CN115197321A - 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 technical problem to be solved by the invention is to provide an IL-2 non-blocking anti-CD 25 antibody or a fragment thereof. The present invention provides novel anti-CD 25 antibodies. The anti-CD 25 antibody has good affinity, specificity and thermal stability, does not block an IL-2 downstream signal path, can specifically inhibit regulatory T cells (Tregs) without influencing T effector cells (Teffs), achieves the anti-tumor effect by enhancing tumor immunity, and shows 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 an antibody targeting CD25 and application thereof.

Background

The antibody is a biological macromolecule composed of heavy chains and light chains, is secreted by B lymphocytes, and plays an important role in humoral immunity of an organism. The heavy chain or the light chain of the antibody molecule respectively consists of a variable region and a constant region, wherein the variable region mainly plays a role in combining target antigens, and the constant region mainly plays an immune regulation effect. Antibodies can be classified into IgG, igM, igE, igA, igD, etc., according to spatial structure and amino acid sequence characteristics, and heavy and light chains can be further subdivided into multiple subtypes. For example, human IgG heavy chains can be classified as IgG1, igG2, igG3, and IgG4, and light chains can be classified as κ and λ. Because the antibody can specifically and efficiently bind to a target molecule or a target cell, regulate and control a signal path downstream of the target molecule, or kill the target cell through an immune effect, the antibody can be developed into a medicament for treating diseases. Currently, antibodies have been developed as an important biotechnological drug, and monoclonal antibodies account for a large majority of them, mainly antibodies of the IgG type, so that the monoclonal antibodies are often referred to as IgG-type antibodies.

The IgG type antibody molecule is a tetramer consisting of 2 heavy chains and 2 light chains through interchain disulfide bonds, and has a molecular weight of about 150kD. Antibody molecules can be divided into variable and constant regions according to their 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 Regions (CDRs) and Framework Regions (FRs), wherein each of the heavy or light chains 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 (FR 1, FR2, FR3, FR 4) flanking the CDR regions. The loop formed by the CDR region is the main part for combining the antibody molecule with the antigen, and the FR region forms the support structure of the CDR region through space folding. The specific recognition of different antigen molecules by antibodies is mainly realized by the amino acid polymorphism of 6 CDR regions (VH- CDR 1, 2, 3 and VL-CDR1, 2, 3) and the conformation polymorphism of loop. Because the structural similarity of the FR regions of different antibodies is higher, after the CDR region of one antibody is replaced with the CDR region of other antibody molecules, if the FR regions of different antibody molecules are matched properly, the conformational change of the CDR region before and after replacement is smaller, so that the new variable region formed after replacement can still retain the antigen binding capacity, and the characteristic is the basis of CDR grafting (CDR grafting) technology. CDR regions of a mouse antibody can be replaced with CDRs of a human antibody by a CDR-grafting technique, and thus, the CDR regions can be recombined with human FR regions to become humanized antibodies (humanized antibodies), and if the FR regions are properly matched between human and mouse antibodies, the binding ability of the antigen can still be retained.

Hybridoma technology is an important technology in the process of antibody discovery at present, and the antibody molecules generated by the technology complete an affinity maturation process in mice, so that the antibody molecules can effectively reduce nonspecific binding with self or similar proteins. Due to 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 generated when murine antibodies are used for human therapy, resulting in the development of ADA (anti-drug antibody). The neutralizing anti-drug antibody not only can influence the target accessibility of the antibody drug and reduce the treatment effect of the antibody drug, but also can increase the risk of side effects due to immune complexes formed by the anti-drug antibody. The use of CDR grafting techniques to transform murine antibodies into humanized antibodies has been shown to reduce the risk of HAMA reactions and reduce the incidence of ADA. At present, dozens of humanized antibodies are on the market, including trastuzumab, pembrolizumab and bevacizumab, which indicates 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 soluble ligand outside cells, block the binding of the ligand and a receptor, and cut off downstream signal transmission induced by the ligand, so that antibody drugs developed by taking immune cytokines as targets can improve inflammatory diseases, such as adalimumab, belimumab, siltuximab and other antibodies are approved to be on the market. The antibody constant region can play an immune regulation role, including antibody-dependent cellular cytotoxicity (ADCC), 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 commercially available antibodies of rituximab, trastuzumab, cetuximab and the like, and great success is achieved. In addition, the development of new antibody technologies such as bispecific antibodies, antibody-drug conjugates (ADCs) and the like derived based on monoclonal antibodies is also fast in recent years, and breakthrough is achieved to different degrees. By far, cancer and inflammatory diseases are the disease areas where antibody drugs are most used.

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 of cancer immunotherapy, it is found that although cancer immunotherapy has a high remission rate for a few cancer patients such as malignant melanoma, the remission rate for most cancers is not very ideal, and one of the important reasons is that the tumor immunity is weakened due to immunosuppressive factors existing in the microenvironment in which tumor cells are located. Tumor Microenvironment (TME) refers to the surrounding tissue environment upon which tumor cells depend for survival, with immune cells of various types being an important component of the tumor microenvironment. Immune cells in the tumor microenvironment are largely divided into two main categories by function: one is immune effector cells, mainly including effector T cells (Teffs) for killing tumor cells, NK cells, dendritic Cells (DC) for presenting antigens, and the like; another class is immunosuppressive cells, mainly including regulatory T cells (Tregs), tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and the like. The immunosuppressive cell can inhibit the function of immune effector cell, lead to the immune escape of tumor, promote the occurrence and development of tumor. Tregs, an important immunosuppressive cell, is of interest due to their potent immunosuppressive effects and infiltration into a wider range of tumor tissues.

Tregs are T cell subsets with immune suppression function, and the detection phenotype of the Tregs is generally considered to be CD4+ CD25+ FoxP3+, so that the Tregs play an important role in maintaining immune homeostasis of an organism and preventing autoimmune diseases. With the development of tumor immunology, the action mechanism of Tregs in tumor immune escape and immunotherapy drug 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 transcutaneously endocytose CD80 and CD86 molecules of DC cells, and reduce the antigen presenting capacity of the DC cells and the activation capacity of the DC cells on Teffs. Tregs highly express metabolic enzymes such as CD39 and CD73, and produce Adenosine (ADO) to suppress immune functions of immune cells such as Teffs, NK cells, and macrophages. In addition, tregs also highly express CD25, compete for IL-2 that predates Teffs, attenuating Teffs proliferation and activation. Tregs infiltrate into various tumor tissues such as lung cancer, liver cancer, ovarian cancer, breast cancer, gastrointestinal tumors and the like, so that the ratio of Teffs to Tregs is low, which is also an important reason for low response and drug resistance of cancer immunotherapy, and therefore, the removal of Tregs or the reversal of the immunosuppressive function of Tregs becomes a cancer immunotherapy strategy.

Constitutively high expressing CD25 is an important feature of Tregs. CD25, the IL-2 receptor alpha chain (IL-2R alpha), can form an intact IL-2 receptor complex (IL-2R alpha. Beta. Gamma.) together with the IL-2 receptor beta chain (CD 122) and the IL-2 receptor gamma chain (CD 132). IL-2R is largely classified into 3 types: IL-2R alpha beta gamma heterothain trimer constitutes IL-2 high affinity receptor (KD = 10) ^-11 M), IL-2R β γ constitutes a moderate affinity receptor (KD = 10) ^-9 M), IL-2 ra alone constitutes a low affinity receptor (KD = 10) ^-8 M). Although the IL-2R alpha chain is not responsible for signal transduction, the IL-2R alpha beta gamma and IL-2 which are composed of the IL-2R alpha chain form a complex which is more stable, and the activation effect of intracellular Jak3 signals is stronger, so that the proliferation and the activation of T cells can be more effectively started. CD25 is constitutively highly expressed in Tregs, and is less expressed in Teffs and NK cells, so that IL-2 high affinity receptor IL-2R alpha beta gamma is mainly expressed in Tregs. Unlike Tregs, teffs primarily express the affinity receptor IL-2R β γ in IL-2, while expressing less of the high affinity receptor IL-2R α β γ. Since the high affinity receptor IL-2R α β γ mediates IL-2 signaling much more efficiently than the medium affinity receptor IL-2R β γ, CD25, although expressed less at Teffs, is important for the proliferation and activation of Teffs. The FDA approved CD25 antibody (e.g., basiliximab) on the market in 1998 can block the binding of IL-2 to CD25 on T lymphocytes, thereby inhibiting T cells, and is mainly used for preventing organ transplant rejection.

Since the constitutive high expression of CD25 in Tregs is also considered to be an important marker of Tregs, the use of CD25 as an antibody target for inhibiting Tregs is also a reasonable choice. The expression of CD25 in Teffs is low, and the anti-CD 25 antibody can be more combined with Tregs and preferentially kills the Tregs through ADCC, CDC and the like. In contrast, teffs bind less anti-CD 25 antibody, and thus the anti-CD 25 antibody is difficult to exert an effective killing effect, and has a limited Teffs inhibitory effect. anti-CD 25 antibodies can be classified as IL-2 competitive and non-competitive antibodies, depending on whether the antibody binding epitope can compete for IL-2 binding to CD25. When the anti-CD 25 antibody blocks IL-2/IL-2R binding and a downstream signal path thereof, teffs can be blocked from receiving IL-2 signals, so that the blocking type anti-CD 25 antibody is unfavorable for the anti-tumor function of Teffs. In contrast, when the anti-CD 25 antibody does not block IL-2/IL-2R binding, the IL-2R of Teffs can still bind IL-2 and exert normal anti-tumor effect, so that the non-blocking antibody can inhibit Tregs without affecting the anti-tumor function of Teffs. The anti-CD 25 antibodies basiliximab and daclizumab which are approved to be marketed by the FDA in the United states are IL-2 blocking antibodies and are used for preventing immune diseases such as organ transplant rejection and multiple sclerosis.

Although CD25 is a good target for the modulation of tregs, IL-2 blocking and non-blocking antibodies differ in the modulation of T cell immunity and in the treatment of diseases. IL-2 blocking antibodies are advantageous for inhibiting T cell immunity, while IL-2 non-blocking antibodies are advantageous for activating T cell immunity, and therefore screening strategies for antibodies are required depending on the disease to be treated. For cancer immunotherapy, tregs are required to be inhibited without affecting Teffs functions, so that an IL-2 non-blocking antibody may be a reasonable choice, and is more favorable for achieving an anti-tumor effect by enhancing tumor immunity.

Disclosure of Invention

The technical problem to be solved by the invention is to provide an IL-2 signal non-blocking 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 solution of the present invention for solving the above 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 respectively shown in 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 in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.

The heavy chain variable region VH of the anti-CD 25 antibody can be 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 light chain variable region VL can be 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 represented by SEQ ID No.13 and SEQ ID No.14, respectively.

The anti-CD 25 antibody or fragment thereof provided by the present invention may 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 respectively shown in 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 are respectively shown in SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO. 12.

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

Further, the 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, igM, igE, igA, igD heavy chains, and the light chain constant region may be derived from the constant region of human immunoglobulin kappa or lambda light chains.

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-described anti-CD 25 antibodies or fragments thereof.

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

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

The invention also provides the 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 invention can be prepared into various forms of pharmaceutical preparations according to the conventional pharmaceutical technology, and preferably liquid injections and freeze-dried injections.

The antibodies of the invention may be combined with other drugs to form pharmaceutical compositions that may be used to treat diseases in conjunction with other therapeutic methods, including chemotherapy, radiation therapy, biological therapy, and the like.

The invention has the beneficial effects that:

the present invention provides an anti-CD 25 antibody, wherein a preferred anti-CD 25 antibody is a humanized antibody obtained by splicing the CDR region of a murine monoclonal antibody and the FR region of a human antibody by a CDR-grafting technique. The anti-CD 25 antibody has good affinity, specificity and functionality, does not block an IL-2 downstream signal path, is an IL-2 non-blocking anti-CD 25 antibody, can inhibit Tregs highly expressed by CD25, does not influence Teffs to receive IL-2 signals, achieves the anti-tumor effect by enhancing tumor immunity, and shows good application potential in the aspect of cancer treatment.

Drawings

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

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

Figure 3 elisa assay of anti-CD 25 hybridoma antibodies for binding activity to human, monkey, murine CD25.

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

FIG. 5 analysis of the binding kinetics 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 competitive 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 STAT5 phosphorylation. (a) NK-92 cells; (B) human PBMC.

FIG. 10 species cross-reactivity of antibodies F10-2 and 7B7. (A) ELISA; (B) analysis of Biacore binding kinetics of antibody 7B7 to monkey CD25.

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

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

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

Figure 14 antibodies F10-2 and 7B7 reduced Tregs infiltration in tumor tissues.

Figure 15. Body weight change after mice administration.

FIG. 16H & E staining of important organ tissues after mouse administration.

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 analysis of the effect of h7B7 humanized antibodies on IL-2 induced phosphorylation of PBMC STAT 5.

Figure 19.H7b7 humanized antibody specifically reduces Tregs in PBMC.

FIG. 20 cell binding activity of antibodies h7B7-15S to h7B 7-15.

FIG. 21 Tregs inhibitory 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 mouse monoclonal antibody and a FR region of a human antibody by adopting a CDR grafting technology, has good affinity, specificity and functionality, does not block IL-2/IL-2R combination, is an IL-2 non-blocking anti-CD 25 antibody, can inhibit Tregs highly expressed by CD25, and does not influence the acceptance of IL-2 signals by Teffs.

The original murine antibody of the anti-CD 25 antibody of the invention is obtained by screening by a hybridoma technology. Mixing the human CD25 protein with an adjuvant, immunizing a mouse, separating a mouse spleen cell after the serum titer is qualified, fusing the mouse spleen cell with a mouse myeloma cell in vitro, and culturing to obtain a cell culture supernatant containing the antibody. First, hybridoma antibodies having CD25 binding activity were screened by ELISA, antibodies with good binding kinetics were further screened by SPR (Biacore), and then IL-2/CD25 noncompetitive antibodies were screened by competitive ELISA. The cell binding activity of the hybridoma antibody is detected by adopting flow cytometry, and the IL-2 signal non-blocking type functional antibody is further screened by STAT5 phosphorylation and PBMC proliferation. To investigate the specificity of the antibody, it was ensured by ELISA and flow cytometry that no binding signal was present between the antibody and the CD25 negative cell component. The invention also detects the cross-reactivity of the antibody to human, monkey and mouse CD25 proteins, and finds that the antibodies B7 and H6 can better recognize monkey CD25, and the antibodies F10 and E5 have weaker binding action on monkey CD25. Furthermore, none of antibodies B7, H6, F10, E5 recognized murine CD25.

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

The obtained recombinant monoclonal antibody is verified in the aspects of binding kinetics, competitiveness, cell binding activity, STAT5 phosphorylation, specificity, species cross-reactivity and the like according to a detection method for screening the hybridoma, so that the activity of the recombinant monoclonal antibody in all aspects is consistent with that of the corresponding hybridoma antibody. In order to detect whether the antibody has an inhibiting effect on Tregs, human peripheral blood PBMCs are separated, anti-CD 25 recombinant monoclonal antibodies are added for incubation, and flow cytometry is adopted to confirm that anti-CD 25 antibodies F10-2 (from F10) and 7B7 (from B7) can specifically reduce Tregs in the PBMCs, and the influence on Teffs is small. In order to investigate the in vivo anti-tumor effect of the antibody, a mouse transplantation tumor model is established by adopting a CD25 humanized mouse, IL-2 blocking type and non-blocking type anti-CD 25 antibodies are given to the mouse, and the non-blocking type antibodies F10-2 and 7B7 can be confirmed to effectively inhibit tumor growth and reduce Tregs infiltration in tumor tissues; the IL-2 blocking antibody basiliximab cannot inhibit the growth of the tumor and cannot reduce Tregs infiltration in the tumor tissue, so that the rationality of the antibody screening thought and the treatment concept is verified.

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

Recombinant plasmids containing the 20 humanized antibody genes are constructed and transfected into HEK293 cells for transient expression. Cell culture supernatants were collected for SPR binding kinetics screening. Among them, 5 h7B7 humanized antibodies, including h7B7-6, h7B7-7, h7B7-14, h7B7-15, and h7B7-19, showed good affinity, indicating that the CDR regions of antibody 7B7 have good transplantability.

In the humanization of the antibody F10-2, no binding activity was detected with the 20 humanized antibodies after CDR-grafting, and thus the present invention continued with back mutation (back mutation). Through analyzing the structural characteristics of the antibody variable region and multiple back mutation screens, it is found that R79 in the antibody light chain variable region FR3 is very critical for maintaining the antibody affinity. The humanized antibody may show good affinity retaining murine residue R79 in VL-FR 3.

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

The invention continuously confirms the specific inhibition effect of the h7B7 humanized antibody on Tregs. Separating PBMC from human peripheral blood, adding an anti-CD 25 antibody for incubation, and confirming that the antibodies h7B7-6, h7B7-7, h7B7-14, h7B7-15 and h7B7-19 can specifically reduce Tregs in the PBMC by adopting flow cytometry, but have little influence on Teffs.

The invention further adopts a UNcle multi-parameter high-throughput protein stability analysis system to detect the melting temperature (Tm) and 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 superior.

To reduce the risk of antibody heterogeneity, the present invention further mutates the NYS glycosylation motif in the humanized antibody h7B7 heavy chain variable region CDR 2. SPR (surface plasmon resonance) binding kinetic analysis shows that the antibody (named h7B 7-15S) obtained after N is mutated into S can still keep better binding kinetic characteristics, cell binding activity, tregs inhibition function and thermal stability, and is basically consistent with the 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), scFv (single-chain antibody) and the like by a conventional genetic recombination technique. Antibody fragments such as Fab, scFv and the like have small volume and strong tissue permeability, and have unique advantages in some application fields. Fab is a heterodimer consisting of heavy chain variable region-constant region 1 (VH-CH 1) and light chain variable region-constant region (VL-CL) and has a molecular size of 1/3 of that of IgG molecules. Due to the absence of the Fc segment, fab-induced immune effects were significantly reduced compared to IgG and cytokine release was weaker. Currently, antibody drugs with Fab as the structure, such as abciximab, ranibizumab and the like, have been approved to be on the market. The scFv is formed by fusing VH and VL and a connecting peptide linker between the VH and VL, has the molecular size of only 1/6 of that of IgG, has the characteristics of strong tissue permeability, short half-life and the like, has unique advantages in the fields of imaging diagnosis and some treatment, and a bispecific antibody blinatumomab based on the scFv is also approved to be marketed. The antibody fragment can be further fused with other proteins or coupled with other small molecules, and can be used for diagnosis and treatment of diseases through targeted delivery.

The anti-CD 25 antibody of the present invention can be mutated for amino acids in the CDR regions by genetic engineering techniques to further improve affinity. The CDR regions of antibodies play a key role in the binding of antibodies to antigens, where amino acids can interact with the amino acids of the antigen through hydrogen bonds, ionic bonds, 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 a mutant antibody is carried out, and the antibody affinity evolution is realized in vitro.

The antibody of the invention can be expressed by stable cell lines, so that the antibody can be used for large-scale production of a large amount of protein. The gene coding the amino acid of the antibody can be obtained by the conventional gene recombination technology, and can be inserted into an expression vector after DNA sequence optimization, synthesis and PCR amplification. The vector used may be a plasmid, virus or gene fragment commonly used in 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 out of cells. The vector sequence contains elements such as a promoter for gene expression, protein translation initiation and termination signals, and poly A (PolyA). The vectors contain antibiotic resistance genes and replication elements to facilitate replication of the vectors in host cells, such as bacteria, for use in vector preparation. In addition, the vector may also contain selective gene to facilitate the selection of stably transfected host cell for constructing stably expressed cell strain.

After the vector containing the antibody-encoding DNA sequence is constructed, the vector can be used to transfect or transform a host cell to express the corresponding protein. There are many expression systems that can be used to express antibodies, including eukaryotic cells and prokaryotic cells, including mammalian cells, insect cells, yeast, bacteria, and the like. Mammalian cells are the preferred system for expressing the protein, since prokaryotic cells readily form inclusion bodies when expressing 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., and are included among the cells that can be used in the present invention. The recombinant vector containing the gene encoding the antibody 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 expression using stably transfected host cells containing the selectable gene. For example, after stably transfecting host cells lacking Neomycin resistance with a recombinant vector containing a Neomycin (Neomycin) resistance gene, the concentration of Neomycin may be increased in a cell culture solution to select a stable cell line with high expression; alternatively, for example, after stably transfecting host cells lacking DHFR with a recombinant vector containing the dihydrofolate reductase (DHFR) gene, the cell culture broth can be increased in the concentration of Methotrexate (MTX) to select for stable cell lines with high expression.

Expression systems other than mammalian cells, such as insect cells, yeast, bacteria, etc., may also be used to express the antibodies or fragments thereof of the present invention, which are also encompassed by the list of host cells that can be used with the present invention. These expression systems express proteins in higher amounts than mammalian cells in some cases, but are likely to form inclusion bodies, and therefore further protein renaturation is required.

The antibodies of the present invention may also be carried and expressed using viral vectors, including, but not limited to, adenoviral vectors (adenovirus vectors), adeno-associated viral vectors (adeno-associated viral vectors), retroviral vectors (retroviral vectors), herpes simplex viral vectors (herpes simplex viral-based vectors), lentiviral vectors (lentivirus 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 between the anti-CD 25 antibodies of the invention and CD25 negative cellular components from a variety of different tissue sources as analyzed by ELISA and flow cytometry. The anti-CD 25 antibody of the present invention can also recognize human and monkey CD25 proteins, which is useful for pharmacokinetic studies and safety evaluation in primates such as cynomolgus monkeys.

Human PBMC and NK-92 cell models show that the preferred anti-CD 25 antibodies of the invention do not block IL-2 induced cellular STAT5 phosphorylation and are IL-2 non-blocking antibodies. The in vitro PBMC model shows that the IL-2 non-blocking anti-CD 25 antibodies of the invention can specifically reduce Tregs in PBMCs with little effect 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 has no obvious toxic or side effect. The IL-2 non-blocking anti-CD 25 antibody does not block an IL-2 downstream signal channel, can specifically inhibit Tregs without affecting Teffs, achieves the anti-tumor effect by enhancing tumor immunity, and shows good application potential in the aspect of cancer treatment.

The antibody of the invention can be prepared into various forms of pharmaceutical preparations according to the conventional pharmaceutical technology, and liquid injections and freeze-dried injections are preferred.

The antibodies of the invention may be combined with other drugs to form pharmaceutical compositions that may be used to treat diseases in conjunction 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 in accordance with the present invention. The content and use of the invention is not limited to the scope of the embodiments.

Example 1 immunization of mice

A female BALB/c mouse which is 7-8 weeks old and has the weight of about 20g is taken as an immune host, and antigen immunity is carried out after adaptive feeding for one week. The expression-purified CD25-His (C-terminal fusion 6 XHis tag of human CD25 ectodomain) was prepared to 1mg/mL with PBS (pH 7.4), and after filtration through a 0.22 μm filter, 50 μ L was mixed well with 50 μ L of immunoadjuvant (QuickAntibody), and injected into the calf muscle of the hind leg of the mouse. On day 21, the immunization was boosted 1 time in the same manner, and the tail vein blood was collected on day 35 to determine the serum antibody titer. CD25-hIgG1Fc (Fc fragment of human IgG1 fused to C-terminal of human CD25 ectodomain) was coated onto an ELISA plate (50 ng/well), and the mouse serum antibody titer was determined by ELISA. Mice with an antibody titer greater than 30000 were given an antigen challenge once and splenocyte fusion was performed 3 days later.

Example 2 spleen cell fusion

After euthanasia of mice, spleens were isolated under sterile conditions and prepared using a 70 μm mesh screenSpleen cell suspensions and washed 2 times with basal medium for cell counting. SP2/0 was mixed with splenocytes at a ratio of 1:3, centrifuged, and the supernatant discarded, 1mL of PEG preheated at 37 ℃ was added dropwise over 1min, left to stand at 37 ℃ for 90s, followed by addition of 20mL of basal medium preheated at 37 ℃ over 6 min. Cells were harvested by centrifugation (RT, 800rpm, 3min), and 20mL HAT medium pre-warmed at 37 ℃ was added to resuspend the cells, following a 1X 10 protocol ⁵ And (3) adding the fused cells into a 96-hole cell culture plate according to the density of each splenocyte/hole, placing the 96-hole cell culture plate in a carbon dioxide cell culture box for culture, and taking culture supernatant for ELISA detection when the cell confluency reaches more than 70%.

Example 3 affinity ELISA screening

A CD25-hIgG1Fc solution was prepared at a concentration of 1. Mu.g/mL using PBS, and an ELISA plate (50. Mu.L/well) was added and coated overnight at 4 ℃. PBST washing plate 3 times, adding 5% BSA blocking solution (200 u L/hole), at 37 degrees C were incubated for 2h. The plates were washed 3 times with PBST, hybridoma cell culture supernatant (50. Mu.L/well) was added, and incubated at 37 ℃ for 1h. The plates were washed 3 times with PBST, and 1. The plates were washed 3 times with PBST, ready-to-use TMB color developing solution (100. Mu.L/well) was added, and incubated at 37 ℃ for 10min in the absence of light. 2M H was added ₂ SO ₄ The color development was stopped (100. Mu.L/well) and the OD value was measured at 450 nm. OD value of mouse IgG (1. Mu.g/mL) control well<0.3, taking the positive well (OD value)>2.0 Culture supernatants of corresponding hybridomas were screened for neutrality.

Example 4 SPR screening

The appropriate amount of coupling was calculated according to the formula RL = (Rmax. Times. MWligand d)/(Sm. Times. MWanalyte), and the anti-mouse antibody was coupled to the CM5 chip using the amine coupling kit. Hybridoma cell culture supernatants were captured onto chips and the response to CD25-His flow through the channel was detected using Biacore 8K. Data fitting was performed by Evaluation Software to obtain binding curves and kinetic parameters. Preferably, the hybridoma cells with better binding kinetics are subcloned.

Example 5 hybridoma subcloning

The hybridoma cells were subcloned by limiting dilution. Collecting hybridoma cells secreting neutralizing antibody, counting, diluting the hybridoma cells with complete culture medium, adding the hybridoma cells into a 96-well cell culture plate at the cell density of 0.5 per well, continuously culturing, and preserving the seeds of the remaining cells after expanding culture. After 10 days of subclone culture, culture supernatants from the single cloning wells were subjected to affinity ELISA and SPR verification, respectively, and competitive ELISA screening was performed. Taking the positive clone, 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

The enzyme plate (25 ng/well) was coated with CD25-hIgG1Fc overnight at 4 ℃. PBST plates were washed 3 times, 5% BSA blocking solution (200. Mu.L/well) was added, and incubated at 37 ℃ for 2h. The plates were washed 3 times with PBST, hybridoma cell culture supernatant (50. Mu.L/well), isotype control mIgG (2. Mu.g/ml, 50. Mu.L/well), and the competitive antibody basiliximab (2. Mu.g/ml, 50. Mu.L/well) were added and incubated at 37 ℃ for 1h. PBST washing plate 3 times, adding PBS preparation concentration of 0.2 u g/mL IL-2-His solution (50 u L/hole), at 37 degrees C were incubated for 1h. PBST washing plate 3 times, adding 5% BSA diluted 10000 times of anti-His-HRP (50 u L/hole), at 37 degrees C were incubated for 1h. The plates were washed 3 times with PBST, ready-to-use TMB color developing solution (100. Mu.L/well) was added, and incubated at 37 ℃ for 10min in the absence of light. 2M H was added ₂ SO ₄ The color development was stopped (100. Mu.L/well) and the OD value was measured at 450 nm. Compared with the competitive antibody basiliximab control with weaker color development, the antibody with stronger color development signal is the non-competitive hybridoma antibody.

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

CD 25-positive SU-DHL-1 cells were collected at 1X 10 cells per group ⁶ And (4) respectively. 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 incubation was complete, cells were harvested by centrifugation, washed with 500 μ L PBS and repeated 2 times. To the cell pellet, 100 μ L of Alexa Fluor 488-labeled goat anti-mouse IgG (H + L) (1 dilution 200) was added, resuspended, mixed well, and incubated on ice for 40min in the absence of light. After incubation was complete, cells were harvested by centrifugation, washed with 500 μ L PBS and repeated 2 times. Finally using 300 muL PBS resuspended cells and analyzed by flow cytometry.

Example 8 purification of hybridoma antibodies

And (3) carrying out expanded culture on the hybridoma cells, collecting culture supernatant, centrifuging at 4000rpm for 10min, and taking the supernatant for later use. The antibody in the culture supernatant of hybridoma cells was purified using an anti-mouse IgG affinity filler. After the antibody bound to the filler was eluted 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 antibody in the sample was determined by Nanodrop and the purity of the antibody was further checked by SDS-PAGE. The purified antibody was filtered through a 0.22 μm filter and stored in portions at-20 ℃ until use.

Example 9 cellular STAT5 phosphorylation Screen

NK-92 cells were added to 6-well plates (1X 10) ⁶ One/well, 2 mL/well), incubated overnight. Different anti-CD 25 antibodies or isotypic antibodies (10. Mu.g/mL) were added and incubated at 37 ℃ for 30min. Except for the blank control group, rhIL-2 was not added, and rhIL-2 was added to each of the other groups (final concentration: 10 IU/mL) and stimulated at 37 ℃ for 10min. A pre-cooled RIPA lysate containing protease inhibitors and phosphatase inhibitors is prepared and placed on ice for future use. After the rhIL-2 stimulation is finished, collecting each group of cells, adding a freshly prepared RIPA lysate, adopting an ultrasonic cell disrupter to disrupt the cells, and placing on ice for lysis for 30min. After completion of lysis, the cells were centrifuged (13000 rpm) at 4 ℃ for 20min, and the supernatant was collected and assayed for protein concentration by BCA method. Each group was subjected to Western-blot detection using an equivalent amount of 50. Mu.g of total protein, an anti-pSTAT 5 antibody and an anti- β -actin antibody as primary antibodies, and an HRP-labeled antibody as a secondary antibody, and a signal was detected by chemiluminescence. In addition, PBMCs from human peripheral blood were isolated using lymphocyte separation tubes, and STAT5 phosphorylation was examined for each PBMC group using the method described above. Combining the results of the two models of NK-92 and PBMC, hybridoma antibodies E5, B7, A6, F10, F8 had minimal effect on rhIL-2-induced STAT5 phosphorylation and were the preferred IL-2 non-blocking anti-CD 25 antibody (see FIG. 1). In contrast, hybridoma antibody H6 significantly inhibited STAT5 phosphorylation (see fig. 1), an IL-2 blocking type anti-CD 25 antibody, and was used as a control for subsequent related studies.

Example 10 PBMC cell proliferation screening

PBMC of human peripheral blood were isolated using lymphocyte separation tubes and added to 6-well plates (1X 10) ⁴ One/well, 100. Mu.L/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 different concentrations (0.05. Mu.g/mL, 0.5. Mu.g/mL, 5. Mu.g/mL, 50. Mu.g/mL) and incubated at 37 ℃ for 72 hours. Cell proliferation was detected using CellTiter-Glo Luminescent Cell Viability Assay kit. The results show that hybridoma antibodies E5, B7, F10, at each concentration, had minimal inhibitory effect on human PBMC proliferation, and are the preferred IL-2 non-blocking anti-CD 25 antibody (see figure 2). In contrast, the IL-2 blocking anti-CD 25 antibody H6 significantly inhibited PBMC proliferation (see FIG. 2).

Example 11 specificity screening

Culturing and collecting various cell lines, taking different anti-CD 25 antibodies as primary antibodies, taking Alexa Fluor 488-labeled goat anti-mouse IgG (H + L) as secondary antibodies, and detecting the binding condition of the antibodies and cell surface proteins by adopting Flow Cytometry (FCM). The results showed that the antibodies E5, B7, F10, H6 showed binding signals to CD 25-positive human diffuse tissue lymphoma cell SU-DHL-1 and human histiocytic lymphoma cell U937, but not to CD 25-negative human embryonic kidney cell 293T, human hepatoma cell HepG2, human gastric carcinoma cell MGC-803, human pancreatic carcinoma cell PANC-1, human colorectal carcinoma epithelial cell DLD-1 (see Table 1). On the other hand, various cell lines were cultured and collected, and cell lysis samples were prepared using RIPA lysate and an ultrasonic cell disruptor, coated onto an elisa plate (500 ng/well), and coated with CD25-hIgG1Fc 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 adopted to detect the combination condition of the antibodies and cell lysis components. The results showed that none of the antibodies E5, B7, F10, H6 showed binding signals to CD25 negative 293T, hepG, MGC-803, PANC-1, DLD-1 cells, but to coated CD25-hIgG1Fc (see Table 1).

TABLE 1 anti-CD 25 hybridoma antibody specificity detection

Note: "●" represents binding, "-" represents no binding, "/" represents no detection.

Example 12 species Cross-reactivity assay

Mixing human CD25 (homemade human CD 25-His), monkey CD25 (Yi Qiao Shenzhou), and mouse CD25 (R)&Company D) were coated onto an enzyme plate (50 ng/well) and incubated overnight at 4 ℃. PBST washing plate 3 times, adding 5% BSA blocking solution (200 u L/hole), at 37 degrees C were incubated for 2h. The plates were washed 3 times with PBST, hybridoma cell culture supernatant (50. Mu.L/well) was added, and incubated at 37 ℃ for 1h. The plates were washed 3 times with PBST, and 1. The plates were washed 3 times with PBST, ready-to-use TMB color developing solution (100. Mu.L/well) was added, and incubated at 37 ℃ for 10min in the absence of light. 2M H was added ₂ SO ₄ The color development was stopped (100. Mu.L/well) and the OD was measured at 450 nm. The results show that antibodies B7, H6 recognize monkey CD25 well, with binding similar to human CD25, whereas antibodies F10, E5 bind weakly to monkey CD25 (see figure 3). Furthermore, none of the antibodies B7, H6, F10, E5 recognized murine CD25 (see fig. 3).

Example 13 antibody subtype identification

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

Example 14 antibody competitive ELISA assay

Competition curves for anti-CD 25 antibodies were generated using a competition ELISA assay. CD25-hIgG1Fc coated enzyme plates (25 ng/well) were added with 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) to detect competition of the anti-CD 25 antibodies for IL-2-His (0.2. Mu.g/mL, 50. Mu.L/well). Inhibition was calculated for ELISA data and competition curves were plotted using GraphPad Prism8 software. The results show that the antibodies E5, B7, F10 are less competitive than the competitive antibody basiliximab, and belong to the non-competitive antibody (see fig. 4).

Example 15 determination of antibody binding kinetics

The appropriate coupling amount was calculated according to the formula RL = (Rmax. Times. MWligand)/(Sm. Times. MWanalyte), and the anti-mouse antibody was coupled to the CM5 chip using amine coupling kit. Different anti-CD 25 antibodies were captured onto the chip and the response values of different concentrations of CD25-His flowed 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 obtaining antibody variable region sequences

Collecting the hybridoma cell subclones, and extracting RNA by using a Trizol method. And carrying out reverse transcription by taking the extracted RNA as a template to obtain cDNA. The heavy and light chain variable regions of the antibody were PCR amplified using degenerate primers (Novagen Ig-Primer Sets), respectively, and the PCR amplification products were detected by agarose gel electrophoresis. And obtaining a target DNA fragment by adopting a gel recovery kit, and then carrying out TA cloning to construct a recombinant plasmid. And transforming the recombinant plasmid into competent cells by adopting a heat shock method, and coating a plate for blue-white screening. And (3) picking a white single colony to 0.5mL of LB liquid culture medium, performing shake culture for 3 hours at 37 ℃ and 220rpm, and sampling and sequencing a bacterial liquid. The amino acid sequences of the variable regions of the anti-CD 25 antibodies 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 segments and the light chain variable region gene segments with signal peptides and mouse heavy chain (IgG 2 a) and light chain (kappa) constant region gene segments respectively by adopting overlapping PCR (polymerase chain reaction), and sequencing and identifying. And respectively inserting the correctly spliced heavy chain and light chain genes of the antibody into pTT5 plasmid, transfecting the recombinant plasmid to HEK293 cells by adopting a PEI method, performing serum-free suspension culture, and transiently expressing the antibody. Cell supernatants from 7 days of culture were collected, filtered through 0.22 μm filters, and the antibodies were purified by protein G affinity chromatography. And (3) performing ultrafiltration replacement on the antibody to a PBS solution, identifying the purity and concentration of the antibody by reducing SDS-PAGE and NanoDrop 2000, subpackaging, and storing at-80 ℃ for later use. Preparation of human murine chimeric antibodies (IgG 1. Kappa. Type) was carried out in a similar manner as described above.

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

According to the detection method of hybridoma screening, the binding kinetics (see figure 6), the competition (see figure 7), the cell binding activity (see figure 8), the cell STAT5 phosphorylation (see figure 9), the specificity, the species cross reactivity (see figure 10) and the like of the recombinant monoclonal antibodies F10-2 and 7B7 are respectively verified. The results show that the activity of antibodies F10-2 and 7B7 is essentially identical to that of 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 for 3 days by the addition of anti-CD 3 antibody (1. Mu.g/mL) and anti-CD 28 antibody (1. Mu.g/mL). PBMCs with good growth conditions were collected and added to 6-well plates (2X 10) ⁶ One/well), cultured overnight. 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 the mixture was incubated overnight. Cells were harvested by centrifugation, the Fc receptor blocked by adding Human BD Fc Block, and dead cells were distinguished with dead and live cell Stain (Fixable visualization Stain), and then flow cytometric analysis was performed by labeling the cells with Hu CD45 APC-Cy7, hu CD3 FITC, hu CD4 PE-Cy7, hu CD8 PerCP-Cy5.5, hu CD25PE, hu Foxp3 AF647, respectively. The results showed that IL-2 non-blocking anti-CD 25 antibodies F10-2 and 7B7 specifically reduced Tregs in PBMCs compared to IL-2 blocking anti-CD 25 antibody basiliximab, with little effect on Teffs (see FIG. 11).

Example 20 anti-tumor Effect of antibodies F10-2 and basiliximab in mice

Mouse colon cancer cell MC38 (5X 10) ⁵ One/one) of 6-8 weeks old CD25 (IL 2 RA) humanized mice (C57 BL/6-Il2 RA) ^{tm1(hIL2RA)Smoc} South mochi). When the tumor volume reaches 100-150mm ³ In time, mice were performedDivided into groups (6/group) and administered in tail vein (10 mg/kg) on days 0, 3, 6, 9, 12, and 14, respectively. The antibodies F10-2 and basiliximab used were of the mouse IgG2a kappa type (mIgG 2a kappa) with mouse IgG (mIgG) as control. Tumor volume was measured every 3 days (calculated as V =1/2 × major axis × minor axis) ² ) And the weight and status of the mice 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 the IL-2 non-blocking anti-CD 25 antibody F10-2 was able to significantly inhibit tumor growth (see FIG. 12).

Example 21 anti-tumor Effect of antibodies F10-2 and 7B7 in mice

Mouse colon cancer cell MC38 (5X 10) ⁵ One/one) of 6-8 weeks old CD25 (IL 2 RA) humanized mice (C57 BL/6-Il2 RA) ^{tm1(hIL2RA)Smoc} South mochi). When the tumor volume reaches 100-150mm ³ Mice were divided into groups (6 mice/group) and administered to the tail vein on days 0, 3, 6, 10, and 13 (10 mg/kg), respectively. The antibodies F10-2 and 7B7 used were of the mouse IgG2a kappa type (mIgG 2a kappa) with mouse IgG (mIgG) as control. Tumor volume was measured every 3 days (calculated as V =1/2 × major axis × minor axis) ² ) And the weight and status of the mice were monitored. The results showed that both antibodies 7B7 and F10-2 were effective in inhibiting tumor growth, and that the tumor-inhibiting effects were 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, separating and shearing tumor tissues, adding a DMEM medium containing 1mg/mL IV type collagenase to digest the tumor tissues, filtering by a 70-micron screen to obtain cell suspension, and further adopting Histopaque separating medium to obtain immune cells. After blocking the Mouse Fc Block, single and multiple staining with PBS, fixable visualization Stain 575V, ms CD45 APC-Cy 7-F11, ms CD3 BB 700-2C 11, 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 is unable to reduce Treg infiltration in tumor tissues. However, IL-2 non-blocking anti-CD 25 antibodies F10-2 and 7B7 can significantly reduce Treg infiltration in tumor tissues and both effects are similar (see fig. 14).

Example 23 in vivo safety Studies of antibodies F10-2 and 7B7 in mice

Throughout the course of treatment in the mouse antitumor test, the mice were behaviorally and normally active, survived well and showed no significant change in body weight (see FIG. 15). After the experiment, 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 staining with hematoxylin and eosin (H & E), the tissue sections were observed under a microscope. The results showed that no significant lesions were observed in the tissues of each of the important organs after the treatment with antibodies F10-2 and 7B7 (see FIG. 16).

Example 24 humanization of antibody 7B7

The variable region of antibody 7B7 was homologously modeled based on antibody structure in the PDB database and CDR regions were determined based on amino acid primary sequence features and variable region spatial conformation. The variable region of the antibody is compared with the amino acid sequences coded by human antibody germline genes V and J, and according to the factors of consistency, similarity, conservation and the like of the FR of the framework region, 5V genes and 1J gene in an IGHV library are preferably grafted and spliced with the CDR of the heavy chain to form 5 different humanized heavy chain variable regions. Similarly, it is preferable that 4V genes and 1J gene in the IGKV library are graft-spliced with the light chain CDRs to constitute 4 different humanized light chain variable regions. The 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, VL-CDR3 of humanized antibody h7B7 are shown in Table 4. The humanized antibodies h7B7-6, h7B7-7, h7B7-14, h7B7-15, h7B7-19 have superior binding kinetics by Biacore binding kinetics analysis (see FIG. 17), with h7B7-15 being more superior (see Table 5). The amino acid sequence of the h7B7-15 heavy chain variable region is shown in SEQ ID NO.13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 14.

TABLE 3 genes of human antibody germline for use with h7B7 humanized antibodies

		Light chain 1	Light chain 2	Light chain 3	Light chain 4
							Human being germ li ne Form panel	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 h7B7 humanized antibody complementarity determining region CDR amino acid sequence

CDR region	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 according to the method for humanizing antibody 7B7. 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, VL-CDR3 of humanized antibody hF10-2 are shown in Table 6. After CDR grafting, since no binding activity was detected by 20 humanized antibodies, back mutation (back mutation) was continued. Antibody structures from homology modeling were analyzed, and key amino acid residues that directly interact with CDRs at the VH/VL binding interface and support CDRs were selected as canonical residues (canonical residues), and humanized antibody mutants were constructed by back-mutating some of the canonical residues. Further analysis of the antibody variable region structure revealed that VH-CDR3 is shorter in length and directed to the conformationally flattened VL-CDR2, while the charged long side chain adjacent to R79 in VL-FR3 is more prominent, suggesting that R79 may be involved in antigen binding. R79 in VL-FR3 was confirmed to be critical for maintaining antibody affinity by constructing antibody mutants and performing Biacore assays (see Table 7). The humanized antibody retained the murine residue R79 in the FR3 light chain variable region and showed good affinity (see Table 8), with hF10-2-4 being preferred (see Table 8), hF10-2-4 heavy chain variable region amino acid sequence shown in SEQ ID NO.15 and light chain variable region amino acid sequence shown in SEQ ID NO. 16.

TABLE 6 hF10-2 humanized antibody CDR amino acid sequences

CDR region	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 germline genes and amino acid sequences for hF10-2 humanized antibody

Note: dark residues are the difference residues between the two heavy chains and between the 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: "/" indicates not measured.

Example 26 Effect of humanized antibodies on cellular STAT5 phosphorylation

Human peripheral blood PBMC were added to 6-well plates (1X 10) ⁶ One/well, 2 mL/well), incubated overnight. Different anti-CD 25 antibodies or isotypes of antibodies (10. Mu.g/mL) were added and incubated at 37 ℃ for 30min. Except for the blank control group without rhIL-2, other groups were added with rhIL-2 (final concentration is 10 IU/mL) respectively, and pricked at 37 deg.CActivate for 10min. And (3) cracking the cells, taking 50 mu g of total protein in each group in an equivalent amount, carrying out Western-blot, and detecting STAT5 phosphorylation conditions. The results show that none of the humanized antibodies h7B7-6, h7B7-7, h7B7-14, h7B7-15, h7B7-19 was able to effectively block rhIL-2 induced PBMC STAT5 phosphorylation compared to the IL-2 blocking anti-CD 25 antibody basiliximab, with the blocking effect of h7B7-15 being the weakest (see FIG. 18).

Example 27 in vitro Tregs inhibition assay for humanized antibodies

PBMCs from human peripheral blood were isolated using lymphocyte separation tubes and activated for 3 days by the addition of anti-CD 3 antibody (1. Mu.g/mL) and anti-CD 28 antibody (1. Mu.g/mL). PBMC with good growth status were collected and added to 6-well plates (2X 10) ⁶ One/well), cultured overnight. mu.g/mL of human IgG isotype antibody, basiliximab, and h7B7 humanized antibody were added, and the mixture was incubated overnight. Cells were collected by centrifugation, the Fc receptor was blocked by adding Human BD Fc Block, dead cells were differentiated by adding dead and live cell dye (Fixable viatility Stain), and the cells were labeled with Hu CD45 APC-Cy7, hu CD3 FITC, hu CD4 PE-Cy7, hu CD8 PerCP-Cy5.5, hu CD25PE, and Hu Foxp3 AF647, respectively, for analysis by flow cytometry. The results showed that the humanized antibodies h7B7-6, h7B7-7, h7B7-14, h7B7-15, h7B7-19 specifically reduced Tregs in PBMCs compared to the IL-2 blocking anti-CD 25 antibody basiliximab (see FIG. 19).

Example 28 analysis of thermal stability of humanized antibody

The melting temperature (Tm) and aggregation onset temperature (Tagg) of the antibody were determined using a UNcle multiparameter high throughput protein stability assay system. The buffer used was PBS (pH 7.4) and the concentration of the antibody sample was 1mg/mL. The Tm value is determined by reflecting the conformational change of the protein by the change in fluorescence of the endogenous aromatic amino acids of the protein. And detecting aggregates with different sizes by adopting two wavelengths of 266nm and 473nm, monitoring the aggregation condition of the protein in the temperature rise process, and determining the Tagg value. The UNcle anlysis software analysis showed that the thermal stability of 5 h7B7 humanized antibodies was not very 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	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 the NYS glycosylation motif, in an attempt to reduce the potential heterogeneity of the antibody molecule by mutating the glycosylation site N therein. It was found by residue analysis and antibody germline gene alignment that N in NYS glycosylation motif is generated by somatic high frequency mutation (SHM), so Q (side chain similarity high), a (no side chain), S (mouse germline gene residue corresponding to this site), and Y (human germline gene residue corresponding to this site) were selected respectively to replace N in NYS, and 4 mutants were constructed respectively for Biacore binding kinetics screening (see table 10). The results show that the antibody (h 7B7-15S, shown in SEQ ID NO.17 as the heavy chain variable region sequence) retains better affinity after the S substitution (N53S).

TABLE 10 binding kinetics of h7B7 mutants

Antibodies	ka(1/Ms)	kd(1/s)	KD(M)	Mutations
					h7B7-15	1.53E+05	3.56E-04	2.33E-09	Not mutated
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: "/" indicates no detected affinity.

Example 30 detection of antibody Activity and thermostability after glycosylation site mutation

And performing functional verification and thermal stability analysis on the antibody h7B7-15S after glycosylation site mutation. The results showed that the antibody h7B7-15S was substantially identical to h7B7-15 before glycosylation mutation in terms of cell binding activity (see FIG. 20), tregs inhibitory function (see FIG. 21), thermostability (see Table 11), and the like.

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

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

Sequence listing

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

GYSITSDYAWN

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

YINYSGSTSYNPSLKS

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

KGGFFDV

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

SASQGISNYLN

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

YTSSLHS

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

LQYSKLPWT

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

GFSLTSYGVH

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

VIWRGGSTDYNAAFMS

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

NERFYGFDY

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

RSSKSLLHSNGITYLY

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

QMSNLAS

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

AQNLELPT

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

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

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

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> Sichuan university

<120> CD 25-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 (11)

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 in 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 in 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 in 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 in 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 a humanized antibody framework region FR, the light chain variable region VL is formed by splicing the VL-CDR1, the VL-CDR2 and the VL-CDR3 with the humanized antibody framework region FR, wherein the amino acid sequences of the heavy chain variable region VH and the light chain variable region VL of the antibody are respectively shown in SEQ ID NO.13 and SEQ ID NO. 14.

4. 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 a humanized antibody framework region FR, the light chain variable region VL is formed by splicing the VL-CDR1, the VL-CDR2 and the VL-CDR3 with the humanized antibody framework region FR, wherein the amino acid sequences of the heavy chain variable region VH and the light chain variable region VL of the antibody are respectively shown in SEQ ID NO.15 and SEQ ID NO. 16.

5. The anti-CD 25 antibody or fragment thereof according to claim 3, wherein: n in a glycosylation motif NYS in the antibody heavy chain variable region VH-CDR2 can be mutated into Q, A or S, wherein S is a preferred mutation, and the amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO. 17.

6. anti-CD 25 antibody or fragment thereof according to any one of claims 1 to 5, characterized in that the antibody fragment can be a Fab or scFv.

7. The anti-CD 25 antibody or fragment thereof according to any one of claims 1 to 6, wherein the heavy chain constant region is from the constant region of a human immunoglobulin IgG1, igG2, igG3, igG4, igM, igE, igA or IgD heavy chain and the light chain constant region is from the constant region of a human immunoglobulin kappa or lambda light chain.

8. A nucleic acid molecule encoding the anti-CD 25 antibody or fragment thereof of claims 1-7.

9. A recombinant vector comprising the nucleic acid molecule of claim 8.

10. A cell comprising the recombinant vector of claim 9.

11. Use of the anti-CD 25 antibody or fragment thereof according to any one of claims 1 to 7 for the preparation of an anti-tumor medicament.

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