Patent Description:
Regulatory T cells (Treg) play a vital role in mediating immune homeostasis, and can promote the establishment and maintenance of peripheral tolerance. However, their role becomes more complex in the context of cancer. Because cancer cells express their own tumor-associated antigens, the presence of Treg that suppresses the response of effector cells can promote tumor progression. Therefore, the infiltration of Treg in established tumors is one of major obstacles for efficacy. Treg that uses inhibitory mechanisms is believed to make a significant contribution to the limitations and even failures of current therapies, especially immunotherapies that rely on inducing or enhancing anti-tumor responses (<NPL>). The tumor infiltration of Treg is also associated with several human cancers with poor prognosis (<NPL>). It has been proven that Treg cells contribute to the establishment and progression of tumors in mouse models and their absence leads to a delay in tumor progression. In humans, a high proportion of the infiltration of tumor Treg cells, more importantly, a lower ratio of effector T cells (Teff) to Treg cells, is associated with the poor prognosis of various human cancers (Shang et al.

CD25 is one of the potential molecular targets to achieve Treg depletion. CD25 is also known as interleukin-<NUM> high affinity receptor alpha chain (IL-2Rα). CD25 is expressed at high levels on Treg, but CD25 is not present or expressed at low levels on Teff.

In the prior art, there are antibodies that bind to CD25 but do not block the binding of IL2 to CD25, such as MA251 (<NPL>, <NPL>), but they still exist defects such as insufficient CD25 binding activity, inhibition of PBMC activation, and general pharmacokinetic performance.

In the face of the patient's demand for medicines for disease treatment, especially demands for antibody drugs, there is still an urgent clinical need to provide an anti-CD25 antibody with higher binding activity.

<CIT> relates to Isolated human monoclonal antibodies which bind to and inhibit human CD25, and related antibody-based compositions and molecules, are disclosed. The human antibodies can be produced by a hybridoma, a transfectoma or in a nonhuman transgenic animal, e.g., a transgenic mouse, capable of producing multiple isotypes of human monoclonal antibodies by undergoing V-D-J recombination and isotype switching. Also disclosed are pharmaceutical compositions comprising the human antibodies, nonhuman transgenic animals, hybridomas and transfectomas which produce the human antibodies, and therapeutic and diagnostic methods for using the human antibodies.

<CIT> discloses Monoclonal antibodies to the CD25 antigen are characterized by the amino acid sequence of their hypervariable regions. Initially produced in murine form, they may be converted to chimeric or humanized forms, immunoconjugates or antibody fragments (generally described as binding molecules). The products are useful for the prophylaxis or treatment of transplant rejection, paticularly in combination with other antibodies to activated T-cells, for example CD7 antibodies.

<CIT> disclosure relates to use of an anti-CD25 antibody, not inhibiting IL-<NUM> - CD25 interaction, with enhanced binding to activating Fc gamma Rs that lead to effective depletion of tumor-infiltrating Treg cells and improved control of established tumors. Combination with anti-programmed cell death protein-<NUM> antibodies further improves tumor rejection.

The invention is defined by the claims and any other aspects, configurations, instances or embodiments set forth herein not falling within the scope of the claims are for information only.

Any references in the description to methods of treatment (or diagnosis) refer to the compounds, pharmaceutical compositions and medicaments of the present invention for use in a method of treatment of the human (or animal) body by therapy (or for diagnosis).

In the full text of the present invention, but not encompassed by the wording of the claims, various embodiments regarding VL (light chain variable region), VH (heavy chain variable region), LCDR (light chain complementarity determining region), HCDR (heavy chain complementarity determining region), LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 may be implemented individually or in any combination.

An aspect, not encompassed by the wording of the claims, relates to an antibody or antigen-binding fragment thereof including three heavy chain complementarity determining regions, wherein the HCDR1 amino acid sequence is represented by SEQ ID NO: <NUM>, the HCDR2 amino acid sequence is represented by SEQ ID NO: <NUM>, and the HCDR3 amino acid sequence is represented by SEQ ID NO: <NUM>. Further, the antibody or antigen-binding fragment thereof further includes three light chain complementarity determining regions, wherein the LCDR1 amino acid sequence is represented by SEQ ID NO: <NUM>, the LCDR2 amino acid sequence is represented by SEQ ID NO: <NUM>, and the LCDR3 amino acid sequence is represented by SEQ ID NO: <NUM>.

In an aspect of the present invention, the antibody provided in the present invention includes a heavy chain variable region represented by SEQ ID NO: <NUM>; further includes a light chain variable region represented by SEQ ID NO: <NUM>.

An aspect, not encompassed by the wording of the claims, relates to an antibody or antigen-binding fragment thereof including three light chain complementarity determining regions, wherein the LCDR1 amino acid sequence is represented by SEQ ID NO: <NUM>, the LCDR2 amino acid sequence is represented by SEQ ID NO: <NUM>, and the LCDR3 amino acid sequence is represented by SEQ ID NO: <NUM>; and/or three heavy chain complementarity determining regions, wherein the HCDR1 amino acid sequence is represented by SEQ ID NO: <NUM>, the HCDR2 amino acid sequence is represented by SEQ ID NO: <NUM>, and the HCDR3 amino acid sequence is represented by SEQ ID NO: <NUM>.

An aspect, not encompassed by the wording of the claims, relates to an antibody or antigen-binding fragment thereof including the light chain variable region of the amino acid sequence represented by SEQ ID NO: <NUM>, and/or the heavy chain variable region of the amino acid sequence represented by SEQ ID NO: <NUM>.

According to an aspect of the present invention, the sequence of the light chain constant region of the antibody or antigen-binding fragment thereof of anyone of the preceding aspects is SEQ ID NO: <NUM>.

According to an aspect of the present invention, the sequence of the heavy chain constant region of the antibody or antigen-binding fragment thereof of any one of the preceding aspects is SEQ ID NO: <NUM>.

Specifically, the antibody provided in the present invention includes the light chain variable region of the amino acid sequence represented by SEQ ID NO: <NUM>, the heavy chain variable region of the amino acid sequence represented by SEQ ID NO: <NUM>, the light chain constant region of the amino acid sequence represented by SEQ ID NO: <NUM> and the heavy chain constant region of the amino acid sequence represented by SEQ ID NO: <NUM>.

Specifically, the antibody or antigen-binding fragment thereof provided,not encompassed by the wording of the claims, preferably includes the light chain variable region of the amino acid sequence represented by SEQ ID NO: <NUM>, the heavy chain variable region of the amino acid sequence represented by SEQ ID NO: <NUM>, the light chain constant region of the amino acid sequence represented by SEQ ID NO: <NUM> and the heavy chain constant region of the amino acid sequence represented by SEQ ID NO: <NUM>.

According to an aspect of the present invention, the antibody thereof of the present invention binds to CD25, preferably to human CD25.

An aspect, not encompassed by the wording of the claims, relates to the antibody or antigen-binding fragment thereof of any one of the preceding aspects including a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a Fab fragment, a Fab' fragment, a F(ab')<NUM> fragment, a Fv fragment, a scFv fragment, a dsFv fragment or the like.

According to an aspect of the present invention, the present invention relates to a nucleic acid encoding the antibody of any one of the preceding aspects.

An aspect, not encompassed by the wording of the claims, relates to a vector including the nucleic acid of the preceding aspect, or the vector can express the antibody of anyone of the preceding aspects. Preferably, the vector may be a viral vector; preferably, the viral vector includes but is not limited to a lentiviral vector, an adenovirus vector, an adeno-associated viral vector, a retroviral vector, or the like; preferably, the vector may be a non-viral vector; preferably, the vector may be a mammalian cell expression vector; preferably, the expression vector may be a bacterial expression vector; and preferably, the expression vector may be a fungal expression vector.

According to an aspect of the present invention, the present invention relates to a cell that can express a cell of the antibody of any one of the preceding aspects. Preferably the cell is a bacterial cell; preferably the bacterial cell is an E. coli cell and the like; preferably the cell is a fungal cell; preferably the fungal cell is a yeast cell; preferably the yeast cell is a Pichia pastoris cell and the like; preferably the cell is a mammalian cell; preferably the mammalian cell is a Chinese hamster ovary cell (CHO), a human embryonic kidney cell (<NUM>), a B cell, a T cell, a DC cell, a NK cell, or the like.

According to an aspect of the present invention, the present invention relates to a pharmaceutical composition including the antibody, the nucleic acid, or the cell of anyone of the preceding aspects, preferably the pharmaceutical composition further includes a pharmaceutically acceptable excipient, and the pharmaceutically acceptable vector preferably includes one or more of the following: a solvent, a dispersant, an additive, a plasticizer and the like which are pharmaceutically acceptable.

In some embodiments, ,which are not part of the invention, the pharmaceutical composition may further include other therapeutic agents. The other therapeutic agents include chemotherapeutic agents, immunotherapeutic agents, or hormonal therapeutic agents. The combined administration of the antibody or antigen-binding fragment thereof and the other therapeutic agents can enhance the therapeutic effect of the therapeutic agents.

In some embodiments, which are not part of the invention, the "enhance the therapeutic effect" refers to enhancing the therapeutic effect of other therapeutic agents or therapies. The antibody or antigen-binding fragment can be administered individually or in combination with other therapeutic agents or therapies. The other therapeutic agents or therapies include chemotherapeutic agents, immunotherapeutic agents, hormonal therapeutic agents, radiation therapy and surgery.

According to an aspect of the present invention, there is provided a kit including the antibody of the present invention, or including a nucleic acid encoding the antibody.

According to an aspect of the present invention, the present invention relates to an application of the antibody, the nucleic acid, of any one of the preceding aspects in preparation of medicaments for treatment or prophylaxis of diseases.

An aspect, not encompassed by the wording of the claims, relates to an application of the antibody or the nucleic acid of any one of the preceding aspects in preparation of diagnostic or detection kits.

In an aspect of the present invention, there is provided a method of treating or preventing diseases including administering the antibody, the nucleic acid, of the present invention to subjects in need.

In an aspect of the present invention, there is provided a method of diagnosis or detection including administering the antibody, the nucleic acid of the present invention to subjects or samples in need. Preferably, the method is a method of diagnosing or detecting diseases.

According to an aspect of the present invention, the present invention relates to use of the antibody, the nucleic acid, of any one of the preceding aspects for the treatment or prophylaxis of diseases.

According to an aspect of the present invention, the present invention relates to use of the antibody, the nucleic acid, of any one of the preceding aspects for detection or diagnosis. Preferably, the use is to diagnose or detect diseases.

According to an aspect of the present invention, the disease is a cancer.

According to an aspect of the present invention, the cancer includes gastric cancer, esophageal cancer, head-and-neck cancer, bladder cancer, cervical cancer, sarcoma, cytoma, lung cancer, colon cancer, ovarian cancer, renal cancer, colorectal cancer, pancreatic cancer, liver cancer, melanoma, breast cancer, myeloma, glioma, leukemia, lymphoma and the like.

An aspect, not encompassed by the wording of the claims, relates to a method for preparing the antibody of any one of the preceding aspects, which includes transfecting cells with the above vector, and expressing the antibody by the transfected cells; or includes expressing the antibody with the above cell.

An aspect, not encompassed by the wording of the claims, relates to the following advantages: enhanced CD25 protein binding activity, enhanced CD25 protein affinity, enhanced CD25 expressing cell killing ability, reduced PBMC activation inhibition, enhanced in vivo tumor growth inhibition ability, enhanced in vivo tumor killing ability, enhanced ability to reduce the number of Treg cells, or enhanced ability to increase the number of effector T cells.

In order to more clearly explain the present invention or the technical solutions in the prior art, the drawings or the description of the prior art will be briefly described below. Obviously, those of ordinary skill in the art can obtain other drawings based on these drawings without the exercise of inventive faculty.

The technical solutions of the present invention will be described clearly and completely below in connection with the drawings, and obviously, the described embodiments are part of the embodiments of the present invention, but not all the embodiments.

The technical features involved in the different embodiments described throughout the full text of the present invention can be implemented in combination with each other.

The CD25 (Sino Biological, catalog NO. <NUM>-H08H) is emulsified with Freund's adjuvant to immunize fully human antibody transgenic mouse BoAn-hMab1 of Boan bio (prepared according to the method described in Chinese Patent <CIT>). The first immunization uses Freund's complete adjuvant (Sigma, catalog number: F5881-<NUM>), the second immunization to the sixth immunization use Freund's incomplete adjuvant (Sigma, catalog number: F5506-<NUM>), total of <NUM> mice were immunized at this time. <NUM> mice with higher serum titers were selected for booster immunization, and the mice were sacrificed <NUM> days later to remove the spleen for subsequent experiments. The serum titers (<NUM>-fold dilution) of the mice are shown in <FIG>.

RNA is extracted from the spleen cells of the immunized mice in <NUM>, and then reverse-transcribed into cDNA, the steps for establishing the phage library are performed referring to the method described in Carlos F. Barbas III, Phage display: A laboratory manual, the variable regions of the heavy and light chains are obtained from the cDNA by PCR method, and then scFv is obtained by overlapping extension PCR of the variable regions of the heavy chain and the light chain, scFv is ligated with plasmid pCOMB3x after digesting, and then the ligation product is electrotransfected into E. coli TG1 competent cells, add the phage to infect TG1 after being incubated, and the supernatant of the culture concentrated is the phage library of the present invention.

The screened phage clones express scFv, and detect the binding of scFv and CD25, and detect the blocking of scFv on IL2/CD25 binding, and select scFv that binds well to CD25 and does not block CD25 for subsequent construction.

ELISA detection of binding of scFv and CD25: Preparation of CBS buffer: <NUM> of Na<NUM>CO<NUM> (Sinopharm, <NUM>) and <NUM> of NaHCO<NUM> are weighed, and the distilled water was added to <NUM> to prepare CBS buffer. The CD25 (<NUM>-H08H, Sino Biological) protein was diluted to <NUM>µg/mL with pH <NUM> CBS, coated with enzyme-labeled plate, <NUM>µL/well, and incubated overnight at <NUM>; <NUM>% defatted milk powder was used for sealing at <NUM> for <NUM> after washing the plate; <NUM>µL of PBST (PBS + <NUM>%Tween20) is added after washing the plate, and then <NUM>µL of scFv periplasm was added to incubate at <NUM> for <NUM>. An anti-flag secondary antibody (Proteintech, catalog number: HRP-<NUM>) was added after washing the plate to incubate at <NUM> for <NUM>. <NUM>µL of TMB (Makewonder, catalog NO. <NUM>) substrate was added to each well for color development after washing the plate, <NUM>µL of <NUM> H<NUM>SO<NUM> was added to each well to stop the color development after <NUM> mins, and OD450 was read with a microplate reader.

ELISA detection of blocking of scFv on CD25/IL2 binding: the CD25 (<NUM>-H08H, Sino Biological) protein was diluted to <NUM>µg/mL with pH <NUM> CBS, and coated with enzyme-labeled plate, <NUM>µL/well, and incubated overnight at <NUM>; <NUM>% defatted milk powder was used for sealing at <NUM> for <NUM> after washing the plate. <NUM>µL of scFv periplasm was added to each well after washing the plate. Then, biotin-labeled IL2 protein (final concentration is <NUM>µg/mL) was added, <NUM>µL/well, and incubated at <NUM> for <NUM>; STREP/HRP diluted with PBST was added after washing the plate, <NUM>µL/well, and incubated at <NUM> for <NUM>. <NUM>µL of TMB is added to each well for color development after washing the plate, and <NUM>µL of <NUM> H<NUM>SO<NUM> was added to each well to stop the color development after <NUM> mins, OD450 was read with a microplate reader.

Magnetic bead screened clones CD25Q2-BA3\BA9\BA125, CD25Q8-BT942, CD25Q11-BA402\BA406\BA410\BA415\BA422\BA428 and CD25Q14-BA443\BA448\BA458 and plate screened clones CD25Q11-CA35\CA36\CT848 and CD25Q14-CA705\CA707\CA721 were sent to Invitrogen Biotechnology Ltd for sequencing. The amino acid sequences of the light chain variable region and the heavy chain variable region of each clone are set forth in Table <NUM>.

The nucleotide sequence fragment encoding VH was finally inserted into the vector pCDNA3. <NUM> (Life Technology) with the nucleotide sequence encoding the heavy chain constant region amino acid sequence SEQ ID NO: <NUM> of the antibody, the nucleotide sequence fragment encoding VL was inserted into the vector pCDNA3. <NUM> (Life Technology) with the nucleotide sequence encoding the light chain constant region amino acid sequence (SEQ ID NO: <NUM>) of the antibody, through variable region gene amplification (<NUM>*Phanta Max Master Mix, manufacturer: Vazyme, Item No.: P515-AA, Lot No.: TE211GB), signal peptide and variable region overlap extension, homologous recombination (ClonExpress II One Step Cloning Kit, manufacturer: Vazyme, Item No.: C112-<NUM>, Lot No.: TE211L8) and the like, performed by conventional molecular biology techniques. The linked vector is transfected into HEK293 cells and incubated in <NUM>\<NUM>% CO2\<NUM> rpm shaker, and after transiently expressing <NUM>-7days, the supernatant was purified by Protein A affinity chromatography to obtain anti-CD25 antibody, and the antibody concentration was determined by UV280 binding extinction coefficient.

Production of control antibody: MA251 antibody is an anti-human CD25 antibody that does not block the binding of IL2 and CD25 in the prior art, has a high affinity for human CD25, and has a good performance of not blocking the binding of IL2 and CD25. The MA251 antibody is a classic antibody studying the binding of IL2 and CD25. The nucleotide sequence encoding the variable region of the MA251 antibody is synthesized by the complete gene and then inserted into the vector pCDNA3. <NUM> and expressed by HEK293 cells, and the produced antibody is named CD25-MA251-IgG1 (the sequence of the heavy chain variable region is SEQ ID NO: <NUM>, the sequence of the light chain variable region is SEQ ID NO: <NUM>, the sequence of the light chain constant region is SEQ ID NO: <NUM>, and the sequence of the heavy chain constant region is SEQ ID NO: <NUM>).

The CD25 protein (<NUM>-H08H, Sino Biological) was diluted to different concentrations (<NUM>µg/mL,<NUM>µg/mL,<NUM>µg/mL,<NUM>µg/mL,<NUM>µg/mL,<NUM>µg/mL) with CBS, was coated with enzyme-labeled plate, <NUM>µL/well, and incubated overnight at <NUM>; <NUM>% defatted milk powder was used for sealing at <NUM> for <NUM> after washing the plate; <NUM>µL of candidate antibody that was diluted to <NUM>µL/mL with PBST (PBS+<NUM>% Tween20) was added to each well, and incubated at <NUM> for <NUM>; then the goat anti-human IgG/HRP (KPL, catalog number: <NUM>-<NUM>) was added and incubated at <NUM> for <NUM>, and after color developing for <NUM>, OD450 was read on a microplate reader to obtain EC<NUM> by calculating. The results are shown in <FIG> and Table <NUM> to Table <NUM>.

As shown in Table <NUM>, a EC<NUM> value of the binding of the candidate antibody CD25Q2-BA9-IgG1 and antigen CD25 is <NUM>, which is significantly lower than the EC<NUM> value of the control group CD25-MA251-IgG1 that is <NUM>, which indicates that the antigen binding capacity of the candidate antibody is significantly better than that of the control group CD25-MA251-IgG1.

It is predicted that the candidate antibodies CD25Q2-BA9-IgG1 and CD25Q8-BT942-IgG1 have a stronger targeting and binding effect on the Treg cells expressing CD25, have better killing effect, reduce inhibition of the Treg cells on the Teff cells, and have better pharmaceutical effects as compared with the control group CD25-MA251-IgG1.

FBS (Gibco, catalog number: <NUM>-<NUM>) and RPMI-<NUM> (Gibco, catalog number: <NUM>-<NUM>) were mixed according to <NUM>:<NUM> to prepare <NUM>% FBS RPMI-<NUM>, SU-DHL-<NUM> target cells were collected, and was diluted to <NUM>×<NUM><NUM> cells/mL by using <NUM>% FBS RPMI-<NUM>, appropriate candidate antibody was taken and diluted to 25µg/mL by using <NUM>% FBS RPMI-<NUM>, this concentration was used as an initial concentration; diluted <NUM> times in gradient in sequence to a total of <NUM> points for future use; effector cells Jurkat (G7011, Promega) were collected, and diluted to <NUM>×<NUM><NUM> cells/mL by using <NUM>% FBS RPMI-<NUM>, target cells were added in the white <NUM>-well plate with 25µL/well; the antibody diluted in gradient was added in the wells covered with target cells, with 25µL/well; effector cells Jurkat were added with 25µL/well, and the <NUM>-well plate was placed into the cell incubator to culture <NUM>; and the <NUM>-well plate was removed and placed at room temperature to enable the temperature thereof to equilibrate to room temperature; Bio-Gl chromogenic solution (G7940, Promega) was added with 75µL/well, reacting for <NUM> mins, Luminescense was read from a Tecan microplate reader to obtain a value. The results are shown in <FIG> and Table <NUM> to Table <NUM>.

As shown in Table <NUM>, the EC<NUM> value of simulated killing activity detection of the candidate antibody CD25Q2-BA9-IgG1 is <NUM>, which is lower than the EC<NUM> value of the control group CD25-MA251-IgG1 that is <NUM>, which indicates that the killing ability of the candidate antibody to SU-DHL-<NUM> is better than that of the control group CD25-MA251-IgG <NUM>.

The above results indicate that the candidate antibody CD25Q2-BA9-IgG1 has a good killing effect on cells expressing CD25, which predicts that the candidate antibody can reduce the Treg cells expressing CD25 and their inhibition on Teff cells, thereby having better pharmaceutical effects.

Antibody binding kinetics uses BIAcore8K instrument based on surface plasmon resonance (SRP) technology to measure. Anti-human IgG antibody amino was coupled to a CM5 biosensor chip by the GE anti Human IgG Fc amino coupling kit (GE, cat # BR-<NUM>-<NUM>) to obtain approximately <NUM> response units (RU). For kinetic measurements, the CD25 protein (Sino Biological, <NUM>-H08H) was diluted <NUM>-fold continuously with HBS-EP + <NUM> × (GE, BR-<NUM>-<NUM>) buffer, starting at <NUM>, being diluted <NUM>-fold for <NUM> concentration gradients and setting <NUM> concentration. The antibody to be detected: <NUM>µg/ml, sample injection time <NUM>, flow rate <NUM>µL/min, stable for <NUM>; CD25 protein: binding for <NUM>, flow rate <NUM>µL/min, dissociation for <NUM>; regeneration: regeneration was performed for <NUM> with <NUM> MgCl<NUM> buffer, Startup <NUM> times. The association constant (ka) and dissociation constant (kd) were calculated using a simple one-to-one Languir binding model (BIAcore Evaluation Software version <NUM>), and the equilibrium dissociation constant (KD) was calculated by the ratio kd/ka. The affinity data of each antibody is shown in Table <NUM>.

As shown in Table <NUM>, the equilibrium dissociation constant KD value of the candidate antibody CD25Q2-BA9-IgG1 is <NUM>. 43E-<NUM>, which is lower than the KD value of the control group CD25-MA251-IgG1 that is <NUM>. 19E-<NUM>, which indicates that the affinity of CD25 protein of the candidate antibody CD25Q2-BA9-IgG1 is better than that of the control group CD25-MA251-IgG1.

As shown in Table <NUM>, the equilibrium dissociation constant KD value of the candidate antibody CD25Q8-BT942-IgG1 is <NUM>. 79E-<NUM>, which is lower than the KD value of the control group CD25-MA251-IgG1 that is <NUM>. 19E-<NUM>, which indicates that the affinity of CD25 protein of the candidate antibody CD25Q8-BT942-IgG1 is better than that of the control group CD25-MA251-IgG1.

The above results predict that the candidate antibodies CD25Q2-BA9-IgG1 and CD25Q8-BT942-IgG1 have a stronger targeting and binding effect on the Treg cells expressing CD25, have better killing effect, reduce inhibition of the Treg cells on the Teff cells, and have better pharmaceutical effects as compared with the control group CD25-MA251-IgGl.

The frozen PBMC (peripheral blood mononuclear cells, manufacturer: ALLCELLS, item no. : PB003F-C) were recovered and then co-cultured with 10µg/mL of CD25 antibody on the <NUM>-U bottom plate for <NUM> mins, no antibody was added to the control group, the control group was labeled as NoAb, and then IL2 (<NUM>. 1U/mL, 1U/mL, 10U/mL) was added to incubate for <NUM> mins (working medium: <NUM>+<NUM>% FBS, containing <NUM> L-glutamine and 10000U/mL Pen-Strep), to prepare cell suspension: after the last washing, the supernatant was discarded and the sample was vortexed on pulse to completely dissociate the pellet; <NUM>µL Foxp3 fixation/permeabilization working solution was added to each well. It was incubated at <NUM>-<NUM> or room temperature in the dark for <NUM>-<NUM> minutes; the sample was centrifuged at <NUM>-<NUM> for <NUM> minutes at room temperature, and the supernatant was discarded; <NUM>µL of 1X membrane breaking solution was added to each well, the sample was centrifuged at <NUM>-<NUM> for <NUM> minutes at room temperature, the supernatant was discarded, and is washed twice; (BD Phosflow™ Perm Buffer III is preliminarily put at -<NUM> to pre-cool) washed with PBS once, centrifuged and the supernatant being discarded; ice-cold Phosflow™ Perm Buffer III was added slowly while being vortexed, and incubated on ice for <NUM> minutes; the cells were washed twice with PBS, centrifuged at <NUM> for <NUM> minutes to discard the supernatant; the cells were resuspend in PBS to <NUM><NUM> cells/mL, contained separately in <NUM>µL/well, the antibody staining fluorescently labeled was continued and the flow cytometry was performed; living cells were distinguished, and the CD3 positive T cells were further distinguished. The higher the percentage of phosphorylated signal transducers and transcription activator <NUM> (PSTAT5), the lower the blocking rate. The results are shown in <FIG> and Table <NUM>.

As shown in Table <NUM>, the %PSTAT5 value of the candidate antibody CD25Q2-BA9-IgG1 is <NUM>, which is significantly higher than the %PSTAT5 value of the control group CD25-MA251-IgG1 that is <NUM>, which indicates that the candidate antibody is significantly better than the control group CD25-MA251-IgG1 in not blocking the binding of IL2 and PBMC. It indicates that the candidate antibody CD25Q2-BA9-IgG1 can inhibit the activation of PBMC less than the control group CD25-MA251-IgG1, which predicts that the candidate antibody CD25Q2-BA9-IgG1 can better achieve the PBMC immune effect and have better pharmaceutical effect/anti-tumor effect.

As shown in Table <NUM>, the %PSTAT5 value of the candidate antibody CD25Q8-BT942-IgG1 is <NUM>, and the %PSTAT5 value of CD25-MA251-IgG1 is <NUM>, which indicates that the candidate antibody basically equivalent to the control group CD25- MA251-IgG1 in not blocking the binding of IL2 and PBMC. It predicts that the candidate antibody CD25Q8-BT942-IgG1 can well achieve the PBMC immune effect and have good pharmaceutical effect/anti-tumor effect.

CD25Q2-BA9-IgG1 antibody was administered intravenously to <NUM> rhesus monkeys at a dose of <NUM>/kg, flow cytometer (CytomicsTM FC500) was used to detect the content of the Treg cells (CD3+CD4+CD25+FoxP3+) at different time points before and after the administration according to flow cytometry, and the detection time points were: (±<NUM> minute) before and after the administration, <NUM> hours (±<NUM> minute), <NUM> hours (±<NUM> minutes), <NUM> hours (±<NUM> minutes), <NUM> hours (±<NUM> minutes), <NUM> hours (±<NUM> minutes, <NUM> days), <NUM> hours (±<NUM> minutes, <NUM> days), <NUM> hours (±<NUM> hour, <NUM> days), <NUM> hours (±<NUM> hour, <NUM> days) after the administration, the experimental results are shown in <FIG>.

As can be seen from <FIG>, after administration of CD25Q2-BA9-IgG1, it can significantly reduce the content of Treg cells in rhesus monkeys and can effectively regulate the immune microenvironment.

There are two cynomolgus monkeys in each group, they were intravenously injected with different CD25 antibodies, and whole blood samples were taken out intravenously at (<NUM>) before the administration and <NUM>, <NUM>, <NUM>, <NUM>, 1d, 2d, 4d, 7d, 10d, and 14d after the administration, put in a blood sample collection tube, and coagulated naturally in the ice box, the blood sample was put in the centrifuge within <NUM> after being taken out, centrifuged at <NUM>~<NUM> for <NUM> mins, the serum was separated and put in the sample storage tube, and the cynomolgus monkeys were intravenously injected with different CD25 antibodies again on the 14th day, the whole blood samples were taken out intravenously at (<NUM>) before the administration and <NUM> (14d+<NUM>), <NUM> (14d+<NUM>), <NUM> (14d+<NUM>), <NUM> (14d+<NUM>), 1d (15d), 2d(16d), 4d(18d) and 7d(21d) after the administration, put in a blood sample collection tube, and coagulated naturally in the ice box, the blood sample were put in the centrifuge within <NUM> after being taken out, centrifuged at <NUM>-<NUM> for <NUM> mins, the serum was separated and put in the sample storage tube, the metabolism of antibodies in the cynomolgus monkeys was detected by ELISA, and the results are shown in <FIG> and Table <NUM>.

The results indicate: CD25Q2-BA9-IgG1 and CD25Q8-BT942-IgG1 have a higher bioavailability than that of the control antibody CD25-MA251-IgG1, and have good pharmacokinetic performance.

B-hIL2Rα humanized mice (Biocytogen) were divided into <NUM> groups according to body weight, wherein <NUM> mice was in the G1 negative control group and <NUM> mice were in each of the G2-G4 treatment groups. Individual administration was performed from the day of grouping (<NUM>/kg, I. , BIW) (<NUM>/kg, intraperitoneal injection, twice a week), the next day, the MC38 cells resuspended in PBS was inoculated subcutaneously on the right side of B-hIL2Rα humanized mice at a concentration of <NUM>×<NUM><NUM> cells/<NUM> with <NUM>/mouse. The tumor volume and animal body weight were measured twice per week, and the measurement values were recorded, tumor volume (mm<NUM>) = <NUM> × long diameter × short diameter<NUM>. The results are shown in <FIG> and Table <NUM> and Table <NUM>.

As shown in <FIG>, the body weight of mice increased steadily, which indicates that CD25Q2-BA9-IgG1, CD25Q8-BT942-IgG1 and CD25Q11-CT848-IgG1 (<NUM>/kg, IP, BIW) do not have toxic and side effects on the mice.

As shown in <FIG>, compared with the control group, CD25Q2-BA9-IgG1, CD25Q8-BT942-IgG1 and CD25Q11-CT848-IgG1 can significantly inhibit the growth of tumor of mouse MC38, of which CD25Q8-BT942-IgG1 shows a better anti-tumor effect.

After the 5th administration of the test mice (<NUM> days after grouping) in <NUM>, <NUM> mice were taken from the negative control group and <NUM> mice were taken from each treatment group, the mice were killed and the tumor was cut up, the digestive enzyme was added therein, incubated and digested for <NUM> minutes at <NUM>, and resuspend as a single cell suspension after filtering and washing. <NUM>µL of sealing and death and life dye solution and <NUM>µL of cell suspension were added to each well of a <NUM>-well round bottom plate, mixed well and incubated in the dark for <NUM> mins at <NUM>; <NUM>µL of surface dye was added to each well, mixed well and incubated in the dark for <NUM> mins at <NUM>; <NUM>µL of FACS solution was added to each well and washed twice, <NUM>, <NUM>, centrifuged for <NUM> mins, and the supernatant was discarded; <NUM>µL of the fixed solution resuspended cells were added to each well, fixed for <NUM> mins at room temperature after being mixed well; after fixation, centrifuged at <NUM>, <NUM> for <NUM> mins, and the supernatant was discarded; <NUM>µL of membrane-penetrating solution was added to each well, centrifuged at <NUM>, <NUM> for <NUM> mins, and the supernatant was discarded; <NUM>µL of intracellular dye solution was added to each well, room temperature, incubated in the dark for <NUM> mins; <NUM>µL of membrane-penetrating solution was added to each well and washed twice, <NUM>, centrifuged for <NUM> mins, and the supernatant was discarded; the cells were resuspend with <NUM>µL of PBS, and the content of CD8+cells (Teff) in CD3, the content of CD25+Foxp3+cells (Treg) in CD3 and the content of Foxp3+cells (Treg) in CD4 were detected on machine. The results are shown in <FIG> to DIG.

Claim 1:
An antibody that binds to CD25, comprising a heavy chain variable region, a heavy chain constant region, a light chain variable region, and a light chain constant region, wherein the heavy chain variable region amino acid sequence is represented by SEQ ID NO: <NUM>, the heavy chain constant region amino acid sequence is represented by SEQ ID NO: <NUM>, the light chain variable region is represented by SEQ ID NO: <NUM>, and the light chain constant region is represented by SEQ ID NO: <NUM>.