quantitative detection of CD127 protein;

b) and (3) judging the CD127 protein epitope or functional domain.

In still another embodiment of the present invention, there is provided the antibody or the antigen-binding fragment of the antibody, the nucleic acid molecule, the cell line CD127-7E7, and the use of the test kit in serology, pathological diagnosis, tomography, or positron emission tomography-tomography of immune-related diseases.

The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.