CN115197321B - Antibodies targeting CD25 and uses thereof - Google Patents

Abstract

The invention relates to the technical field of antibodies, in particular to preparation of an anti-CD 25 antibody or a fragment thereof and application thereof. The invention aims to provide an IL-2 non-blocking type anti-CD 25 antibody or a fragment thereof. The technical scheme of the invention provides a novel anti-CD 25 antibody. The anti-CD 25 antibody has good affinity, specificity and thermal stability, does not block an IL-2 downstream signal channel, can specifically inhibit regulatory T cells (Tregs) without affecting T effector cells (Teffs), thereby achieving the anti-tumor effect by enhancing tumor immunity and showing good application potential in the aspect of cancer treatment.

Description

Antibodies targeting CD25 and uses thereof

Technical Field

The invention relates to the technical field of antibodies, in particular to a CD25 targeting antibody and application thereof.

Background

Antibodies are biological macromolecules composed of heavy chains and light chains, are secreted by B lymphocytes, and play an important role in humoral immunity of organisms. The heavy chain or the light chain of the antibody molecule is respectively composed of a variable region and a constant region, wherein the variable region mainly plays a role in binding a target antigen, and the constant region mainly plays an immune regulation effect. Antibodies can be classified into IgG, igM, igE, igA, igD and the like according to spatial structure and amino acid sequence characteristics, and heavy and light chains can be further subdivided into a plurality of subtypes. For example, human IgG heavy chains can be classified as IgG1, igG2, igG3, and IgG4, and light chains can be classified as kappa and lambda. Since antibodies can specifically and efficiently bind to target molecules or target cells, regulate signaling pathways downstream of target molecules, or kill target cells by immune effects, antibodies can be developed as drugs for disease treatment. Antibodies have been developed currently as an important biotechnological drug, where monoclonal antibodies take up a substantial part, which in turn are predominantly of the IgG type, so that so-called monoclonal antibodies are often referred to as IgG type antibodies.

An IgG type antibody molecule is a tetramer consisting of 2 heavy chains and 2 light chains with a molecular weight of about 150kD via interchain disulfide bonds. Antibody molecules can be divided into variable and constant regions according to structural and functional characteristics, with the variable region acting primarily for antigen binding and the constant region acting primarily for immunological effects and transport. The variable region of an antibody can be further divided into complementarity determining region CDRs and framework regions FRs, wherein each of the heavy or light chain contains 3 CDR regions (heavy chain VH-CDR1, VH-CDR2, VH-CDR3, light chain VL-CDR1, VL-CDR2, VL-CDR 3.) and 4 FR regions flanking the CDR regions (FR 1, FR2, FR3, FR 4). The loop formed by the CDR region is the main part of the binding of the antibody molecule to the antigen, and the FR region forms the supporting structure of the CDR region by spatial folding. The specific recognition of antibodies against different antigen molecules is mainly achieved by amino acid polymorphisms of the 6 CDR regions (VH-CDR 1, 2, 3 and VL-CDR1, 2, 3) together with conformational polymorphisms of the loop. Because of the high structural similarity of the FR regions of different antibodies, when the CDR regions of one antibody are substituted for the CDR regions of other antibody molecules, if the FR regions of different antibody molecules are appropriately matched, the conformational change of the CDR regions before and after substitution is small, so that the novel variable region formed after substitution can still retain the antigen binding ability, which is the basis of CDR grafting (CDR grafting) technology. The CDR regions of the murine antibody may be recombined with the human FR regions into a humanized antibody by CDR grafting techniques to replace the CDRs of the human antibody (humanized antibody), and if the FR regions between the human-murine antibody are properly matched, the antigen binding capacity may still be maintained.

Hybridoma technology is an important technology in the current antibody discovery process, and antibody molecules generated by the technology complete the affinity maturation process in mice, so that the antibody molecules can effectively reduce nonspecific binding with self or similar proteins. Because of the differences in amino acid sequences encoded by antibody genes between different species, human anti-mouse antibodies, i.e., HAMA (human anti-mouse antibody) reactions, are produced when murine antibodies are used in human therapy, resulting in the occurrence of ADA (anti-drug antibody). The neutralizing anti-drug antibody not only can influence the accessibility of the target point of the antibody drug and reduce the treatment effect of the antibody drug, but also can increase the side effect risk due to immune complexes formed by the anti-drug antibody. The modification of murine antibodies to humanized antibodies using CDR-grafting techniques can reduce the risk of HAMA reactions and reduce ADA incidence, which has been demonstrated by clinical studies. Currently, there are tens of humanized antibodies on the market, including trastuzumab, pembrolizumab, bevacizumab and other heavy weight drugs, indicating that the humanized technology is a reliable technology for developing therapeutic antibodies.

Monoclonal antibodies can exert pharmacological effects through a variety of mechanisms. The antibody variable region can bind to extracellular soluble ligand, block the binding of ligand and receptor and cut off downstream signal transmission induced by ligand, so that the antibody medicine developed by taking immune cell factor as target can improve inflammatory diseases, for example, antibodies such as adalimumab, belimumab, siltuximab are already marketed in batches. The antibody constant region can exert immune regulation effects, including Antibody Dependent Cellular Cytotoxicity (ADCC) and Complement Dependent Cytotoxicity (CDC) and the like, so that antibody drugs developed by taking tumor cell surface molecules as targets can kill tumor cells, such as antibodies on the market such as rituximab, trastuzumab, cetuximab and the like, have been greatly successful. In addition, novel antibody technologies such as bispecific antibodies and Antibody Drug Conjugates (ADC) derived based on monoclonal antibodies have been developed rapidly in recent years, and have all achieved breakthrough of different degrees. Cancer and inflammatory diseases are currently the most used areas of disease for antibody drugs.

In recent years, cancer immunotherapy has gained widespread acceptance in the medical community as a successful cancer treatment modality. With the gradual expansion of clinical indications for cancer immunotherapy, it has been found that although cancer immunotherapy has a high remission rate for few cancer patients such as malignant melanoma, remission rate for most cancers is not very ideal, one of the important reasons being that the effect of tumor immunity is impaired due to immunosuppressive factors present in the microenvironment in which tumor cells are located. Tumor microenvironment (tumor microenvironment, TME) refers to the surrounding tissue environment on which tumor cells are dependent for survival, wherein various types of immune cells are an important component of the tumor microenvironment. Immune cells in tumor microenvironments are mainly divided into two main categories according to function: one is immune effector cells, mainly including effector T cells (Teffs) that kill tumor cells, NK cells, and Dendritic Cells (DCs) that present antigens, etc.; the other class is immunosuppressive cells, mainly including regulatory T cells (Tregs), tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and the like. The immunosuppressive cells can inhibit the functions of immune effector cells, so that tumors can escape immunologically and the occurrence and development of tumors can be promoted. Tregs are of interest as an important immunosuppressive cell because of their powerful immunosuppressive effect and infiltration into a broader range of tumor tissues.

Tregs are a subset of T cells with immunosuppressive function, and the detection phenotype of Tregs is commonly considered to be CD4+CD25+FoxP3+, and plays an important role in maintaining the immune homeostasis of an organism and preventing autoimmune diseases. With the development of tumor immunology, the mechanism of action of Tregs in tumor immune escape and immunotherapy resistance is gradually revealed. Tregs can secrete cytokines such as TGF-beta, IL-10, IL-35 and the like to inhibit activation of Teffs and NK cells. Tregs highly express CTLA-4, can trans-endocytose CD80 and CD86 molecules of DC cells, and reduce antigen presenting capacity of the DC cells and activation capacity of the DC cells to Teffs. Tregs highly express metabolic enzymes such as CD39 and CD73, and suppress immune functions of immune cells such as Teffs, NK cells and macrophages by generating Adenosine (ADO). In addition, tregs also express CD25 in high levels, competing for IL-2, which deprives Teffs, and attenuating proliferation and activation of Teffs. The infiltration of Tregs in various tumor tissues such as lung cancer, liver cancer, ovarian cancer, breast cancer, gastrointestinal tumor and the like results in low ratio of Teffs to Tregs, which is also an important reason for low response and drug resistance of cancer immunotherapy, so that the removal of Tregs or the reversion of the immunosuppressive function of Tregs becomes a strategy of cancer immunotherapy.

Constitutive high expression of CD25 is an important feature of Tregs. CD25, the IL-2 receptor alpha chain (IL-2Rα), canTogether with the IL-2 receptor beta chain (CD 122) and the IL-2 receptor gamma chain (CD 132) form the complete IL-2 receptor complex (IL-2Rαβγ). IL-2R is largely classified into 3 classes: IL-2rαβγ heterotrimers constitute IL-2 high affinity receptors (kd=10) ^-11 M), IL-2rβγ constitutes a medium affinity receptor (kd=10) ^-9 M), IL-2 ra alone constitutes a low affinity receptor (kd=10) ^-8 M). Although the IL-2Rα chain is not responsible for signal transduction, the IL-2Rαβγ composed of the IL-2Rα chain forms a complex with IL-2 more stably, the activation of intracellular Jak3 signals is stronger, and proliferation and activation of T cells can be more effectively started. CD25 is constitutively high in Tregs, but is lower in Teffs, NK cells, so IL-2 high affinity receptor IL-2Rαβγ is mainly expressed in Tregs. Unlike Tregs, teffs expresses predominantly the affinity receptor IL-2rβγ in IL-2, while expressing less of the high affinity receptor IL-2rαβγ. Since the high affinity receptor IL-2Rαβγ mediates IL-2 signaling with far higher efficiency than the medium affinity receptor IL-2Rβγ, CD25 is less expressed in Teffs, but is important for proliferation and activation of Teffs. The FDA approved the market for CD25 antibodies (e.g., basiliximab) in 1998, blocking the binding of IL-2 to CD25 on T lymphocytes, thereby inhibiting T cells, and was used primarily for the prevention of organ transplant rejection.

Since CD25 is constitutively highly expressed in Tregs and is also considered as an important marker of Tregs, CD25 as an antibody target for inhibiting Tregs is also a reasonable choice. CD25 is expressed less in Teffs and anti-CD 25 antibodies bind more to Tregs, preferentially killing them by ADCC, CDC etc. In contrast, since Teffs bind less anti-CD 25 antibodies, anti-CD 25 antibodies have difficulty in exerting an effective killing effect, and have limited inhibitory effect on Teffs. anti-CD 25 antibodies can be classified into IL-2 competitive and non-competitive antibodies, depending on whether the antibody binding epitope can compete for IL-2 binding to CD 25. When anti-CD 25 antibodies block IL-2/IL-2R binding and its downstream signaling pathways, it also blocks the acceptance of IL-2 signaling by Teffs, and thus blocking anti-CD 25 antibodies are detrimental to the anti-tumor function of Teffs. In contrast, when anti-CD 25 antibodies do not block IL-2/IL-2R binding, the IL-2R of Teffs can still bind IL-2 and exert normal anti-tumor effects, so that non-blocking antibodies can inhibit Tregs and do not affect the anti-tumor functions of Teffs. The FDA in the United states has approved that both the anti-CD 25 antibodies, basiliximab and daclizumab, are IL-2 blocking antibodies, useful for the prevention of immune disorders such as organ transplant rejection, multiple sclerosis, and the like.

Although CD25 is a good target for modulating Treg, IL-2 blocking antibodies and non-blocking antibodies differ in modulating T cell immunity and treating disease. IL-2 blocking antibodies are beneficial for inhibiting T cell immunity, whereas IL-2 non-blocking antibodies are beneficial for activating T cell immunity, thus screening strategies for antibodies are needed to be determined according to the disease to be treated. For cancer immunotherapy, it is necessary to inhibit Tregs without affecting the function of Teffs, so that IL-2 non-blocking antibodies may be a reasonable choice, which is more beneficial to achieve anti-tumor effects by enhancing tumor immunity.

Disclosure of Invention

The invention aims to solve the technical problem of providing an IL-2 signal non-blocking type anti-CD 25 antibody or antibody fragment, which has good affinity, specificity and functionality and can achieve the anti-tumor effect by enhancing tumor immunity.

The technical scheme for solving the technical problems is to provide an anti-CD 25 antibody or a fragment thereof. The amino acid sequences of the heavy chain complementarity determining regions VH-CDR1, VH-CDR2 and VH-CDR3 of the anti-CD 25 antibody or the fragment thereof are shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively, and the amino acid sequences of the light chain complementarity determining regions VL-CDR1, VL-CDR2 and VL-CDR3 of the antibody or the fragment thereof are shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively.

The anti-CD 25 antibody may be a humanized antibody, wherein the variable region VH of the heavy chain is formed by splicing the VH-CDR1, VH-CDR2 and VH-CDR3 shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 with the framework region FR of the humanized antibody, and the variable region VL of the light chain is formed by splicing the VL-CDR1, VL-CDR2 and VL-CDR3 shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 with the framework region FR of the humanized antibody. Preferably, the amino acid sequences of the heavy chain variable region VH and the light chain variable region VL of the anti-CD 25 humanized antibody are shown as SEQ ID NO.13 and SEQ ID NO.14 respectively.

The anti-CD 25 antibody or fragment thereof provided by the invention can also have the following structural units: the amino acid sequences of the heavy chain complementarity determining regions VH-CDR1, VH-CDR2 and VH-CDR3 are shown as SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9 respectively, and the amino acid sequences of the light chain complementarity determining regions VL-CDR1, VL-CDR2 and VL-CDR3 are shown as SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12 respectively.

Similarly, the anti-CD 25 antibody may be a humanized antibody, wherein the heavy chain variable region VH is formed by splicing the VH-CDR1, VH-CDR2 and VH-CDR3 shown in SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9 with the framework region FR of the humanized antibody, and the light chain variable region VL is formed by splicing the VL-CDR1, VL-CDR2 and VL-CDR3 shown in SEQ ID No.10, SEQ ID No.11 and SEQ ID No.12 with the framework region FR of the humanized antibody. Preferably, the amino acid sequences of the heavy chain variable region VH and the light chain variable region VL of the anti-CD 25 humanized antibody are shown as SEQ ID NO.15 and SEQ ID NO.16 respectively. Further, the N in glycosylation motif NYS in the anti-CD 25 humanized antibody VH-CDR2 can be mutated to Q, A or S, where S is the preferred mutation. Correspondingly, the amino acid sequence of the heavy chain variable region VH is shown as SEQ ID NO. 17.

Further, the above antibody fragment may be Fab (antigen binding fragment) or scFv (single-chain fragment variable).

The heavy chain constant region of the anti-CD 25 antibody may be derived from the constant region of human immunoglobulin IgG1, igG2, igG3, igG4, or IgM, igE, igA, igD heavy chain, and the light chain constant region may be derived from the constant region of human immunoglobulin kappa or lambda light chain.

The invention also provides nucleic acid molecules encoding the above anti-CD 25 antibodies or fragments thereof.

The invention also provides recombinant vectors comprising nucleic acid molecules encoding the above anti-CD 25 antibodies or fragments thereof.

Further, the recombinant vector may be a plasmid or a viral vector. The virus vector can be at least one of adenovirus vector (adenoviral vectors), adeno-associated virus vector (adeno-associated viral vectors), retrovirus vector (retroviral vectors), herpes simplex virus vector (herpes simplex virus-based vectors) or lentivirus vector (lentiviral vectors).

The invention also provides a cell containing the recombinant vector. Further, the cells may be eukaryotic cells or prokaryotic cells. Wherein the cells can be selected from mammalian cells, insect cells, yeast or bacteria, etc.

The invention also provides application of the anti-CD 25 antibody or the fragment thereof in inhibiting Tregs cells and achieving an anti-tumor effect by enhancing tumor immunity.

The antibody of the present invention can be prepared into various forms of pharmaceutical preparations according to conventional techniques of pharmacy, and liquid injections and freeze-dried injections are more preferable.

The antibodies of the invention may be formed into pharmaceutical compositions with other drugs that may be used in the treatment of diseases along with other therapeutic methods including chemotherapy, radiation therapy, biological therapy, and the like.

The invention has the beneficial effects that:

the invention provides an anti-CD 25 antibody, wherein the preferred anti-CD 25 antibody is a humanized antibody formed by splicing a CDR region of a murine monoclonal antibody and an FR region of a humanized antibody by adopting a CDR grafting technology. The anti-CD 25 antibody disclosed by the invention has good affinity, specificity and functionality, does not block an IL-2 downstream signal channel, is an IL-2 non-blocking anti-CD 25 antibody, can inhibit Tregs with high expression of CD25, does not influence Teffs to accept IL-2 signals, achieves an anti-tumor effect by enhancing tumor immunity, and has good application potential in the aspect of cancer treatment.

Drawings

FIG. 1 Western blot detection of the effect of anti-CD 25 hybridoma antibodies on IL-2 induced STAT5 phosphorylation. (a) NK-92 cells; (B) human PBMC.

FIG. 2 effects of anti-CD 25 hybridoma antibodies on proliferation of PBMC.

FIG. 3 ELISA detection of binding activity of anti-CD 25 hybridoma antibodies to human, monkey, murine CD 25.

FIG. 4 competitive binding of anti-CD 25 hybridoma antibodies to IL-2/CD 25.

FIG. 5 binding kinetics analysis of anti-CD 25 hybridoma antibodies.

FIG. 6 Biacore binding kinetics analysis of antibodies F10-2 and 7B7. (a) antibody F10-2; (B) antibody 7B7.

FIG. 7 competing binding of antibodies F10-2 and 7B7 to IL-2/CD 25.

FIG. 8 flow cytometry detects the cell binding activity of antibodies F10-2 and 7B7. (a) SU-DHL-1 cells; (B) U937 cells.

FIG. 9 Western blot detection of the effect of antibodies F10-2 and 7B7 on IL-2 induced phosphorylation of STAT 5. (a) NK-92 cells; (B) human PBMC.

FIG. 10 species cross-reactivity of antibodies F10-2 and 7B7. (A) ELISA; (B) Biacore binding kinetics analysis of antibody 7B7 to monkey CD 25.

FIG. 11 antibodies F10-2 and 7B7 specifically reduce Tregs in PBMC.

FIG. 12 anti-tumor effects of antibodies F10-2 and basiliximab in mice.

FIG. 13 anti-tumor effects in mice of antibodies F10-2 and 7B7.

FIG. 14 antibodies F10-2 and 7B7 reduce Tregs infiltration in tumor tissue.

Figure 15 change in body weight after mice were dosed.

FIG. 16H & E staining of vital organ tissues after mouse dosing.

Figure 17 binding kinetics analysis of h7b7 humanized antibodies. (A) h7B7-6; (B) h7B7-7; (C) h7B7-14; (D) h7B7-15; (E) h7B7-19.

FIG. 18 Western blot detection of the effect of h7B7 humanized antibodies on IL-2 induced PBMC STAT5 phosphorylation.

Figure 19 h7b7 humanized antibody specifically reduced Tregs in PBMCs.

FIG. 20 cell binding Activity of antibodies h7B7-15S with h7B 7-15.

FIG. 21 Tregs inhibition function of antibodies h7B7-15S and h7B 7-15.

Detailed Description

The invention provides an anti-CD 25 antibody, wherein the preferred anti-CD 25 antibody is a humanized antibody formed by splicing a CDR region of a murine monoclonal antibody and an FR region of a humanized antibody by adopting a CDR grafting technology, has good affinity, specificity and functionality, does not block IL-2/IL-2R binding, is an IL-2 non-blocking anti-CD 25 antibody, can inhibit Tregs with high expression of CD25, and does not influence Teffs to accept IL-2 signals.

The original murine antibody of the anti-CD 25 antibody is obtained by screening by adopting a hybridoma technology. The human CD25 protein is mixed with an adjuvant and immunized into a mouse, after the serum titer is qualified, spleen cells of the mouse are separated, and after the spleen cells are fused with myeloma cells of the mouse in vitro, the spleen cells are cultured, and cell culture supernatant containing antibodies is obtained. First, hybridoma antibodies having CD25 binding activity were screened by ELISA, antibodies having good binding kinetics were further screened by SPR (Biacore), and then IL-2/CD25 non-competitive antibodies were screened by competitive ELISA. Cell binding activity of the hybridoma antibodies was detected by flow cytometry, and further screening for functional antibodies that are non-blocking in IL-2 signaling by STAT5 phosphorylation and PBMC proliferation. To investigate the specificity of the antibodies, no binding signal was seen by ELISA and flow cytometry to ensure that the antibodies did not bind to CD25 negative cellular components. The invention also detects the cross reactivity of the antibodies to human, monkey and mouse CD25 proteins, and discovers that antibodies B7 and H6 can better recognize monkey CD25, and antibodies F10 and E5 have weaker binding effect on monkey CD25. In addition, none of antibodies B7, H6, F10, E5 recognized murine CD25.

In order to obtain the amino acid sequence of the hybridoma antibody, the invention sequences the antibody mRNA of the hybridoma to obtain the variable region sequence of the hybridoma antibody, prepares a recombinant monoclonal antibody and performs functional activity verification. The antibody variable region mRNA gene is amplified by PCR through an upstream signal peptide primer and a downstream constant region primer, and further sequenced to obtain the antibody variable region gene. The expression vector is constructed by splicing and fusing the murine antibody variable region and the antibody constant region. The HEK293 cells are transfected by expression plasmids containing antibody genes for transient expression, and the purity and the content of the antibodies are confirmed by protein G affinity purification and SDS-PAGE and spectrophotometry for further verification.

The obtained recombinant monoclonal antibody respectively performs verification on binding kinetics, competitiveness, cell binding activity, STAT5 phosphorylation, specificity, species cross reactivity and the like according to a detection method of hybridoma screening so as to ensure that the activity of each aspect of the recombinant monoclonal antibody accords with the corresponding hybridoma antibody. To examine whether the antibodies inhibit Tregs, human peripheral blood PBMCs were isolated, incubated with anti-CD 25 recombinant monoclonal antibodies, and flow cytometry was used to confirm that anti-CD 25 antibodies F10-2 (from F10) and 7B7 (from B7) could specifically reduce Tregs in PBMCs with little impact on Teffs. In order to examine the in vivo anti-tumor effect of the antibody, a CD25 humanized mouse is adopted to establish a mouse transplantation tumor model, IL-2 blocking type anti-CD 25 antibodies and non-blocking type anti-CD 25 antibodies are given to the mouse, and it is confirmed that the non-blocking type antibodies F10-2 and 7B7 can effectively inhibit tumor growth and reduce Tregs infiltration in tumor tissues; the IL-2 blocking antibody basiliximab cannot inhibit tumor growth and also cannot reduce Tregs infiltration in tumor tissues, so that the rationality of the antibody screening thought and treatment concept of the invention is verified.

To achieve humanization of antibody 7B7, antibody 7B7 variable regions were homologously modeled according to antibody structure in PDB database, CDR regions were determined based on amino acid primary sequence characteristics and variable region spatial conformation. The variable region of the antibody 7B7 is compared with the amino acid sequences encoded by human antibody germ line genes V and J, and 5V genes and 1J gene in an IGHV library are preferably transplanted and spliced with the heavy chain CDR of the antibody 7B7 according to the factors such as the consistency, the similarity and the conservation of the frame region FR to form 5 different humanized heavy chain variable regions. Similarly, it is preferred that 4V genes and 1J gene in IGKV library are grafted with antibody 7B7 light chain CDRs to form 4 different humanized light chain variable regions. Humanized heavy and light chain variable regions were fused to human IgG1 kappa heavy and light chain constant regions, respectively, to form complete heavy and light chains, and 5 heavy chains were combined with 4 light chains to construct 20 different humanized antibodies.

Constructing recombinant plasmid containing the 20 humanized antibody genes, and transfecting HEK293 cells for transient expression. Cell culture supernatants were collected for SPR binding kinetics screening. Wherein 5 h7B7 humanized antibodies, including h7B7-6, h7B7-7, h7B7-14, h7B7-15, h7B7-19, exhibit good affinity, indicating that the CDR regions of antibody 7B7 have good portability.

In the humanization of antibody F10-2, no binding activity was detected with the 20 humanized antibodies after CDR-grafting, and therefore the present invention continued to be back mutated. By analyzing the structural characteristics of the antibody variable region, the R79 in the FR3 of the light chain variable region of the antibody is found to be critical for maintaining the affinity of the antibody through repeated back mutation screening. The humanized antibody may exhibit good affinity by retaining the murine residue R79 in VL-FR 3.

To confirm the cellular functional activity of the humanized antibodies, PBMC of human peripheral blood were incubated with the humanized antibodies, followed by IL-2 stimulation, confirming that humanized antibodies h7B7-6, h7B7-7, h7B7-14, h7B7-15, h7B7-19 did not block IL-2 induced PBMC STAT5 phosphorylation, with the blocking effect of antibody h7B7-15 being minimal.

The invention further confirms the specific inhibition effect of the h7B7 humanized antibody on Treg. Human peripheral blood PBMC were isolated, incubated with anti-CD 25 antibody, and flow cytometry was used to confirm that antibodies h7B7-6, h7B7-7, h7B7-14, h7B7-15, h7B7-19 could specifically reduce Tregs in PBMC with little impact on Teffs.

The invention further adopts a unclle multiparameter high-throughput protein stability analysis system to detect the melting temperature (Tm) and the aggregation initiation temperature (Tagg) of the antibody, and analyzes the thermal stability of the humanized antibody. The anti-CD 25 humanized antibodies h7B7-6, h7B7-7, h7B7-14, h7B7-15 and h7B7-19 all show good thermal stability, wherein the thermal stability of the antibodies h7B7-7, h7B7-15 and h7B7-19 is slightly better.

To reduce the risk of heterogeneity of the antibodies, the invention further mutates the NYS glycosylation motif in CDR2 of the heavy chain variable region of the humanized antibody h7B 7. SPR binding kinetics analysis shows that the antibody (named as h7B 7-15S) obtained after N mutation is S can still keep better binding kinetics characteristics, cell binding activity, tregs inhibition function and thermal stability, and is basically consistent with h7B7-15 before glycosylation mutation.

The anti-CD 25 antibody of the present invention can be modified into antibody fragments such as Fab (antigen binding fragment, antigen-binding fragment), scFv (single-chain fragment variable, single-chain antibody) and the like by conventional genetic recombination techniques. Antibody fragments such as Fab, scFv and the like have smaller volume and strong tissue permeability, and have unique advantages in some application fields. Fab is a heterodimer consisting of a heavy chain variable region-constant region 1 (VH-CH 1) and a light chain variable region-constant region (VL-CL) with a molecular size of 1/3 of an IgG molecule. Because of the absence of the Fc segment, the Fab-induced immune effect is significantly reduced compared to IgG and cytokine release is weaker. Antibody drugs such as abciximab, ranibizumab, etc. having Fab as structures are currently approved for the market. The scFv is formed by fusing VH and VL and a connecting peptide linker between the VH and the VL, the molecular size is only 1/6 of that of the IgG, and the scFv has the characteristics of strong tissue permeability, short half-life and the like, has unique advantages in the fields of imaging diagnosis and some treatments, and a bispecific antibody blinatumomab based on the scFv is also approved to be marketed. The antibody fragments may further be fused to other proteins, or conjugated to other small molecules, for diagnosis and treatment of diseases by targeted delivery.

The anti-CD 25 antibodies of the invention may be further improved in affinity by engineering techniques to mutate amino acids in the CDR regions. The antibody CDR regions play a critical role in the binding of antibodies to antigens, wherein the amino acids may interact with the amino acids of the antigen via hydrogen bonding, ionic bonding, van der waals forces, and the like. By mutating amino acids in the CDR regions of an antibody, the interaction of the CDRs with the antigen can be further enhanced, thereby increasing the affinity of the antibody. The application of the antibody library technology in the aspect of antibody affinity evolution is mature, an antibody mutation library can be established through strategies such as alanine hot spot mutation, error-prone PCR and the like, high-throughput screening of mutant antibodies is carried out, and the affinity evolution of the antibodies is realized in vitro.

The antibodies of the invention can be expressed using stable cell lines for large-scale production of large amounts of proteins. The gene encoding the antibody amino acid can be obtained by conventional gene recombination technology, and can be inserted into an expression vector after DNA sequence optimization, synthesis and PCR amplification. The vectors used may be plasmids, viruses or gene fragments which are customary for molecular biology. A protein secretion signal peptide gene is added at the front end of a DNA sequence for encoding the antibody so as to ensure that the antibody can be secreted outside cells. The vector sequence contains a promoter for gene expression, a protein translation initiation and termination signal, polyadenylation (polyA) and other elements. The vector contains antibiotic resistance genes and replication elements to facilitate replication of the vector in a host cell, such as a bacterium, for vector preparation. In addition, a selectable gene may be included in the vector to facilitate selection of stably transfected host cells for construction of stably expressed cell lines.

After construction of the vector containing the DNA sequence encoding the antibody, the vector may be used to transfect or transform a host cell for expression of the corresponding protein. There are various expression systems that can be used to express antibodies, which can be eukaryotic cells, or prokaryotic cells, including mammalian cells, insect cells, yeast, bacteria, and the like. Mammalian cells are the preferred system for expressing the protein because of the ease of inclusion bodies when prokaryotic cells express intact antibodies. There are various mammalian cells that can be used for large-scale expression of antibodies, such as CHO cells, HEK293 cells, NS0 cells, COS cells, etc., all of which are included among the cells that can be used in the present invention. Recombinant vectors containing genes encoding antibodies can be transfected into host cells by a variety of methods including electroporation, lipofection, and calcium phosphate transfection.

A preferred method of protein expression is by stably transfected host cell expression comprising a selectable gene. For example, after stably transfecting a host cell lacking Neomycin resistance with a recombinant vector containing a Neomycin (Neomycin) resistance gene, the concentration of Neomycin may be increased in the cell culture broth to select a stable cell strain with high expression; for example, after stably transfecting a host cell lacking DHFR with a recombinant vector containing the dihydrofolate reductase (DHFR) gene, the concentration of Methotrexate (MTX) may be increased in the cell culture medium to select for stable cell lines with high expression.

Other expression systems besides mammalian cells, such as insect cells, yeast, bacteria, etc., may also be used to express the antibodies or fragments thereof of the present invention, and they are also encompassed by the host cells that can be used in accordance with the present invention. The protein expression level of these expression systems is in some cases higher than that of mammalian cells, but inclusion bodies are easily formed, and thus further protein renaturation is required.

Antibodies of the invention may also be carried and expressed using viral vectors including, but not limited to, adenovirus vectors (adenoviral vectors), adeno-associated virus vectors (adeno-associated viral vectors), retrovirus vectors (retroviral vectors), herpes simplex virus vectors (herpes simplex virus-based vectors), lentivirus vectors (lentiviral vectors), and the like.

The anti-CD 25 antibody can be used for detecting CD25, including ELISA and flow cytometry, and has good specificity. No binding signal was observed for the anti-CD 25 antibodies of the invention as analyzed by ELISA and flow cytometry with CD25 negative cellular components of various tissue sources. The anti-CD 25 antibody of the invention can also identify human and monkey CD25 proteins, which is beneficial to the pharmacokinetic study and safety evaluation by primates such as cynomolgus monkey.

Human PBMC and NK-92 cell models show that the preferred anti-CD 25 antibodies of the invention do not block IL-2 induced phosphorylation of cellular STAT5 and are IL-2 non-blocking antibodies. In vitro PBMC models show that the IL-2 non-blocking anti-CD 25 antibodies of the invention can specifically reduce Tregs in PBMC with little impact on Teffs. The in vivo tumor-bearing mouse model also shows that the IL-2 non-blocking anti-CD 25 antibody can inhibit tumor growth, reduce Tregs infiltration in tumor tissues, and no obvious toxic or side effect is observed. The IL-2 non-blocking type anti-CD 25 antibody does not block the IL-2 downstream signal channel, can specifically inhibit Tregs without affecting Teffs, thereby achieving the anti-tumor effect by enhancing tumor immunity and showing good application potential in the aspect of cancer treatment.

The antibody of the present invention can be prepared into various forms of pharmaceutical preparations according to conventional techniques of pharmacy, and liquid injections and freeze-dried injections are more preferable.

The antibodies of the invention may be formed into pharmaceutical compositions with other drugs that may be used in the treatment of diseases along with other therapeutic methods including chemotherapy, radiation therapy, biological therapy, and the like.

The following examples illustrate the discovery, preparation, testing and use of antibodies of the invention. The content and use of the invention is not limited to the scope of the embodiments.

Example 1 mouse immunization

Female BALB/c mice, 7-8 weeks old and weighing about 20g, were used as immunization hosts and were subjected to antigen immunization after one week of adaptive feeding. Purified CD25-His (C-terminal fusion 6 XHis tag of human CD25 extracellular domain) was formulated to 1mg/mL with PBS (pH 7.4), and after filtration through a 0.22 μm filter, 50. Mu.L was mixed well with 50. Mu.L of immunoadjuvant (QuickAntibody) and injected into the calf muscle of the hind leg of the mouse. The immunization was boosted 1 time on day 21 in the same manner, and serum antibody titer was measured by tail vein blood sampling on day 35. CD25-hIgG1Fc (C-terminal fusion of human IgG1Fc fragment of human CD25 extracellular domain) protein was coated onto an ELISA plate (50 ng/well) and mouse serum antibody titer was determined by ELISA. Mice with antibody titers greater than 30000 were given an antigen impact and spleen cell fusion was performed after 3 days.

Example 2 spleen cell fusion

After the mice were euthanized, spleens were isolated under aseptic conditions, spleen cell suspensions were prepared using a 70 μm screen, and washed 2 times with basal medium for cell counting. SP2/0 was mixed with splenocytes in a 1:3 ratio, the supernatant was discarded after centrifugation, 1mL of PEG preheated at 37℃was added dropwise over 1min, the mixture was allowed to stand at 37℃for 90s, and then 20mL of basal medium preheated at 37℃was added over 6 min. Cells were collected by centrifugation (at room temperature, 800rpm,3 min), resuspended in 20mL HAT medium pre-warmed at 37℃and then grown according to 1X 10 ⁵ The density of individual spleen cells/holes is that the fused cells are added into a 96-hole cell culture plate and placed into a carbon dioxide cell culture box for culture, when the cell reaches more than 70% confluence, the culture supernatant is taken for ELISA detection.

Example 3 affinity ELISA screening

CD25 was formulated at a concentration of 1 μg/mL using PBSAn hIgG1Fc solution was added to the plate (50. Mu.L/well) and coated overnight at 4 ℃. PBST plates were washed 3 times, 5% BSA blocking solution (200. Mu.L/well) was added and incubated for 2h at 37 ℃. PBST plates were washed 3 times, hybridoma cell culture supernatants (50. Mu.L/well) were added, and incubated at 37℃for 1h. PBST plates were washed 3 times, added with HRP-goat anti-mouse solution (50. Mu.L/well) diluted 1:5000, and incubated for 1h at 37 ℃. PBST plates were washed 3 times, added with ready-to-use TMB color development solution (100. Mu.L/well), and incubated at 37℃for 10min in the absence of light. Add 2M H ₂ SO ₄ The development was stopped (100. Mu.L/well) and the OD was measured at 450 nm. OD value of control well of mouse IgG (1. Mu.g/mL)<0.3, taking positive wells (OD value>2.0 Corresponding hybridoma cell culture supernatants were subjected to neutralization screening.

Example 4 SPR screening

The appropriate coupling amount was calculated according to the formula rl= (rmax×mwligand)/(sm×mwanalyte), and anti-mouse anti was coupled to CM5 chip using amine coupling kit. Hybridoma cell culture supernatants were captured on chip and the response to CD25-His flow in the channels was detected using Biacore 8K. Data fitting was performed by Evaluation Software to obtain binding curves and kinetic parameters. Preferably, the subcloning is performed on hybridoma cells having good binding kinetics.

Example 5 hybridoma subcloning

The hybridoma cells were subcloned by limiting dilution. Hybridoma cells secreting neutralizing antibodies were collected and counted, diluted with complete medium, added to 96-well cell culture plates at a cell density of 0.5 cells/well for continued culture, and the remaining cells were expanded for seed retention. After 10 days of subcloning culture, culture supernatants of the monoclonal wells were taken for affinity ELISA and SPR validation, respectively, and competition ELISA screening was performed. And taking positive clones, continuing to perform secondary subcloning, and continuing to verify the secondary subcloning according to the screening mode of the primary subcloning.

Example 6 competitive ELISA screening

CD25-hIgG1Fc coated ELISA plates (25 ng/well) at 4℃overnight. PBST plates were washed 3 times, 5% BSA blocking solution (200. Mu.L/well) was added and incubated for 2h at 37 ℃. PBST plate was washed 3 times and hybridoma cell culture supernatant (50. Mu.L/well) was addedIsotype control mIgG (2. Mu.g/ml, 50. Mu.L/well), competitive antibody basiliximab (2. Mu.g/ml, 50. Mu.L/well) and incubated for 1h at 37 ℃. PBST plates were washed 3 times, IL-2-His solution (50. Mu.L/well) in PBS at a concentration of 0.2. Mu.g/mL was added, and incubated at 37℃for 1h. PBST plates were washed 3 times, 10000-fold diluted anti-His-HRP (50. Mu.L/well) with 5% BSA, and incubated at 37℃for 1h. PBST plates were washed 3 times, added with ready-to-use TMB color development solution (100. Mu.L/well), and incubated at 37℃for 10min in the absence of light. Add 2M H ₂ SO ₄ The development was stopped (100. Mu.L/well) and the OD was measured at 450 nm. The developed signal was significantly stronger for the non-competitive hybridoma antibody compared to the less developed competitive antibody basiliximab control.

Example 7 detection of cell binding Activity of hybridoma antibodies by flow cytometry

CD25 positive SU-DHL-1 cells were collected at a cell number of 1X 10 per group ⁶ And each. Cells were washed 1 time with PBS and centrifuged at 3500rpm for 3min. The cell pellet was resuspended in 100. Mu.L of hybridoma cell culture supernatant, PBS, isotype antibody mIgG (10. Mu.g/mL), anti-CD 25 positive antibody 7G7B6 (10. Mu.g/mL), and incubated on ice for 60min. After the incubation was completed, the cells were collected by centrifugation, washed with 500 μl PBS, and repeated 2 times. To the cell pellet was added 100. Mu.L of Alexa Fluor 488-labeled goat anti-mouse IgG (H+L) (1:200 dilution), resuspended, and incubated on ice for 40min in the absence of light. After the incubation was completed, the cells were collected by centrifugation, washed with 500 μl PBS, and repeated 2 times. Finally, the cells were resuspended in 300. Mu.L PBS and analyzed by flow cytometry.

Example 8 purification of hybridoma antibodies

The hybridoma cells were grown by expansion, and the culture supernatant was collected and centrifuged at 4000rpm for 10min, and the supernatant was collected for use. Antibodies in hybridoma cell culture supernatants were purified using anti-mouse IgG affinity fillers. After elution of the filler-bound antibody with 1% acetic acid (pH 2.5), the eluted sample was adjusted to pH 6.5 with 1M Tris-HCl (pH 9.0). The concentration of the antibodies in the samples was determined using Nanodrop, and further the purity of the antibodies was detected using SDS-PAGE. The purified antibody was filtered through a 0.22 μm filter and stored in aliquots at-20℃until use.

Example 9, cell STAT5 phosphorylation screening

NK-92 cells were added to 6-well plates (1X 10) ⁶ Individual/well, 2 mL/well), and incubated overnight. Different anti-CD 25 antibodies or isotype antibodies (10. Mu.g/mL) were added and incubated at 37℃for 30min. Other components except the blank group without rhIL-2 were added with rhIL-2 (final concentration 10 IU/mL) and stimulated at 37℃for 10min. Preparing precooled RIPA lysate containing protease inhibitor and phosphatase inhibitor, and placing on ice for use. After rhIL-2 stimulation is completed, collecting each group of cells, adding freshly prepared RIPA lysate, crushing the cells by adopting an ultrasonic cytoclasis instrument, and putting the crushed cells on ice for 30min for cracking. After completion of lysis, the supernatant was collected by centrifugation (13000 rpm) at 4℃for 20min and the protein concentration was determined by BCA method. Western-blot detection is carried out on 50 mug total protein in equal amount in each group, an anti-pSTAT 5 antibody and an anti-beta-actin antibody are used as primary antibodies, an HRP-marked antibody is used as secondary antibodies, and a chemiluminescent method is adopted to detect signals. In addition, PBMC from human peripheral blood were isolated using lymphocyte separation tubes and STAT5 phosphorylation was detected in each group of PBMC using the method described above. Combining the results of both NK-92 and PBMC models, hybridoma antibodies E5, B7, A6, F10, F8 have minimal effect on rhIL-2-induced STAT5 phosphorylation, and are preferred IL-2 non-blocking anti-CD 25 antibodies (see FIG. 1). In contrast, hybridoma antibody H6 significantly inhibited STAT5 phosphorylation (see fig. 1), an anti-CD 25 antibody that was blocked by IL-2, and was used as a control for subsequent study.

Example 10 screening for proliferation of PBMC cells

PBMC from human peripheral blood were isolated using lymphocyte separation tubes and added to 6-well plates (1X 10) ⁴ Per well, 100 μl/well). Anti-human CD3 antibody (1. Mu.g/mL) and anti-human CD28 antibody (1. Mu.g/mL) were added to PBMC for stimulation, and anti-CD 25 antibody or mIgG was added at various concentrations (0.05. Mu.g/mL, 0.5. Mu.g/mL, 5. Mu.g/mL, 50. Mu.g/mL) and incubated at 37℃for 72h. Cell proliferation was detected using CellTiter-Glo Luminescent Cell Viability Assay cell viability detection kit. The results showed that hybridoma antibodies E5, B7, F10 had minimal inhibition of human PBMC proliferation at each concentration, and were the preferred IL-2 non-blocking anti-CD 25 antibodies (see fig. 2). In contrast, IL-2 blocking anti-CD 25 antibody H6 significantly inhibited PBMC proliferation (see FIG. 2).

Example 11 specific screening

Multiple cell lines were cultured and collected, and binding of the antibodies to cell surface proteins was detected by Flow Cytometry (FCM) using different anti-CD 25 antibodies as primary antibodies and Alexa Fluor 488-labeled goat anti-mouse IgG (h+l) as secondary antibodies. The results showed that antibodies E5, B7, F10, H6 showed binding signals to CD25 positive human diffuse tissue lymphoma cell SU-DHL-1 and human tissue cell lymphoma cell U937, whereas no binding signals were shown to CD25 negative human embryonic kidney cell 293T, human liver cancer cell HepG2, human gastric cancer cell MGC-803, human pancreatic cancer cell PANC-1, human colorectal cancer epithelial cell DLD-1 (see Table 1). On the other hand, a variety of cell lines were cultured, collected, cell-lysed samples were prepared using RIPA lysate and an ultrasonic cytoclasis apparatus, coated onto an elisa plate (500 ng/well), and coated with CD25-hig 1Fc protein as a positive control. Different anti-CD 25 antibodies are used as primary antibodies, HRP-goat anti-mouse antibodies are used as secondary antibodies, and ELISA is used for detecting the binding condition of the antibodies and cell lysis components. The results showed that antibodies E5, B7, F10, H6 showed no binding signals to CD25 negative 293T, hepG2, MGC-803, PANC-1, DLD-1 cells, and binding signals to coated CD25-hIgG1Fc (see Table 1).

TABLE 1 anti-CD 25 hybridoma antibody specificity detection

Note that: "++" represents binding "-" represents non-binding "/" represents undetected.

Example 12 species Cross-reactivity detection

Human CD25 (homemade human CD 25-His), monkey CD25 (Yiqiaoshenzhou), mouse CD25 (R)&Company D) was coated onto an elisa plate (50 ng/well) and incubated overnight at 4 ℃. PBST plates were washed 3 times, 5% BSA blocking solution (200. Mu.L/well) was added and incubated for 2h at 37 ℃. PBST plates were washed 3 times, hybridoma cell culture supernatants (50. Mu.L/well) were added, and incubated at 37℃for 1h. PBST plates were washed 3 times, added with HRP-goat anti-mouse solution (50. Mu.L/well) diluted 1:5000, and incubated for 1h at 37 ℃. PBST plate is washed 3 times and added for useTMB color development (100. Mu.L/well) was incubated at 37℃for 10min in the dark. Add 2M H ₂ SO ₄ The development was stopped (100. Mu.L/well) and the OD was measured at 450 nm. The results showed that antibodies B7, H6 recognized monkey CD25 better, binding was similar to human CD25, while antibodies F10, E5 bound less to monkey CD25 (see fig. 3). Furthermore, none of antibodies B7, H6, F10, E5 recognized murine CD25 (see fig. 3).

Example 13 antibody subtype identification

The subtype of antibodies H6, E5, B7 and F10 is identified by using a mouse monoclonal antibody subtype identification detection kit (Yiqiaoshenzhou). The method is ELISA double-antibody sandwich method, rabbit anti-mouse IgG1/IgG2a/IgG2b/IgG3/IgM antibody is used as coating antibody, HRP-rabbit anti-mouse antibody is used as detection antibody. The results showed that antibodies H6 and F10 were of the mIgG2a type and antibodies E5 and B7 were of the mIgG1 type.

Example 14, antibody competitive ELISA assay

Competition curves for anti-CD 25 antibodies were plotted using competition ELISA analysis. CD25-hIgG1Fc coated ELISA plates (25 ng/well), different anti-CD 25 antibodies (100. Mu.g/mL, 10. Mu.g/mL, 1. Mu.g/mL, 0.1. Mu.g/mL, 0.01. Mu.g/mL, and 0. Mu.g/mL) were added, and competition of anti-CD 25 antibodies for IL-2-His (0.2. Mu.g/mL, 50. Mu.L/well) was examined. Inhibition was calculated for ELISA data and competition curves were plotted using GraphPad Prism8 software. The results showed that antibodies E5, B7, F10 were less competitive than the competitive antibody basiliximab, belonging to the non-competitive antibody (see fig. 4).

Example 15, antibody binding kinetics assay

The appropriate coupling amount was calculated according to the formula rl= (rmax×mwligand)/(sm×mwanalyte), and anti-mouse anti was coupled to CM5 chip using amine coupling kit. Different anti-CD 25 antibodies were captured to the chip and the response values of different concentrations of CD25-His flowing through the channel were detected using Biacore 8K. Data fitting was performed by Evaluation Software to obtain binding curves and kinetic parameters (see fig. 5).

Example 16 acquisition of antibody variable region sequences

Hybridoma subclones were collected and RNA was extracted using Trizol method. Reverse transcription is carried out by taking the extracted RNA as a template to obtain cDNA. The heavy and light chain variable regions of the antibodies were PCR amplified using degenerate primers (Novagen Ig-Primer Sets), respectively, and the PCR amplified products were detected by agarose gel electrophoresis. And obtaining a target DNA fragment by adopting a gel recovery kit, and then performing TA cloning to construct a recombinant plasmid. And (3) transforming the recombinant plasmid into competent cells by adopting a heat shock method, and plating to perform blue and white spot screening. White single colony is picked up to 0.5mL LB liquid culture medium, shake culture is carried out for 3h at 37 ℃ and 220rpm, and bacterial liquid is taken and sent for sequencing. The amino acid sequences of the variable regions of the anti-CD 25 antibody 7B7 (from B7) and F10-2 (from F10) are shown in the following Table (see Table 2).

TABLE 2 variable region amino acid sequences of anti-CD 25 antibodies

EXAMPLE 17 construction and preparation of recombinant monoclonal antibodies

And splicing the heavy chain variable region gene fragment and the light chain variable region gene fragment with the signal peptide, the mouse heavy chain (IgG 2 a) constant region gene fragment and the light chain (kappa) constant region gene fragment by adopting overlap PCR, and sequencing and identifying. And (3) inserting the correctly spliced heavy chain genes and light chain genes of the antibody into pTT5 plasmid respectively, transfecting the recombinant plasmid into HEK293 cells by adopting a PEI method, performing serum-free suspension culture, and transiently expressing the antibody. Cell supernatants cultured for 7 days were collected, filtered through a 0.22 μm filter, and the antibodies were purified by protein G affinity chromatography. The antibody is ultrafiltered and replaced to PBS solution, reduced SDS-PAGE and NanoDrop 2000 are adopted to identify the purity and concentration of the antibody, and the antibody is subpackaged and stored at-80 ℃ for standby. The preparation of human murine chimeric antibodies (IgG 1 kappa type) was performed using a similar procedure as described above.

Example 18 functional verification of antibodies F10-2 and 7B7

According to the detection method of hybridoma screening, the recombinant monoclonal antibodies F10-2 and 7B7 are respectively verified in terms of binding kinetics (see FIG. 6), competitiveness (see FIG. 7), cell binding activity (see FIG. 8), cell STAT5 phosphorylation (see FIG. 9), specificity, species cross-reactivity (see FIG. 10) and the like. The results showed that the activity of antibodies F10-2 and 7B7 in each aspect was substantially identical to the corresponding hybridoma antibodies, as expected.

Example 19 in vitro Tregs inhibition assay for antibodies F10-2 and 7B7

Human peripheral blood PBMC were isolated using lymphocyte separation tubes and activated by the addition of anti-CD 3 antibody (1. Mu.g/mL) and anti-CD 28 antibody (1. Mu.g/mL) for 3 days. PBMC with good growth state were collected and added to 6-well plate (2×10) ⁶ Individual/well), and incubated overnight. 10. Mu.g/mL of human IgG isotype antibody, basiliximab (hIgG 1. Kappa. Type), antibody F10-2 (hIgG 1. Kappa. Type), and antibody 7B7 (hIgG 1. Kappa. Type) were added, respectively, and incubated overnight. Cells were collected by centrifugation, blocked with Human BD Fc Block and differentiated with dead living cell dye (Fixable Viability Stain), and flow cytometry analysis was performed using Hu CD45 APC-Cy7, hu CD3 FITC, hu CD4 PE-Cy7, hu CD8 PerCP-Cy5.5, hu CD25PE, hu Foxp3 AF647 labeled cells, respectively. The results show that IL-2 non-blocking anti-CD 25 antibodies F10-2 and 7B7 can specifically reduce Tregs in PBMCs compared to IL-2 blocking anti-CD 25 antibody basiliximab, with little impact on Teffs (see figure 11).

Example 20 in vivo antitumor Effect of antibodies F10-2 and basiliximab in mice

Mouse colon cancer cells MC38 (5×10) ⁵ Individual/individual) was inoculated into 6-8 week old CD25 (IL 2 RA) humanized mice (C57 BL/6-IL2 RA) ^{tm1(hIL2RA)Smoc} South mold organism). When the tumor volume reaches 100-150mm ³ At this time, mice were grouped (6 mice/group) and administered (10 mg/kg) at the tail vein on days 0, 3, 6, 9, 12, 14, respectively. The antibodies F10-2 and basiliximab used were mouse IgG2aκ (mIgG 2aκ) and mouse IgG (mIgG) was used as a control. Tumor volume was measured every 3 days (formula v=1/2×long diameter×short diameter ² ) And mice body weight and status were monitored. The results showed that there was no significant difference in tumor size between the IL-2 blocking anti-CD 25 antibody basiliximab group and the mIgG control group, whereas IL-2 non-blocking anti-CD 25 antibody F10-2 was able to significantly inhibit tumor growth (see fig. 12).

Example 21 in vivo antitumor Effect of antibodies F10-2 and 7B7 in mice

Mouse colon cancer cells MC38 (5×10) ⁵ And/or) were inoculated into 6-8-week-old CD25 (IL 2 RA) humanized mice (C57 BL/6-Il2ra ^{tm1(hIL2RA)Smoc} South mold organism). When the tumor volume reaches 100-150mm ³ At this time, mice were grouped (6 mice/group) and administered (10 mg/kg) at the tail vein on days 0, 3, 6, 10, 13, respectively. Antibodies F10-2 and 7B7 used were of the mouse IgG2aκ type (mIgG 2aκ) with mouse IgG (mIgG) as control. Tumor volume was measured every 3 days (formula v=1/2×long diameter×short diameter ² ) And mice body weight and status were monitored. The results show that both antibodies 7B7 and F10-2 can effectively inhibit tumor growth, and the tumor inhibition effect of both antibodies is similar (see FIG. 13).

Example 22 antibodies F10-2 and 7B7 can reduce Tregs infiltration in tumor tissue

After the mouse anti-tumor test is finished, tumor tissues are separated and sheared, the tumor tissues are digested by adding a DMEM medium containing 1mg/mL type IV collagenase, cell suspension is obtained by filtering through a 70 mu m screen, and immune cells are further obtained by using a Histopaque separating liquid. After Mouse BD Fc Block blocking, single and multiple staining of PBS, fixable Viability Stain 575V, ms CD45 APC-Cy7 30-F11, ms CD3 BB700 145-2C11, ms CD4 FITC RM4-5, ms CD8a PE-Cy7 53-6.7, hu CD25 BV421 2A3, ms Foxp3 PE MF23 were performed, respectively, for flow cytometry analysis. The results show that the IL-2 blocking anti-CD 25 antibody basiliximab does not reduce Treg infiltration in tumor tissue. However, IL-2 non-blocking anti-CD 25 antibodies F10-2 and 7B7 significantly reduced Treg infiltration in tumor tissue and were similar in effect (see fig. 14).

In vivo safety study in mice of example 23, antibodies F10-2 and 7B7

Throughout the course of treatment in the anti-tumor test of mice, mice were behaving normally, survived well, and had no significant change in body weight (see figure 15). After the test, heart, liver, spleen, lung, kidney and lymph node tissues of each group of mice are taken and fixed in neutral formalin fixing solution, paraffin embedding is carried out by a conventional method, and tissue sections are prepared. After hematoxylin and eosin (H & E) staining, the tissue sections were observed under a microscope. The results showed that no significant lesions were seen in the vital organ tissues after antibody F10-2 and 7B7 treatment (see FIG. 16).

Example 24 humanization of antibody 7B7

The variable region of antibody 7B7 was homologously modeled according to the antibody structure in the PDB database, and CDR regions were determined based on the amino acid primary sequence characteristics and the spatial conformation of the variable region. The antibody variable region is compared with amino acid sequences encoded by human antibody germ line genes V and J, and 5V genes and 1J gene in an IGHV library are preferably transplanted and spliced with heavy chain CDR according to the factors of the consistency, the similarity, the conservation and the like of the frame region FR to form 5 different humanized heavy chain variable regions. Similarly, it is preferred that 4V genes and 1J gene in the IGKV library be graft spliced with the light chain CDRs to form 4 different humanized light chain variable regions. Humanized heavy and light chain variable regions were fused to human IgG1 kappa heavy and light chain constant regions, respectively, to form complete heavy and light chains, and 5 heavy chains were combined with 4 light chains to construct 20 different humanized antibodies (see table 3). The amino acid sequences of the heavy chain complementarity determining regions VH-CDR1, VH-CDR2, VH-CDR3 and light chain complementarity determining regions VL-CDR1, VL-CDR2 and VL-CDR3 of the humanized antibody h7B7 are shown in Table 4. By Biacore binding kinetics analysis, humanized antibodies h7B7-6, h7B7-7, h7B7-14, h7B7-15, h7B7-19 have superior binding kinetics (see FIG. 17), with h7B7-15 being more preferred (see Table 5). The amino acid sequence of the h7B7-15 heavy chain variable region is shown as SEQ ID NO.13, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 14.

TABLE 3 humanized antibody germ line Gene for H7B7 humanized antibody

		Light chain 1	Light chain 2	Light chain 3	Light chain 4
							Human body germ li ne Template	IGKV1-27*01	IGKV1-39*01	IGKV1-33*01	IGKV1-12*01
Heavy chain 1	IGHV4-30-4*01	h7B7-1	h7B7-2	h7B7-3	h7B7-4
						Heavy chain 2	IGHV4-31*02	h7B7-5	h7B7-6	h7B7-7	h7B7-8
Heavy chain 3	IGHV4-59*01	h7B7-9	h7B7-10	h7B7-11	h7B7-12
						Heavy chain 4	IGHV4-34*09	h7B7-13	h7B7-14	h7B7-15	h7B7-16
Heavy chain 5	IGHV4-61*02	h7B7-17	h7B7-18	h7B7-19	h7B7-20

TABLE 4 CDR amino acid sequences of H7B7 humanized antibodies

CDR regions	Amino acid sequence	Sequence numbering
			VH-CDR1	GYSITSDYAWN	SEQ ID NO.1
VH-CDR2	YINYSGSTSYNPSLKS	SEQ ID NO.2
			VH-CDR3	KGGFFDV	SEQ ID NO.3
VL-CDR1	SASQGISNYLN	SEQ ID NO.4
			VL-CDR2	YTSSLHS	SEQ ID NO.5
VL-CDR3	LQYSKLPWT	SEQ ID NO.6

TABLE 5 binding kinetics of H7B7 humanized antibodies

Example 25 humanization of antibody F10-2

Antibody F10-2 was humanized following the method for humanization of antibody 7B 7. The amino acid sequences of the heavy chain complementarity determining regions VH-CDR1, VH-CDR2, VH-CDR3 and light chain complementarity determining regions VL-CDR1, VL-CDR2 and VL-CDR3 of humanized antibody hF10-2 are shown in Table 6. After CDR grafting, no binding activity was detected in any of the 20 humanized antibodies, and thus back mutation (back mutation) was continued. The antibody structure from homologous modeling is analyzed, key amino acid residues which have direct interaction with the CDRs and support the CDRs on a VH/VL binding interface are selected as classical residues (canonical residues), and part of the classical residues are subjected to back mutation to construct humanized antibody mutants. Further analysis of the antibody variable region structure found that VH-CDR3 was shorter in length and directed to conformationally flattened VL-CDR2, while the charged long side chain of R79 was more prominent adjacent to VL-FR3, presumably with R79 involved in antigen binding. By constructing antibody mutants and performing Biacore assays, it was confirmed that R79 in VL-FR3 was critical for maintaining antibody affinity (see table 7). The humanized antibody can show good affinity by retaining the murine residue R79 in the light chain variable region FR3 (see Table 8), wherein hF10-2-4 is preferred (see Table 8), the amino acid sequence of the heavy chain variable region of hF10-2-4 is shown as SEQ ID NO.15, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 16.

TABLE 6 CDR amino acid sequences of hF10-2 humanized antibody complementarity determining regions

CDR regions	Amino acid sequence	Sequence numbering
			VH-CDR1	GFSLTSYGVH	SEQ ID NO.7
VH-CDR2	VIWRGGSTDYNAAFMS	SEQ ID NO.8
			VH-CDR3	NERFYGFDY	SEQ ID NO.9
VL-CDR1	RSSKSLLHSNGITYLY	SEQ ID NO.10
			VL-CDR2	QMSNLAS	SEQ ID NO.11
VL-CDR3	AQNLELPT	SEQ ID NO.12

TABLE 7 humanized antibody germ line Gene and amino acid sequence for hF10-2 humanized antibody

Note that: dark residues are residues that differ between two heavy chains and between two light chains.

TABLE 8 binding kinetics of hF10-2 humanized antibodies

	ka(1/Ms)	kd(1/s)	KD(M)
				hF10-2-1(BM4+BM)	/	/	/
hF10-2-2(BM5+BM)	/	/	/
				hF10-2-3(BM4+BM3)	3.18E+05	3.33E-03	1.05E-08
hF10-2-4(BM5+BM3)	6.99E+04	1.26E-03	1.81E-08

Note that: "/" indicates undetected.

EXAMPLE 26 Effect of humanized antibodies on cell STAT5 phosphorylation

Human peripheral blood PBMC were added to 6-well plates (1X 10) ⁶ Individual/well, 2 mL/well), and incubated overnight. Different anti-CD 25 antibodies or isotype antibodies (10. Mu.g/mL) were added and incubated at 37℃for 30min. Other components except the blank group without rhIL-2 were added with rhIL-2 (final concentration 10 IU/mL) and stimulated at 37℃for 10min. Cells were lysed and 50. Mu.g of total protein was taken in equal amounts from each group for Western-blot to detect STAT5 phosphorylation. The results showed that none of the humanized antibodies h7B7-6, h7B7-7, h7B7-14, h7B7-15, h7B7-19 effectively blocked rhIL-2 induced phosphorylation of PBMC STAT5, with the blocking of h7B7-15 being the weakest, compared to the IL-2 blocking anti-CD 25 antibody basiliximab (see FIG. 18).

Example 27 in vitro Tregs inhibition assay of humanized antibodies

Human peripheral blood PBMC were isolated using lymphocyte separation tubes and activated by the addition of anti-CD 3 antibody (1. Mu.g/mL) and anti-CD 28 antibody (1. Mu.g/mL) for 3 days. PBMC with good growth state were collected and added to 6-well plate (2×10) ⁶ Individual/well), and incubated overnight. Adding 10 mug/mL human IgG isotype antibody and basilixix respectivelymab, h7B7 humanized antibody, were incubated overnight. Cells were collected by centrifugation, blocked with Human BD Fc Block, and differentiated with a dead living cell dye (Fixable Viability Stain), and flow cytometry analysis was performed using Hu CD45 APC-Cy7, hu CD3 FITC, hu CD4 PE-Cy7, hu CD8 PerCP-Cy5.5, hu CD25 PE, hu Foxp3 AF647 labeled cells, respectively. The results show that humanized antibodies h7B7-6, h7B7-7, h7B7-14, h7B7-15, h7B7-19 can specifically reduce Tregs in PBMC compared to the IL-2 blocking anti-CD 25 antibody basiliximab (see FIG. 19).

Example 28 thermal stability analysis of humanized antibodies

The melting temperature (Tm) and aggregation initiation temperature (Tagg) of the antibodies were determined using a unclle multiparameter high-throughput protein stability analysis system. The buffer used was PBS (pH 7.4) and the antibody sample concentration was 1mg/mL. The Tm value is determined by reflecting conformational changes in the protein by fluorescent changes in the endogenous aromatic amino acids of the protein. Aggregate with different sizes is detected by adopting two wavelengths of 266nm and 473nm, aggregation of protein in the heating process is monitored, and Tagg value is determined. Analysis by unclle analysis showed that the thermal stability of the 5 h7B7 humanized antibodies was not greatly different, with h7B7-7, h7B7-15, and h7B7-19 being slightly superior (see Table 9).

TABLE 9 thermal stability analysis of H7B7 humanized antibodies

Antibodies to	Tm(℃)	Tagg(℃)
			h7B7-6	71.15	79.20
h7B7-7	71.30	79.81
			h7B7-14	71.43	77.94
h7B7-15	71.32	79.57
			h7B7-19	71.39	79.63

EXAMPLE 29 glycosylation site mutation

The present invention contemplates that the VH-CDR2 of humanized antibody h7B7 contains a NYS glycosylation motif, in an attempt to reduce potential heterogeneity of the antibody molecule by mutating glycosylation site N therein. Through residue analysis and alignment of the germline genes of the antibodies, it was found that N in the NYS glycosylation motif was generated by somatic high frequency mutation (SHM), so that Q (high side chain similarity), a (no side chain), S (murine germline gene residue corresponding to this site), Y (human germline gene residue corresponding to this site) were each selected to replace N in NYS, and 4 mutants were constructed, respectively, for Biacore binding kinetics screening (see table 10). The results show that after S substitution (N53S), the antibody (h 7B7-15S, heavy chain variable region sequence shown as SEQ ID NO. 17) can maintain better affinity.

TABLE 10 binding kinetics of H7B7 mutants

Antibodies to	ka(1/Ms)	kd(1/s)	KD(M)	Mutation
					h7B7-15	1.53E+05	3.56E-04	2.33E-09	Unmutated
h7B7-15Q	2.38E+05	5.02E-03	2.11E-08	N53Q
					h7B7-15A	1.21E+05	1.03E-03	8.55E-09	N53A
h7B7-15S	1.15E+05	5.54E-04	4.83E-09	N53S
					h7B7-15Y	/	/	/	N53Y

Note that: "/" indicates no affinity was detected.

Example 30 antibody Activity and thermal stability assay after glycosylation site mutation

Functional verification and thermal stability analysis were performed on the antibody h7B7-15S after glycosylation site mutation. The results showed that antibodies h7B7-15S were substantially identical to h7B7-15 prior to glycosylation mutation in terms of cell binding activity (see fig. 20), tregs inhibition function (see fig. 21), thermostability (see table 11), etc.

TABLE 11 thermal stability analysis of antibodies h7B7-15S and h7B7-15

Humanized antibodies	Tm(℃)	Tagg(℃)
			h7B7-15S	71.13	79.55
h7B7-15	71.32	79.57

Sequence listing

SEQ ID NO.1 antibody 7B7 heavy chain variable region VH-CDR1 amino acid sequence

GYSITSDYAWN

SEQ ID NO.2 antibody 7B7 heavy chain variable region VH-CDR2 amino acid sequence

YINYSGSTSYNPSLKS

SEQ ID NO.3 antibody 7B7 heavy chain variable region VH-CDR3 amino acid sequence

KGGFFDV

SEQ ID NO.4 antibody 7B7 light chain variable region VL-CDR1 amino acid sequence

SASQGISNYLN

SEQ ID NO.5 antibody 7B7 light chain variable region VL-CDR2 amino acid sequence

YTSSLHS

SEQ ID NO.6 antibody 7B7 light chain variable region VL-CDR3 amino acid sequence

LQYSKLPWT

SEQ ID NO.7 antibody F10-2 heavy chain variable region VH-CDR1 amino acid sequence

GFSLTSYGVH

SEQ ID NO.8 antibody F10-2 heavy chain variable region VH-CDR2 amino acid sequence

VIWRGGSTDYNAAFMS

SEQ ID NO.9 antibody F10-2 heavy chain variable region VH-CDR3 amino acid sequence

NERFYGFDY

SEQ ID NO.10 antibody F10-2 light chain variable region VL-CDR1 amino acid sequence

RSSKSLLHSNGITYLY

SEQ ID NO.11 antibody F10-2 light chain variable region VL-CDR2 amino acid sequence

QMSNLAS

SEQ ID NO.12 antibody F10-2 light chain variable region VL-CDR3 amino acid sequence

AQNLELPT

SEQ ID NO.13 humanized antibody h7B7-15 heavy chain variable region amino acid sequence

QVQLQESGPGLVKPSQTLSLTCAVYGYSITSDYAWNWIRQPPGKGLEWIGYINYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARKGGFFDVWGQGTTVTVSS

SEQ ID NO.14 humanized antibody h7B7-15 light chain variable region amino acid sequence

DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAPKLLIYYTSSLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQYSKLPWTFGGGTKVEIK

SEQ ID NO.15 humanized antibody hF10-2-4 heavy chain variable region amino acid sequence

QVQLKESGPGLVKPSDTLSLTCTVSGFSLTSYGVHWIRQPPGKGLEWIGVIWRGGSTDYNAAFMSRLSITKDNSKSQVSLKLSSVTAADTAVYYCAKNERFYGFDYWGQGTLVTVSS

SEQ ID NO.16 humanized antibody hF10-2-4 light chain variable region amino acid sequence

DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPTFGGGTKVEIK

SEQ ID NO.17 humanized antibody h7B7-15S heavy chain variable region amino acid sequence

QVQLQESGPGLVKPSQTLSLTCAVYGYSITSDYAWNWIRQPPGKGLEWIGYISYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARKGGFFDVWGQGTTVTVSS

SEQ ID NO.18 antibody 7B7 heavy chain variable region amino acid sequence

EVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYINYSGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARKGGFFDVWGAGTTVTVSS

SEQ ID NO.19 antibody 7B7 light chain variable region amino acid sequence

DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCLQYSKLPWTFGGGTKLEIK

SEQ ID NO.20 antibody F10-2 heavy chain variable region amino acid sequence

QVQLKQSGPGLVQPSQSLSITCTVSGFSLTSYGVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQAGDTAVYYCAKNERFYGFDYWGQGTTLTVSS

SEQ ID NO.21 antibody F10-2 light chain variable region amino acid sequence

DIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPTFGSGTKLEIK

SEQ ID NO.22 IGHV4-59 x 07 (BM 4) heavy chain amino acid sequence

QVQLKQSGPGLVKPSDTLSLTCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVSLKLSSVTAADTAVYYCAKNERFYGFDYWGQGTLVTVSS

SEQ ID NO.23 IGHV4-59 x 07 (BM 5) heavy chain amino acid sequence

QVQLKESGPGLVKPSDTLSLTCTVSGFSLTSYGVHWIRQPPGKGLEWIGVIWRGGSTDYNAAFMSRLSITKDNSKSQVSLKLSSVTAADTAVYYCAKNERFYGFDYWGQGTLVTVSS

SEQ ID NO.24 IGKV2-28 x 01 (BM) light chain amino acid sequence

DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPTFGGGTKVEIK

SEQ ID NO.25 IGKV2-28 x 01 (BM 3) light chain amino acid sequence

DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPTFGGGTKVEIK

Sequence listing

<110> university of Sichuan

<120> CD25 targeting antibodies and uses thereof

<160> 25

<170> SIPOSequenceListing 1.0

<210> 1

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 1

Gly Tyr Ser Ile Thr Ser Asp Tyr Ala Trp Asn

1 5 10

<210> 2

<211> 16

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Tyr Ile Asn Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 3

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 3

Lys Gly Gly Phe Phe Asp Val

1 5

<210> 4

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 4

Ser Ala Ser Gln Gly Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 5

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 5

Tyr Thr Ser Ser Leu His Ser

1 5

<210> 6

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 6

Leu Gln Tyr Ser Lys Leu Pro Trp Thr

1 5

<210> 7

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 7

Gly Phe Ser Leu Thr Ser Tyr Gly Val His

1 5 10

<210> 8

<211> 16

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 8

Val Ile Trp Arg Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Met Ser

1 5 10 15

<210> 9

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 9

Asn Glu Arg Phe Tyr Gly Phe Asp Tyr

1 5

<210> 10

<211> 16

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 10

Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr

1 5 10 15

<210> 11

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 11

Gln Met Ser Asn Leu Ala Ser

1 5

<210> 12

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 12

Ala Gln Asn Leu Glu Leu Pro Thr

1 5

<210> 13

<211> 116

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 13

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Asn Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Lys Gly Gly Phe Phe Asp Val Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 14

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 14

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Ser Lys Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 15

<211> 117

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 15

Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Asp

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Arg Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys Asn Glu Arg Phe Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 16

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 16

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn

85 90 95

Leu Glu Leu Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 17

<211> 116

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 17

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Lys Gly Gly Phe Phe Asp Val Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 18

<211> 116

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 18

Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Ile Asn Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Lys Gly Gly Phe Phe Asp Val Trp Gly Ala Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 19

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 19

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Ser Lys Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 20

<211> 117

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 20

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Arg Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Lys Met Asn Ser Leu Gln Ala Gly Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys Asn Glu Arg Phe Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser

115

<210> 21

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 21

Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly

1 5 10 15

Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn

85 90 95

Leu Glu Leu Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 22

<211> 117

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 22

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Asp

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Arg Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys Asn Glu Arg Phe Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 23

<211> 117

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 23

Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Asp

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Arg Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys Asn Glu Arg Phe Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 24

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 24

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn

85 90 95

Leu Glu Leu Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 25

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 25

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn

85 90 95

Leu Glu Leu Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Claims (16)

1. An anti-CD 25 antibody or fragment thereof, characterized in that: the amino acid sequences of the heavy chain complementarity determining regions VH-CDR1, VH-CDR2 and VH-CDR3 of the antibody or the fragment thereof are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, and the amino acid sequences of the light chain complementarity determining regions VL-CDR1, VL-CDR2 and VL-CDR3 of the antibody or the fragment thereof are respectively shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.

2. An anti-CD 25 antibody or fragment thereof, characterized in that: the amino acid sequences of the heavy chain complementarity determining regions VH-CDR1, VH-CDR2 and VH-CDR3 of the antibody or the fragment thereof are respectively shown as SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9, and the amino acid sequences of the light chain complementarity determining regions VL-CDR1, VL-CDR2 and VL-CDR3 of the antibody or the fragment thereof are respectively shown as SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO. 12.

3. The anti-CD 25 antibody or fragment thereof according to claim 1, wherein: the heavy chain variable region VH is formed by splicing the VH-CDR1, the VH-CDR2 and the VH-CDR3 with the human antibody framework region FR, and the light chain variable region VL is formed by splicing the VL-CDR1, the VL-CDR2 and the VL-CDR3 with the human antibody framework region FR.

4. An anti-CD 25 antibody or fragment thereof according to claim 3, characterized in that: the amino acid sequences of the heavy chain variable region VH and the light chain variable region VL are respectively shown as SEQ ID NO.13 and SEQ ID NO. 14.

5. The anti-CD 25 antibody or fragment thereof according to claim 2, wherein: the heavy chain variable region VH is formed by splicing the VH-CDR1, the VH-CDR2 and the VH-CDR3 with the human antibody framework region FR, and the light chain variable region VL is formed by splicing the VL-CDR1, the VL-CDR2 and the VL-CDR3 with the human antibody framework region FR.

6. The anti-CD 25 antibody or fragment thereof according to claim 5, wherein: the amino acid sequences of the heavy chain variable region VH and the light chain variable region VL are respectively shown as SEQ ID NO.15 and SEQ ID NO. 16.

7. An anti-CD 25 antibody or fragment thereof according to claim 3, characterized in that: the N mutation in glycosylation motif NYS in the antibody heavy chain variable region VH-CDR2 is Q, A or S.

8. The anti-CD 25 antibody or fragment thereof according to claim 7, wherein: the amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO. 17.

9. An anti-CD 25 antibody or fragment thereof according to any one of claims 1 to 8, wherein the antibody fragment is a Fab or scFv.

10. An anti-CD 25 antibody or fragment thereof according to claim 9, characterized in that the heavy chain constant region of the antibody is derived from the constant region of a human immunoglobulin IgG1, igG2, igG3, igG4, igM, igE, igA or IgD heavy chain.

11. An anti-CD 25 antibody or fragment thereof according to claim 9, wherein the light chain constant region of the antibody is derived from the constant region of a human immunoglobulin kappa or lambda light chain.

12. A nucleic acid molecule encoding an anti-CD 25 antibody or fragment thereof according to any one of claims 1 to 11.

13. A recombinant vector comprising the nucleic acid molecule of claim 12.

14. A cell comprising the recombinant vector of claim 13.

15. Use of an anti-CD 25 antibody or fragment thereof according to any one of claims 1 to 11 for the preparation of an anti-tumour medicament, characterized in that the tumour is lung cancer, liver cancer, ovarian cancer, breast cancer or a tumour of the gastrointestinal tract.

16. The use according to claim 15, wherein the gastrointestinal tumour is colon cancer.