Anti-CD25 antibody and use thereof

Abstract

Provided are an anti-CD25 antibody and the use thereof. The provided anti-CD25 antibody or an antigen-binding fragment thereof can specifically bind to CD25, retains IL-2 signaling on Teff cells while eliminating Treg cells, and effectively increases the ratio of Teff / Treg cells, thereby significantly enhancing the efficacy of tumor immunotherapy.

Description

Anti-cd25 antibodies and uses thereof TECHNICAL FIELD

[0001] The present application relates to the field of immunology and oncology, in particular to anti-CD25 antibodies or antigen-binding fragments thereof and uses thereof. BACKGROUND

[0002] CD25 is the alpha chain of the IL-2 receptor, also known as interleukin-2 receptor alpha subunit (IL-2Ra), which is highly expressed in regulatory T cells (Treg cells) and is a marker of Tregs. Treg cells have a significant immunosuppressive effect, both to prevent the body from developing autoimmune diseases and to promote the immune escape of tumor cells, indirectly accelerating the proliferation of tumor cells. The depletion of Treg cells directly promotes tumor regression, and CD25 is a potential target for Treg cell depletion and can be used to develop new immunotherapy methods.

[0003] CD25 is significantly different in expression levels between Treg cells and effector T cells (Teff cells), and anti-CD25 antibodies can selectively deplete Treg cells while retaining IL-2 signaling on Teff cells, effectively increasing the Teff / Treg cell ratio, thereby significantly enhancing the efficacy of tumor immunotherapy.

[0004] CD25 is expressed at elevated levels on certain tumor cells. CD25 expression has been demonstrated on malignant cells in chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL) and acute myelogenous leukemia (AML), non-Hodgkin lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma and peripheral T-cell lymphoma, as well as angioimmunoblastic T-cell lymphoma and anaplastic large cell lymphoma.

[0005] An alternative marker of CD25 expression is the shed surface antigen, soluble CD25 (sCD25). The secretion of sCD25 in malignancies can have multiple biological effects. These can include its effect on binding circulating IL2, thereby prolonging the half-life and potential biological activity of IL2, contributing to the maintenance of an inflammatory state in cancer patients. High serum levels of soluble CD25 can help identify patients with poor prognosis in DLBCL, cutaneous lymphoma, follicular lymphoma, AML, ALL, Hodgkin lymphoma, and are used as a prognostic indicator in a subset of adult T-cell leukemia lymphoma (ATLL).

[0006] In addition, secretion of sCD25 can be an adaptive mechanism mediating tumor immune escape in hepatocellular carcinoma, melanoma or renal cell carcinoma (RCC). Plasma from melanoma or renal cell carcinoma (RCC) patients with elevated sCD25 concentrations can significantly reduce cell growth when included in an IL2-stimulatory assay. Free CD25 can also be detected in the microenvironment of solid tumors such as lung adenocarcinoma, esophageal cancer and head and neck cancer.

[0007] In addition, CD25 is also expressed in some autoimmune diseases, such as rheumatoid arthritis, scleroderma, uveitis, skin diseases (such as psoriasis and atopic dermatitis), etc. The expression of CD25 is also related to allograft rejection and graft-versus-host reaction. SUMMARY

[0008] The present application provides an antibody or antigen binding fragment that specifically binds to CD25, the provided antibody is an IL-2 non-blocking antibody, which can selectively deplete Treg cells while retaining IL-2 signaling on Teff cells, thereby allowing IL-2 to stimulate Teff cells, increase their killing ability, correct the tumor microenvironment, and enhance the efficacy of tumor immunotherapy.

[0009] In some embodiments, the antibody or antigen binding fragment comprises one or more of the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3:

[0010] HCDR1 comprises or consists of a sequence as set forth in SEQ ID NO: 1 or 2,

[0011] HCDR2 comprises or consists of a sequence as set forth in SEQ ID NO: 3 or 4,

[0012] HCDR3 comprises or consists of a sequence as set forth in any one of SEQ ID NOs: 5-8,

[0013] LCDR1 comprises or consists of a sequence as set forth in any one of SEQ ID NOs: 9-11,

[0014] LCDR2 comprises or consists of a sequence as set forth in SEQ ID NO: 12, and LCDR3 comprises or consists of a sequence as set forth in any one of SEQ ID NOs: 13-15.

[0015] In some embodiments, the antibody or antigen binding fragment comprises one or more of the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3:

[0016] HCDR1 comprises or consists of the sequence of SEQ ID NO: 1 or 2,

[0017] HCDR2 comprises or consists of the sequence of SEQ ID NO: 3 or 4,

[0018] HCDR3 comprises or consists of the sequence of any one of SEQ ID NOs: 5-8,

[0019] LCDR1 comprises or consists of the sequence of any one of SEQ ID NOs: 9-11,

[0020] LCDR2 comprises or consists of the sequence of SEQ ID NO: 12,

[0021] LCDR3 comprises or consists of the sequence of any one of SEQ ID NOs: 13-15;

[0022] provided that if the antibody or antigen binding fragment comprises a LCDR1 of the sequence of SEQ ID NO: 9, a LCDR2 of the sequence of SEQ ID NO: 12 and a LCDR3 of the sequence of SEQ ID NO: 13, or if the antibody or antigen binding fragment comprises a LCDR1 of the sequence of SEQ ID NO: 10, a LCDR2 of the sequence of SEQ ID NO: 12 and a LCDR3 of the sequence of SEQ ID NO: 14, then the antibody or antigen binding fragment further comprises one HCDR as described above.

[0023] In some embodiments, the antibody or antigen binding fragment comprises the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3:

[0024] HCDR1 comprises or consists of the sequence of SEQ ID NO: 1 or 2,

[0025] HCDR2 comprises or consists of the sequence of SEQ ID NO: 3 or 4,

[0026] HCDR3 comprises or consists of the sequence of any one of SEQ ID NOs: 5-8,

[0027] LCDR1 comprises or consists of the sequence of any one of SEQ ID NOs: 9-11,

[0028] LCDR2 comprises or consists of the sequence of SEQ ID NO: 12,

[0029] LCDR3 comprises or consists of the sequence set forth as any one of SEQ ID NOs: 13-15.

[0030] In some embodiments, the antibody or antigen-binding fragment comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2, and a LCDR3, wherein:

[0031] (1) the HCDR1 comprises or consists of the sequence set forth as SEQ ID NO: 1, the HCDR2 comprises or consists of the sequence set forth as SEQ ID NO: 3, the HCDR3 comprises or consists of the sequence set forth as SEQ ID NO: 5, the LCDR1 comprises or consists of the sequence set forth as SEQ ID NO: 9, the LCDR2 comprises or consists of the sequence set forth as SEQ ID NO: 12, and the LCDR3 comprises or consists of the sequence set forth as SEQ ID NO: 13; or

[0032] (2) the HCDR1 comprises or consists of the sequence set forth as SEQ ID NO: 1, the HCDR2 comprises or consists of the sequence set forth as SEQ ID NO: 3, the HCDR3 comprises or consists of the sequence set forth as SEQ ID NO: 6, the LCDR1 comprises or consists of the sequence set forth as SEQ ID NO: 9, the LCDR2 comprises or consists of the sequence set forth as SEQ ID NO: 12, and the LCDR3 comprises or consists of the sequence set forth as SEQ ID NO: 13; or

[0033] (3) the HCDR1 comprises or consists of the sequence set forth as SEQ ID NO: 2, the HCDR2 comprises or consists of the sequence set forth as SEQ ID NO: 4, the HCDR3 comprises or consists of the sequence set forth as SEQ ID NO: 7, the LCDR1 comprises or consists of the sequence set forth as SEQ ID NO: 10, the LCDR2 comprises or consists of the sequence set forth as SEQ ID NO: 12, and the LCDR3 comprises or consists of the sequence set forth as SEQ ID NO: 14; or

[0034] (4) the HCDR1 comprises or consists of the sequence set forth as SEQ ID NO: 2, the HCDR2 comprises or consists of the sequence set forth as SEQ ID NO: 4, the HCDR3 comprises or consists of the sequence set forth as SEQ ID NO: 8, the LCDR1 comprises or consists of the sequence set forth as SEQ ID NO: 11, the LCDR2 comprises or consists of the sequence set forth as SEQ ID NO: 12, and the LCDR3 comprises or consists of the sequence set forth as SEQ ID NO: 15.

[0035] The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 of the antibody or antigen binding fragment that specifically binds to CD25 according to the present application can each comprise the amino acid sequence of any one or more of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 listed in Table 1, or any combination of the corresponding amino acid sequences of the CDRs in Table 1.

[0036] Table 1 CDR sequences of anti-CD25 antibodies

[0037] In some embodiments, the antibody or antigen binding fragment comprises a heavy chain variable region comprising a sequence as set forth in any one of SEQ ID NOs: 16-19, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a sequence as set forth in any one of SEQ ID NOs: 16-19, or a sequence having one or more conservative amino acid substitutions compared to a sequence as set forth in any one of SEQ ID NOs: 16-19, or consisting of thereof; and / or

[0038] The antibody or antigen binding fragment comprises a light chain variable region comprising a sequence as set forth in any one of SEQ ID NOs: 20-22, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a sequence as set forth in any one of SEQ ID NOs: 20-22, or a sequence having one or more conservative amino acid substitutions compared to a sequence as set forth in any one of SEQ ID NOs: 20-22, or consisting of thereof.

[0039] In some embodiments, the antibody or antigen binding fragment comprises a heavy chain variable region comprising a sequence as set forth in any one of SEQ ID NOs: 16-19, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a sequence as set forth in any one of SEQ ID NOs: 16-19, or a sequence having one or more conservative amino acid substitutions compared to a sequence as set forth in any one of SEQ ID NOs: 16-19, or consisting of thereof; and / or

[0040] the antibody or antigen-binding fragment comprises a heavy chain variable region comprising a sequence as set forth in any one of SEQ ID NOs: 16-19, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence as set forth in any one of SEQ ID NOs: 16-19, or a sequence that has one or more conservative amino acid substitutions compared to a sequence as set forth in any one of SEQ ID NOs: 16-19, or consists of thereof;

[0041] provided that if the antibody or antigen-binding fragment comprises a light chain variable region as set forth in SEQ ID NO: 20 or SEQ ID NO: 21, then the antibody or antigen-binding fragment further comprises a heavy chain variable region as set forth in any one of SEQ ID NOs: 16-19.

[0042] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising or consisting of a sequence as set forth in any one of SEQ ID NOs: 16-19; and / or

[0043] the antibody or antigen-binding fragment comprises a light chain variable region comprising or consisting of a sequence as set forth in any one of SEQ ID NOs: 20-22.

[0044] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein:

[0045] (1) the heavy chain variable region comprises or consists of a sequence as set forth in SEQ ID NO: 16, and the light chain variable region comprises or consists of a sequence as set forth in SEQ ID NO: 20; or

[0046] (2) the heavy chain variable region comprises or consists of a sequence as set forth in SEQ ID NO: 17, and the light chain variable region comprises or consists of a sequence as set forth in SEQ ID NO: 20; or

[0047] (3) the heavy chain variable region comprises or consists of a sequence as set forth in SEQ ID NO: 18, and the light chain variable region comprises or consists of a sequence as set forth in SEQ ID NO: 21; or

[0048] (4) the heavy chain variable region comprises or consists of a sequence as set forth in SEQ ID NO: 19, and the light chain variable region comprises or consists of a sequence as set forth in SEQ ID NO: 22.

[0049] In some embodiments, the heavy chain variable region, the light chain variable region in the antibody or antigen binding fragment can be any combination of the amino acid sequences of the respective variable regions in Table 2.

[0050] Table 2 Variable region sequences of anti-CD25 antibodies

[0051] (underlined sequences are CDR sequences determined according to the Kabat numbering system)

[0052] In some embodiments, the antibody or antigen binding fragment further comprises a light chain constant region and / or a heavy chain constant region.

[0053] In some embodiments, the light chain constant region of the antibody or antigen binding fragment is a kappa or lambda chain constant region.

[0054] In some embodiments, the antibody or antigen binding fragment is of one of the isotypes IgG, IgM, IgA, IgE or IgD.

[0055] In some embodiments, the antibody or antigen binding fragment is of one of the isotypes IgG1, IgG2, IgG3 or IgG4.

[0056] In some embodiments, the antibody or antigen binding fragment is an IgG1 antibody.

[0057] In some embodiments, the antibody or antigen binding fragment comprises a heavy chain constant region comprising, consisting of or consisting essentially of a sequence as set forth in SEQ ID NO: 23, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence as set forth in SEQ ID NO: 23, or a sequence having one or more conservative amino acid substitutions compared to the sequence as set forth in SEQ ID NO: 23, and / or

[0058] the antibody or antigen binding fragment further comprises a light chain constant region comprising, consisting of or consisting essentially of a sequence as set forth in SEQ ID NO: 24, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence as set forth in SEQ ID NO: 24, or a sequence having one or more conservative amino acid substitutions compared to the sequence as set forth in SEQ ID NO: 24.

[0059] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region that consists of the sequence set forth in SEQ ID NO: 23 and a light chain constant region that consists of the sequence set forth in SEQ ID NO: 24.

[0060] Heavy chain constant region sequence of the anti-CD25 antibody (SEQ ID NO: 23):

[0061] Light chain constant region sequence of the anti-CD25 antibody (SEQ ID NO: 24):

[0062] In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody (including a full-length monoclonal antibody), a multispecific antibody or antigen-binding fragment (e.g., a bispecific antibody or antigen-binding fragment thereof).

[0063] In some embodiments, the antibody or antigen-binding fragment is selected from the group consisting of: a Fab, a Fab', a F(ab')2, a F(ab)2, a Fd, a Fv, a Fab / c, a scFv, a scFv multimer, a disulfide stabilized Fv, a (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a diabody, a disulfide stabilized diabody (ds-Diabody), a single domain antibody (sdAb), a nanobody, a domain antibody (dAb), or a bivalent domain antibody.

[0064] In some embodiments, the antibody has two identical heavy chains and two identical light chains.

[0065] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising, consisting of, or consisting essentially of, a sequence set forth in any one of SEQ ID NOs: 25-28, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a sequence set forth in any one of SEQ ID NOs: 25-28, or a sequence having one or more conservative amino acid substitutions compared to a sequence set forth in any one of SEQ ID NOs: 25-28; and / or

[0066] the antibody or antigen binding fragment comprises a heavy chain comprising a sequence as set forth in any one of SEQ ID NOs: 25-28, a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence as set forth in any one of SEQ ID NOs: 25-28, or a sequence that has one or more conservative amino acid substitutions compared to a sequence as set forth in any one of SEQ ID NOs: 25-28, or consists of thereof; and / or

[0067] In some embodiments, the antibody or antigen binding fragment comprises a heavy chain comprising a sequence as set forth in any one of SEQ ID NOs: 25-28, a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence as set forth in any one of SEQ ID NOs: 25-28, or a sequence that has one or more conservative amino acid substitutions compared to a sequence as set forth in any one of SEQ ID NOs: 25-28, or consists of thereof; and / or

[0068] the antibody or antigen binding fragment comprises a heavy chain comprising a sequence as set forth in any one of SEQ ID NOs: 25-28, a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence as set forth in any one of SEQ ID NOs: 25-28, or a sequence that has one or more conservative amino acid substitutions compared to a sequence as set forth in any one of SEQ ID NOs: 25-28, or consists of thereof; and / or

[0069] provided that if the antibody or antigen binding fragment comprises a heavy chain comprising a sequence as set forth in any one of SEQ ID NOs: 25-28, then the antibody or antigen binding fragment further comprises a light chain comprising a sequence as set forth in any one of SEQ ID NOs: 29-31.

[0070] In some embodiments, the antibody or antigen binding fragment comprises a heavy chain comprising a sequence as set forth in any one of SEQ ID NOs: 25-28, or consists of thereof; and / or

[0071] The antibody or antigen-binding fragment comprises a heavy chain comprising or consisting of the sequence set forth in any one of SEQ ID NOs: 25-28, and a light chain comprising or consisting of the sequence set forth in any one of SEQ ID NOs: 29-31.

[0072] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain and a light chain, wherein:

[0073] (1) the heavy chain comprises or consists of the sequence set forth in SEQ ID NO: 25 and the light chain comprises or consists of the sequence set forth in SEQ ID NO: 29; or

[0074] (2) the heavy chain comprises or consists of the sequence set forth in SEQ ID NO: 26 and the light chain comprises or consists of the sequence set forth in SEQ ID NO: 29; or

[0075] (3) the heavy chain comprises or consists of the sequence set forth in SEQ ID NO: 27 and the light chain comprises or consists of the sequence set forth in SEQ ID NO: 30; or

[0076] (4) the heavy chain comprises or consists of the sequence set forth in SEQ ID NO: 28 and the light chain comprises or consists of the sequence set forth in SEQ ID NO: 31.

[0077] Table 3. Heavy and light chain amino acid sequences of anti-CD25 antibodies

[0078] (underlined sequences are CDR sequences determined according to the Kabat numbering system)

[0079] In some embodiments, the antibody has two identical heavy chains and two identical light chains.

[0080] In some embodiments, the antibody is selected from the group consisting of antibodies 5A2, 3C4, 2A11, and R5-15.

[0081] In some embodiments, the antibody or antigen-binding fragment is a human antibody, a humanized antibody, or a chimeric antibody.

[0082] In some embodiments, the antibody or antigen-binding fragment is an isolated antibody or antigen-binding fragment.

[0083] The present application also provides a nucleic acid encoding the antibody or antigen-binding fragment, or a portion thereof (e.g., encoding the heavy chain variable region or the light chain variable region or one or more CDRs of the antibody or antigen-binding fragment). In some embodiments, the nucleic acid is an isolated nucleic acid.

[0084] The present application also provides a vector comprising the nucleic acid. In some embodiments, the vector comprising the nucleic acid is a nucleic acid fragment, a plasmid, a bacteriophage, or a virus. In some embodiments, the vector is an isolated vector.

[0085] The present application also provides a host cell comprising the nucleic acid or the vector. In some embodiments, the host cell is an isolated host cell. In some embodiments, the host cell is a CHO cell, a HEK cell (such as a HEK293F cell), a BHK cell, a Cosl cell, a Cos7 cell, a CVl cell, or a murine L cell.

[0086] The present application also provides a method of producing the antibody or antigen binding fragment, comprising culturing a host cell comprising a nucleic acid encoding the antibody or antigen binding fragment in a culture medium. In some embodiments, the method further comprises purifying the antibody or antigen binding fragment. Purification can be performed using conventional methods, for example, centrifuging a cell suspension and collecting the supernatant, and further removing impurities by centrifugation again. Methods such as Protein A affinity column and ion exchange column can be used for purifying antibody proteins.

[0087] The present application also provides a pharmaceutical composition comprising the antibody or antigen binding fragment.

[0088] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0089] In some embodiments, the pharmaceutical composition is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, or intralesional injection.

[0090] In some embodiments, the pharmaceutical composition administration method includes, but is not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral injection. The antibody, antigen binding fragment, or composition can be administered by any convenient route, for example, by infusion or bolus injection, absorbed through epithelial or skin mucosa (for example, oral mucosa, rectal and intestinal mucosa, etc.), and can be co-administered with other biologically active agents.

[0091] The present application also provides diagnostic methods and uses. In some embodiments, a method for detecting CD25 expression in a sample is provided, the sample is contacted with the antibody or antigen binding fragment, the antibody or antigen binding fragment binds to CD25, and the binding is detected, i.e., the content of CD25 in the sample. In some embodiments, the use of the antibody or antigen binding fragment in the preparation of a kit for diagnosing or prognosing cancer or tumor is provided.

[0092] In some embodiments, a kit comprising the antibody or antigen binding fragment is provided.

[0093] In some embodiments, the kit further comprises a second antibody that specifically recognizes the anti-CD25 antibody.

[0094] In some embodiments, the second antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, or an enzyme.

[0095] In some embodiments, the kit is used to detect the presence or level of CD25 in a sample.

[0096] In some embodiments, the kit further comprises an antibody or antigen-binding fragment against another antigen.

[0097] In some embodiments, the kit further comprises instructions for use.

[0098] The present disclosure also provides methods and uses for treating a disease. In some embodiments, there is provided a method for treating a disease, comprising administering to a patient an effective dose of the antibody or antigen-binding fragment or the pharmaceutical composition. In some embodiments, there is provided use of the antibody or antigen-binding fragment or the pharmaceutical composition in the treatment of a disease. In some embodiments, there is provided use of the antibody or antigen-binding fragment or the pharmaceutical composition in the manufacture of a medicament for the treatment of a disease.

[0099] In some embodiments, the disease is a CD25 -related disease.

[0100] In some embodiments, the CD25 -related disease is an inflammatory disease, an immune-related disease, a cancer, or a tumor.

[0101] In some embodiments, the immune-related disease comprises transplant rejection, an autoimmune disease, or an infectious disease.

[0102] In some embodiments, the transplant rejection is allograft rejection or xenograft rejection.

[0103] In some embodiments, the inflammatory disease or immune-related disease is selected from at least one of rheumatoid arthritis, ankylosing spondylitis, psoriasis, psoriatic arthritis, type 1 diabetes, insulin-dependent type 2 diabetes, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, inflammatory bowel disease, Crohn’s disease, ulcerative colitis, dermatomyositis, Sjogren’s syndrome, arteritis, aplastic anemia, asthma, scleroderma, uveitis, pustulosis palmoplantar, erosive lichen planus, bullous pemphigoid, epidermolysis bullosa, contact dermatitis, and atopic dermatitis.

[0104] In some embodiments, the cancer or tumor is selected from at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, pancreatic cancer, head and neck cancer, sarcoma, cytoma, melanoma, myeloma, glioma, leukemia, and lymphoma.

[0105] The specific dose and treatment regimen for any particular patient will depend on a variety of factors, including the specific antibody or derivative employed, the age and general health of the patient, the age, weight, and general health of the patient, sex and diet, the time of administration, the frequency of administration, the pharmaceutical combination, and the severity of the particular disease being treated. These factors are within the judgment of the medical care practitioner, which is within the purview of one of ordinary skill in the art. The dose will also depend on the individual patient to be treated, the route of administration, the formulation of the drug, the nature of the compound used, the severity of the disease, and the effect desired. The dose used can be determined by principles of pharmacology and pharmacokinetics well known in the art. In some embodiments, the antibody of the present application is administered to a patient at a dose of 0.01 mg / kg to 100 mg / kg of patient body weight per administration. In some embodiments, the administration is once every 1 week to every 3 months.

[0106] In some embodiments, the antibody or antigen-binding fragment or the pharmaceutical composition described herein can be combined with other therapeutic regimens, including administration of one or more of the antibody or antigen-binding fragment or the pharmaceutical composition described herein, and one or more other therapeutic agents or methods used or combined with the antibody or antigen-binding fragment or the pharmaceutical composition described herein. In some embodiments, the other therapeutic regimen includes, but is not limited to, radiotherapy, chemotherapy, hormone therapy, and surgical therapy, etc. For combination therapy, the antibody or antigen-binding fragment or the pharmaceutical composition can be administered simultaneously or separately with the other therapeutic agent. When administered separately, the antibody or antigen-binding fragment or the pharmaceutical composition described herein can be administered before or after the administration of another other therapeutic agent.

[0107] The present application provides methods and uses of the antibody or antigen-binding fragment or the pharmaceutical composition for depleting Treg cells and / or increasing the Teff / Treg cell ratio.

[0108] In some embodiments, the antibody or antigen-binding fragment or the pharmaceutical composition depletes Treg cells while preserving IL-2 signaling on Teff cells. BRIEF DESCRIPTION OF DRAWINGS

[0109] Figure 1: ELISA detection of antibody binding to CD25 protein.

[0110] Figure 2: Binding curve of antibody to CD25-CHO cells.

[0111] Figure 3: Binding curves of antibodies to SU-DHL-1 cells.

[0112] Figure 4: Detection of antibody blocking of IL-2 protein binding to CD25.

[0113] Figure 5: Epitope detection of antibody 5A2, IL-2 protein binding to CD25.

[0114] Figure 6: Detection of antibody mimic killing activity.

[0115] Figure 7: Detection of antibody cell blocking activity.

[0116] Figure 8: Results of antibody anti-tumor activity in MC38 colon cancer model in hCD25 mice.

[0117] Definitions

[0118] Unless otherwise indicated, each of the following terms shall have the meaning set forth herein.

[0119] Definitions

[0120] It should be noted that the term "a" or "an" entity refers to one or more than one of that entity; for example, "an antibody" shall be understood to mean one or more antibodies, and, accordingly, the terms "a" or "an", "one or more", and "at least one" can be used interchangeably herein.

[0121] The terms "comprising" or "including," as used herein, mean that the compositions and methods comprise the recited elements, e.g., components or steps, but do not exclude other elements from being present. "Consisting essentially of means that the compositions and methods exclude other elements of any essential significance to the combination, but allow for the presence of elements that do not pertain to the essence of the compositions or methods. "Consisting of means that the exclusion of unrecited elements is required.

[0122] "Antibody," "antigen binding fragment" refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen. An antibody can be an intact antibody and any antigen binding fragment thereof or a single chain thereof. Thus, "antibody" includes any protein or peptide that comprises at least a portion of an immunoglobulin molecule that has the ability to specifically bind to an antigen. Antibodies and antigen binding fragments include, but are not limited to, a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a variable region of a heavy chain (VH), a variable region of a light chain (VL), a constant region of a heavy chain (CH), a constant region of a light chain (CL), a framework region (FR), or any portion thereof, or at least a portion of a binding protein. CDR regions include CDR regions of a light chain (LCDR1-3) and CDR regions of a heavy chain (HCDR1-3). An antibody or antigen binding fragment can be a polypeptide or polypeptide complex that specifically recognizes and binds one or more (e.g., two) antigens. An antibody or antigen binding fragment that specifically recognizes and binds multiple (e.g., two) antigens can be referred to as a multispecific (e.g., bispecific) antibody or antigen binding fragment.

[0123] "Single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin. In some aspects, these regions are connected by a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine for flexibility, as well as serine or threonine for solubility, and can connect the N-terminus of the VH to the C-terminus of the VL, or vice versa. Despite the removal of the constant regions and the introduction of the linker, the protein retains the specificity of the original immunoglobulin. scFv molecules are generally known in the art, for example, described in U.S. Patent 5,892,019.

[0124] The class of a heavy chain of an antibody includes gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses thereof (e.g., γ1-γ4). The chain's identity determines the "class" of the antibody as IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, etc., have been well characterized and the functional specificity conferred thereby is known. All immunoglobulin classes are within the scope of the present disclosure. In some embodiments, the immunoglobulin molecule is of the IgG class.

[0125] The light chain of an antibody can be classified as kappa (K) or lambda (l). Each heavy chain can be associated with either a K or a l light chain. Generally, when an immunoglobulin is produced by a hybridoma, B cell, or genetically engineered host cell, its light and heavy chains are bound, by covalent disulfide bonds, at their "tailpiece" portions, and the "tailpiece" portions of the two heavy chains are bound, by covalent disulfide bonds or noncovalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus at the forked end of the Y-shaped end to the C-terminus at the bottom of each chain. The immunoglobulin K light chain variable region is Vκ; the immunoglobulin l light chain variable region is Vl.

[0126] The light chain variable region (VL) and the heavy chain variable region (VH) portions determine antigen recognition and specificity. The constant regions of the light and heavy chains confer important biological properties, such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. By convention, the numbering of the constant regions increases as they become more distal from the antigen binding site or amino-terminal end of the antibody. The N-terminal portion is the variable region, and the C-terminal portion is the constant region; the CH3 and CL domains actually comprise the carboxy terminus of the heavy and light chains, respectively.

[0127] In naturally occurring antibodies, the six "complementarity determining regions" or "CDRs" present in each antigen binding domain are short, non-contiguous amino acid sequences that form the antigen binding domain and specifically bind to an antigen, provided that the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining other amino acids in the antigen binding domain, referred to as "framework" regions, show less intermolecular variability. The framework regions mostly adopt a beta-sheet conformation, and the CDRs form loop structures attached to them, or in some cases form part of a beta-sheet structure. Thus, the framework regions position the CDRs in the correct orientation by forming a scaffold, which positions the CDRs in the correct orientation through interchain noncovalent interactions. The antigen binding domain with CDRs in specific positions forms a surface that is complementary to an epitope on an antigen, which promotes the noncovalent binding of the antibody to its antigenic epitope.

[0128] Generally in an antibody molecule, each heavy and light chain has three CDRs, designated HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. In order of location, generally the heavy chain variable region comprises VH FR1, HCDR1, VH FR2, HCDR2, VH FR3, HCDR3, and VH FR4, and the light chain variable region comprises VL FR1, LCDR1, VL FR2, LCDR2, VL FR3, LCDR3, and VL FR4. For a given heavy or light chain variable region, one of ordinary skill in the art can identify the amino acids that comprise the CDRs and framework regions by known methods (see Kabat, E., et al., U.S. Department of Health and Human Services, Sequences of Proteins of Immunological Interest, (1983) and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

[0129] In cases where the terms used and / or accepted in the art have two or more definitions, the definition of the term used herein includes all such meanings unless specifically indicated to the contrary. One specific example is the use of the term "complementarity determining region" ("CDR") to describe the non-contiguous antigen binding sites found within the variable region of both heavy and light chain polypeptides. This particular region can be defined differently in the art, for example, as described in Kabat et al., U.S. Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and Chothia et al., J. Mol. Biol. 196:901-917 (1987), which are incorporated by reference herein in their entireties.

[0130] The CDRs according to the Kabat and Chothia definitions include overlaps or subsets of amino acid residues when compared to each other. Nonetheless, either definition applied to refer to the CDRs of an antibody is within the scope of the present application. The exact residue numbers encompassing a particular CDR will vary depending on the sequence and size of the CDR. One of ordinary skill in the art can generally determine which particular residues are encompassed by a CDR based on the variable region amino acid sequence of an antibody.

[0131] Kabat et al. also defined a numbering system applicable to the variable region sequences of any antibody. One of ordinary skill in the art can apply this "Kabat numbering" system to any variable region sequence without reliance on experimental data other than the sequence itself. "Kabat numbering" refers to the numbering system set forth by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Antibodies can also be numbered using the EU or Chothia numbering system.

[0132] The antibodies provided herein can be derived from any animal, including birds and mammals. Preferably, the antibodies are human, murine, equine, rabbit, goat, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region can be condricthoid in origin (e.g., from a shark).

[0133] A "heavy chain constant region" includes at least one of a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a fragment thereof. The heavy chain constant region of an antibody can be derived from different immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide can include a CH1 domain derived from an IgGl molecule and a hinge region derived from an IgG3 molecule. In another embodiment, the heavy chain constant region can include a hinge region derived in part from an IgGl molecule and in part from an IgG3 molecule. In another embodiment, a portion of the heavy chain can include a chimeric hinge region derived in part from an IgGl molecule and in part from an IgG4 molecule.

[0134] A "light chain constant region" includes a portion of the amino acid sequence from an antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or a constant lambda domain. A "light-heavy pair" refers to the collection of a light chain and a heavy chain that can dimerize through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain. The four chains are connected by disulfide bonds in a "Y" configuration, with the light chain beginning at the "Y" mouth and continuing around the heavy chain through the variable region.

[0135] A "disulfide bond" refers to a covalent bond formed between two sulfur atoms. The thiol group of a cysteine can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are connected by a disulfide bond.

[0136] A "chimeric antibody" is any antibody whose variable region is obtained or derived from a first species and whose constant region (which can be intact, partial, or modified) is derived from a second species. In certain embodiments, the variable region is from a non-human source (e.g., mouse or primate), and the constant region is from a human source.

[0137] An "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor or portion thereof. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually are formed by structurally discontinuous parts of a molecule and are often conformational in nature.

[0138] "Specific binding" or "specific for" generally refers to the binding of an antibody or antigen-binding fragment to a particular antigen via its antigen-binding domain to the complementarity of an epitope to form a relatively stable complex. "Specificity" can be expressed in terms of the relative affinity of an antibody or antigen-binding fragment for a particular antigen or epitope. For example, if antibody "A" has a greater relative affinity than antibody "B" for the same antigen, antibody "A" can be said to have a higher specificity than antibody "B" for that antigen. Specific binding can be described in terms of the equilibrium dissociation constant (KD), with a smaller KD implying tighter binding. Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, bio-layer interferometry, and the like. An antibody that "specifically binds" an antigen a includes an antibody that has an equilibrium dissociation constant KD for antigen a of less than or equal to about 100 nM, less than or equal to about 10 nM, less than or equal to about 5 nM, less than or equal to about 1 nM.

[0139] Antibodies, antigen-binding fragments of the application include modified derivatives, i.e., modifications by covalent attachment of any type of molecule to the antibody or antigen-binding fragment, wherein the covalent attachment does not prevent the antibody or antigen-binding fragment from binding to an epitope. The antibody or antigen-binding fragment can be glycosylated, acetylated, pegylated, phosphorylated, amidated, derivatized via known protecting / blocking groups, proteolytic cleavage, attachment to a cell ligand or other protein, and the like. Any of numerous chemical modifications have been made to alter the mobility of the molecule in various systems, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like.

[0140] The term "isolated" as used herein with respect to cells, nucleic acids, polypeptides, antibodies, etc., e.g., "isolated" DNA, RNA, polypeptide, antibody, refers to a molecule separated from other components of its natural environment, such as other cellular components, e.g., DNA or RNA. The term "isolated" as used herein also refers to nucleic acids or peptides that are substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques or chemical synthesis, or chemical precursors or other chemicals when chemically synthesized. In addition, "isolated nucleic acid" is intended to include nucleic acid fragments that are not naturally occurring as fragments and would not be found in the natural state. The term "isolated" is also used herein to refer to a cell or polypeptide that is separated from other cellular proteins or tissues. Isolated polypeptides are intended to include purified and recombinant polypeptides. Isolated polypeptides, antibodies, etc., are typically prepared by at least one purification step. In some embodiments, isolated nucleic acids, polypeptides, antibodies, etc., are at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or a range between any two of these values (inclusive of the endpoints) or any value therein.

[0141] An "amino acid" refers to an organic compound containing both an amino group and a carboxyl group, such as an alpha-amino acid, which can be encoded by a nucleic acid, either directly or in the form of a precursor. A single amino acid is encoded by a nucleic acid composed of three nucleotides, a so-called codon or base triplet. Each natural amino acid is encoded by at least one codon. The same amino acid is encoded by different codons is referred to as "degeneracy of the genetic code." Amino acids include natural amino acids and unnatural amino acids. Natural amino acids include alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gin, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0142] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a side chain (R group) of similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. Examples of groups of amino acids that have chemical properties similar include: 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid.

[0143] The number of amino acids in a "conservative amino acid substitution" of a VL, VH, heavy chain constant region, light chain constant region, heavy chain, or light chain is about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15 conservative amino acid substitutions, or a range between any two of these values, inclusive of the endpoints, or any value therein.

[0144] "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing positions in each sequence which can be aligned. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.

[0145] A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), or when the polynucleotide is RNA, thymine is replaced by uracil (U). A "polynucleotide sequence" can be represented in letter form of a polynucleotide molecule. This letter representation can be inputted into databases in computers with central processing units and used for bioinformatics applications, for example, for functional genomics and homology searches.

[0146] "Polynucleotide" and "nucleic acid" are used interchangeably and refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. A polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present in a polynucleotide, the modification can be in the form of a modification to a nucleotide of the polymer, but also to the backbone of the polymer. The polynucleotide can be modified chemically or biochemically post-assembly of the polymer. The sequence of a polynucleotide can be interrupted by non-nucleotide components. A polynucleotide can be further modified, such as by conjugation to an labeling component. The term also refers to both double- and single-stranded molecules. Unless otherwise indicated, the use of "polynucleotide" or "nucleic acid" in the present application is intended to include both the double-stranded form and each of the two complementary single-stranded forms in a known or predicted double-stranded form.

[0147] "Encoding" as applied to polynucleotides refers to the polynucleotide that is said to "encode" a polypeptide, in its natural state or when manipulated by methods known to those skilled in the art, transcribed and / or translated into the polypeptide and / or fragments thereof.

[0148] "Recombinant" with respect to a polypeptide or polynucleotide means a form that is not naturally occurring, non-limiting examples of which can be produced by combining elements of different origin into a polynucleotide or polypeptide that is not naturally found.

[0149] "Polypeptide" is intended to encompass both "polypeptides" in the singular and plural form, and refers to a molecule formed by linear linkage of amino acid monomers by amide bonds (also known as peptide bonds). "Polypeptide" refers to any single chain or multiple chains of two or more amino acids, and does not refer to a particular length of product. Thus, included within the definition of "polypeptide" are peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains", or any other term used to refer to two or more amino acid chains, and "polypeptide" can be used in place of or in alternation with any of the foregoing terms. The term "polypeptide" is also intended to refer to products produced by post-expression modifications of a polypeptide, including but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic processing, or non-naturally occurring amino acid modifications. A polypeptide can be derived from a natural biological source or produced by recombinant techniques, but it need not be translated from a specified nucleic acid sequence, it can be produced in any manner including chemical synthesis.

[0150] "Pharmaceutically acceptable" generally refers to a non-toxic material that is acceptable for use in animals, particularly humans, by the national government or listed in the recognized pharmacopeias. In some embodiments, "pharmaceutically acceptable" as used herein means approved by a regulatory agency of the Federal or a state government or listed in the recognized pharmacopeias for use in animals, and more particularly in humans. In addition, "pharmaceutically acceptable excipient" generally refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation auxiliary of the like.

[0151] "Excipient" refers to a diluent, adjuvant, vehicle, or carrier with which the active ingredient is administered to a patient. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of animal origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, or synthetic oils. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried milk products, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Antibacterial agents such as benzyl alcohol or methyl parabens, antioxidants such as ascorbic acid, or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, and agents for the adjustment of tonicity such as dextrose are also envisioned. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated into a suppository with traditional binders and carriers such as glycerin, sorbitol, or polyethylene glycols. Oral formulations can include standard carriers such as pharmaceutical grades of starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E. W. Martin, the entire disclosure of which is hereby incorporated by reference into this application. Such compositions will contain a therapeutically effective amount of the antibody or antigen binding fragment, preferably in purified form, together with a suitable amount of excipient to provide the desired dosage form. The formulation should suit the mode of administration. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0152] In some embodiments, the compositions are formulated in accordance with routine procedures as pharmaceutical compositions adapted to intravenous injection to humans. Compositions for intravenous administration are typically solutions in sterile isotonic aqueous buffer. Compositions can also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0153] The compositions of the present application can be in a variety of forms. These forms include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), dispersions or suspensions, lipid formulations, and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Common preferred compositions are in the form of injectable solutions or infusible solutions. In some embodiments, the mode of administration is parenteral (e.g., intravenous, subcutaneous, intraabdominal, intramuscular). In some embodiments, the antibody is administered by intravenous infusion or injection. In some embodiments, the antibody is administered by intramuscular or subcutaneous injection.

[0154] "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent, slow down, lessen, ameliorate, or stop the progression, progression of, or progression of, an undesirable physiological change or disorder, such as the progression of a disease, including but not limited to alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration, palliation, remission, lessening or elimination (whether partial or total) of the disease state, prolonging of survival beyond that expected in the absence of such treatment, and the like, whether detectable or undetectable. Patients in need of treatment include those who already have the condition or disorder, those prone to have the condition or disorder, or those in which the condition or disorder is to be prevented, and who can or are expected to benefit from administration of the antibody or pharmaceutical composition provided herein for detection, diagnostic procedures, and / or treatment.

[0155] "Patient" refers to any mammal in need of diagnosis, prognosis, or treatment, including humans, dogs, cats, rabbits, mice, horses, cows, and the like.

[0156] "About" refers to the conventional error range for a given technical field or technical discipline, which will be well understood by those of ordinary skill in the art. In some embodiments, "about" refers to a range of ±10%, ±5%, or ±1% of a value stated.

[0157] "EC 50"EC50" or "half maximal effective concentration" refers to the concentration of a drug or other substance that elicits a response halfway between the baseline and maximum after a certain period of exposure. In other words, it is the concentration of a drug or other substance that produces 50% of the maximal effect achievable.

[0158] Methods of making antibodies and polynucleotides encoding antibodies

[0159] The present application also provides polynucleotides or nucleic acid molecules encoding the antibodies, antigen binding fragments, and derivatives thereof described herein. The polynucleotides provided herein can encode a heavy chain variable region, a light chain variable region, an Fc region, a portion of a heavy chain variable region, a portion of a light chain variable region, a heavy chain, or a light chain, etc. Methods of making antibodies are well known in the art and are described herein.

[0160] In certain embodiments, the antibodies produced do not elicit a deleterious immune response in the animal (e.g., human) to be treated. In some embodiments, the antibodies, antigen binding fragments, or derivatives provided herein are modified using art-recognized techniques to reduce their immunogenicity. For example, the antibodies can be humanized, primatized, deimmunized, or chimeric antibodies can be made. These types of antibodies are derived from non-human antibodies, typically murine or primate antibodies, that retain or substantially retain the antigen binding properties of the parent antibody but are less immunogenic in humans. This can be accomplished by a variety of methods, including (a) grafting the entire non-human variable region onto a human constant region to produce a chimeric antibody; (b) grafting at least a portion of one or more non-human complementarity determining regions (CDRs) into a human framework and constant region, with or without retention of key framework residues; or (c) grafting the entire non-human variable region but "hiding" surface residues by substituting them with parts of a human source. Typically, framework residues in the human framework region will be substituted with the corresponding residues from the CDR donor antibody, such as residues that improve antigen binding. These framework substitutions can be identified by methods known in the art, such as by modeling of the interactions of CDR and framework residues to identify framework residues important for antigen binding and by sequence comparison to identify unusual framework residues at particular positions. (See U.S. Patent No. 5,585,089; the entire contents of which are incorporated herein by reference). Antibodies can be humanized using a variety of techniques known in the art, such as CDR-grafting (U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), resurfacing (EP 592,106; EP 519,596), and chain-shuffling (U.S. Patent No. 5,565,332), the entire contents of which are incorporated herein by reference.

[0161] Deimmunization can also be used to reduce the immunogenicity of the antibody. In the present application, the term "deimmunization" includes altering the antibody to modify T cell epitopes (see, e.g., WO / 9852976A1 and WO / 0034317A2). For example, the heavy chain variable region sequence and the light chain variable region sequence from the starting antibody are analyzed and a human T cell epitope "map" is generated from each variable region showing the location of epitopes relative to the complementarity determining regions (CDRs) and other key residues within the sequence. Individual T cell epitopes from the T cell epitope map are analyzed to identify alternative amino acid substitutions that have a lower risk of altering antibody activity. A series of alternative heavy chain variable region sequences and light chain variable region sequences comprising combinations of amino acid substitutions are designed and these sequences are subsequently incorporated into a series of binding polypeptides. The genes for the complete heavy and light chains comprising the modified variable regions and human constant regions are then cloned into expression vectors and the plasmids are subsequently transfected into cell lines to produce the complete antibodies. The antibodies are then compared using appropriate biochemical and biological assays to identify the best antibody.

[0162] The binding specificity of the antibodies or antigen-binding fragments provided herein can be detected by in vitro assays, such as co-immunoprecipitation, radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA).

[0163] Production of scFv can be performed using techniques for producing single chain units (U.S. Patent No. 4,694,778). Single chain units are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain fusion peptide. Techniques for assembling functional Fv fragments in E. coli can also be used.

[0164] Examples of techniques that can be used to produce single chain Fv (scFv) and antibodies include those described in U.S. Patent Nos. 4,946,778 and 5,258,498. For certain uses, including in vivo use of the antibodies and in vitro detection assays, chimeric antibodies, humanized antibodies, or fully human antibodies can be used. Chimeric antibodies are a class of molecules in which different portions of the antibody are derived from different animal species, such as antibodies having the variable regions of a murine monoclonal antibody and the constant regions of a human immunoglobulin. Methods for producing chimeric antibodies are known in the art, see U.S. Patent Nos. 5,807,715, 4,816,567, and 4,816,397, the entire contents of each of which are incorporated herein by reference.

[0165] In addition, primatized antibodies containing monkey variable regions and human constant region sequences can be produced, as described in U.S. Patent Nos. 5,658,570, 5,693,780, and 5,756,096, the entire contents of each of which are incorporated herein by reference.

[0166] Antibodies can be produced by a variety of methods known in the art, including phage display methods using antibody libraries derived from immunoglobulin sequences. See also U.S. Patents 4,444,887 and 4,716,111, and PCT publications WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33935, and WO 91 / 10741, each of which is incorporated herein by reference in its entirety.

[0167] In another embodiment, DNA encoding the desired monoclonal antibody can be isolated and sequenced using conventional methods (e.g., using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the murine antibody). The isolated and subcloned hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into prokaryotic or eukaryotic host cells such as E. coli cells, Simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulins. Isolated DNA (which can be synthetic as described herein) can also be used to produce sequences for the constant and variable regions of the antibody, as described in U.S. Patent 5,658,570, which is incorporated by reference herein in its entirety. This method extracts RNA from the selected cells and converts it to cDNA, which is then amplified by PCR techniques using Ig-specific primers. Suitable probes for this purpose are also mentioned in U.S. Patent 5,658,570.

[0168] In addition, using conventional recombinant DNA techniques, one or more CDRs of an antibody of the application can be inserted into a framework region, for example, into a human framework region to construct a humanized non-fully human antibody. The framework region can be a naturally occurring or consensus framework region, preferably a human framework region. Some polynucleotides can encode antibodies that, as a result of the combination of framework and CDR regions, specifically bind to at least one epitope of the antigen of interest. One or more amino acid substitutions can be made within the framework region, which can be selected to improve the binding of the antibody to its antigen. In addition, substitutions or deletions of cysteine residues in one or more variable regions that participate in interchain disulfide bonding can be made using this approach, to produce antibody molecules lacking one or more interchain disulfide bonds. Other alterations to the polynucleotides within the skill of the art are also contemplated by the present application.

[0169] Antibodies can be produced by using conventional recombinant DNA technology. Techniques for selecting, constructing and cultivating vectors and cell lines that produce antibodies, etc. can be selected, constructed and cultivated using techniques well known to those skilled in the art. These techniques are described in various laboratory manuals and major publications, for example, Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells, D. L. Hacker, F. M. Wurm, in Reference Module in Life Sciences, 2017, the entire contents of which, including supplements, are incorporated herein by reference in their entirety.

[0170] In some embodiments, DNA encoding the antibody can be designed and synthesized according to the antibody amino acid sequences described herein in a conventional manner, inserted into an expression vector, and then transfected into host cells, and the transfected host cells are cultured in a culture medium to produce the monoclonal antibody. In some embodiments, the antibody expression vector includes at least one promoter element, an antibody coding sequence, a transcription termination signal and a polyA tail. Other elements include enhancers, Kozak sequences and donor and acceptor sites for RNA splicing flanking the inserted sequence. Efficient transcription can be obtained by the early and late promoters of SV40, the long terminal repeat sequences from retroviruses such as RSV, HTLV1, HIVI and cytomegalovirus early promoters, and other cell promoters such as the actin promoter. Suitable expression vectors can include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or Plncx, pcDNA3.1(+ / -), pcDNA3.1 / Zeo(+ / -), pcDNA3.1 / Hygro(+ / -), pcDNA3.1 / G418(+ / -), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI and pCS2, etc. Commonly used mammalian cells include 293 cells, Cos1 cells, Cos7 cells, CV1 cells, murine L cells and CHO cells, etc.

[0171] In some embodiments, the inserted gene fragment needs to contain a screening marker, and common screening markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance and other screening genes, so as to facilitate the screening and isolation of successfully transfected cells. The constructed plasmid is transfected into host cells without the above-mentioned genes, and the successfully transfected cells grow in large quantities after culture in selective medium, producing the desired target protein.

[0172] In addition, mutations can be introduced using standard techniques known to those of skill in the art into the nucleotide sequences encoding the antibodies of the application, including, but not limited to, site-directed mutagenesis and PCR-mediated mutagenesis, which result in amino acid substitutions.

[0173] The relevant descriptions of the publications mentioned herein are all incorporated by reference in their entirety. DETAILED DESCRIPTION

[0174] The technical solutions of the present application are further illustrated below by specific examples, which do not represent a limitation on the scope of protection of the present application. Some non-essential modifications or adjustments made by others according to the concept of the present application still fall within the scope of protection of the present application.

[0175] The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0176] Example 1: FACS screening of scFvs that bind CD25 and do not block CD25 / IL2 binding

[0177] Flow cytometry (FACS) was used to detect the binding of scFvs to CD25 and to detect whether the scFvs blocked the binding of IL-2 to CD25. scFvs with high binding ability to CD25 and without blocking IL-2 binding to CD25 were screened for subsequent construction.

[0178] 1.1 Construction of CD25-CHO cells and preparation of anti-Myc PE

[0179] Construction method of CD25-CHO cells: The nucleotide sequence encoding human CD25 (NM_000417.1) was cloned into an expression vector to construct a recombinant plasmid, which was linearized by enzyme digestion and then electroporated into CHO-K1 cells. After 48 hours (h) of culture, 25 μM MSX (methionine sulfoximine) was added for screening, and a CHO-CD25 cell strain stably expressing human CD25 was obtained.

[0180] Preparation of anti-Myc PE: The heavy chain nucleic acid sequence and light chain nucleic acid sequence encoding the anti-Myc antibody were cloned into an expression vector to obtain a recombinant plasmid. The plasmid was transformed into expression cells HEK293F for expression, and the supernatant was collected and purified to obtain an antibody protein sample. The protein sample was labeled with phycoerythrin (PE) to obtain anti-Myc PE.

[0181] The heavy chain amino acid sequence of the anti-Myc antibody is SEQ ID NO: 40, and the light chain amino acid sequence is SEQ ID NO: 41, as shown below:

[0182] The heavy chain amino acid sequence of the anti-Myc antibody is SEQ ID NO: 40, and the light chain amino acid sequence is SEQ ID NO: 41, as shown below: The heavy chain amino acid sequence of the anti-Myc antibody is SEQ ID NO: 40, and the light chain amino acid sequence is SEQ ID NO: 41, as shown below:

[0183] Light chain amino acid sequence of anti-Myc antibody (SEQ ID NO: 41):

[0184] 1.2 Preparation of scFv

[0185] After Biotin-CD25 (Acrobiosystems, CAT: ILA-H82E6) was combined with streptavidin magnetic beads (Thermo, CAT: 65602), the phage library was incubated, washed, and then trypsin was added to elute the phage specifically bound to the CD25 antigen protein from the magnetic beads, which was used to infect E. coli TG1. After multiple enrichment and screening, the scFv target gene on the phagemid was cloned into a prokaryotic expression plasmid, which was then transformed into E. coli BL21 (DE3) by electroporation. After the colonies were cultured at 37°C to the logarithmic phase, the culture temperature was adjusted to 28°C and cultured overnight. The next day, the supernatant was collected after centrifugation at 4000 rpm and 4°C for 30 min, and was used for FACS detection.

[0186] 1.3 FACS screening of scFv that binds CD25 and does not block CD25 / IL2 binding

[0187] FACS screening of scFv binding to CD25: FACS buffer was prepared by adding 1% fetal bovine serum (FBS) to phosphate buffered saline (PBS). CD25-CHO cells were resuspended with FACS buffer, the cell density was adjusted to 3 million / mL, and 100 μL was added to each well of a 96-well V-shaped plate. After centrifugation at 400g for 5 min, the supernatant was discarded, and 100 μL of the supernatant prepared in Example 1.2 was added. After mixing, the mixture was incubated at 4°C for 1 h. After washing twice with FACS buffer, 100 μL of 1:500 diluted anti-Myc PE diluent was added to each well, and incubated at 4°C for 30 min. After washing twice with FACS buffer, the cell pellets were resuspended with 150 μL of FACS buffer, and the fluorescence intensity (Mean PE-A) was detected using a flow cytometer.

[0188] FACS screening scFv that does not block CD25 / IL2 binding: resuspend CD25-CHO cells with FACS buffer, adjust cell density to 3 million / mL, add to 96-well V-shaped plate, 100 μL per well, 400g centrifugation for 5 min, add supernatant prepared in Example 1.2, 100 μL, mix and incubate at 4°C for 30 min. Take an appropriate amount of Biotin-IL-2 (Proteintech, CAT#HZ-1015-GMP), dilute with PBS to prepare a Biotin-IL-2 dilution solution with a concentration of 150 nM, add to the 96-well V-shaped plate, 100 μL per well, i.e. the final concentration of Biotin-IL-2 per well is 75 nM, incubate at 4°C for 30 min. Wash twice with FACS buffer, add 1:1000 dilution of eBioscience streptavidin PE (Invitrogen, CAT#12-4317-87) dilution, 100 μL per well, incubate at 4°C for 30 min; wash twice with FACS buffer, then resuspend the cell pellet with 150 μL FACS buffer, and detect the fluorescence intensity (Mean PE-A) using a flow cytometer.

[0189] Through the above screening method, scFv that binds to CD25 and does not block the binding of CD25 to IL-2 is screened: scFv (5A2), scFv (3C4), scFv (2A11). The screened scFv is sequenced, and the amino acid sequence composition of its VH and VL is shown in Table 4. The VH and VL in the scFv are connected by a peptide linker, and the amino acid sequence of the peptide linker is: GGGGSGGGGSGGGGS (SEQ ID NO: 32).

[0190] Table 4 Amino acid sequence composition of scFv

[0191] Example 2: Preparation of anti-CD25 antibodies

[0192] The amino acid sequence composition of the variable region and constant region of the anti-CD25 antibody is shown in Table 5, the VH and CH constitute the heavy chain of the antibody, and the VL and CL constitute the light chain of the antibody. The nucleic acid sequence of the anti-CD25 antibody is shown in Table 6. The nucleic acid sequence encoding the heavy chain and the nucleic acid sequence encoding the light chain of each antibody are cloned into an expression vector to obtain a recombinant plasmid, and the correct construction of the recombinant plasmid is verified by sequencing analysis. The above-mentioned recombinant plasmid is transformed into expression cells HEK293F for expression, the supernatant is collected, and the anti-CD25 antibody is obtained by purification. After sequencing, it is consistent with the expected sequence, and is used in the following examples.

[0193] Table 5 Amino acid sequence composition of anti-CD25 antibody

[0194] Table 6 Nucleic acid sequences of anti-CD25 antibodies

[0195] Example 3: ELISA detection of antibody binding to antigen

[0196] The binding ability of the antibodies prepared in Example 2 to CD25 antigen was detected by the ELISA method, and the process is briefly described as follows:

[0197] Antibodies 5A2, 3C4, 2A11, and R5-15 were coated on a 96-well ELISA enzyme plate at a concentration of 2 μg / mL, 100 μL / well, and incubated at 4°C overnight. The next day, after washing 3 times with PBST buffer (0.05% Tween 20, PBS phosphate buffer with a pH of 7.4), 3% bovine serum albumin-containing PBST blocking solution was added for blocking, 200 μL / well, and incubated at 37°C for 2 hours. After washing 3 times with PBST buffer, 100 μL of Biotin-CD25 protein serial dilutions (the initial concentration of Biotin-CD25 protein was 0.5 μg / mL, 3-fold gradient dilution, 12 gradients) were added, and incubated at 37°C for 1 hour. After washing 3 times with PBST buffer, SA-HRP (Jackson Immunoresearch, CAT: 010-030-084) was added, and incubated at 37°C for 1 hour. After washing 8 times with PBST buffer, 100 μL of TMB developing solution (Shanghai Biotech, CAT: E66100) was added, and incubated at room temperature for 15 min. Then, 100 μL of 0.1 M H2SO4 stop solution was added, and the absorbance value was read on an enzyme-labeled instrument at a wavelength of 450 nm.

[0198] The detection results are shown in Figure 1. The EC50 values of antibodies 5A2, 3C4, 2A11, and R5-15 binding to antigen Biotin-CD25 protein were 3.9 ng / mL, 13.5 ng / mL, 38.5 ng / mL, and 1.42 ng / mL, respectively. 50

[0199] Example 4: Detection of antibody binding to CD25-expressing cells

[0200] Flow cytometry was used to detect the binding of antibodies to SU-DHL-1 cells (ATCC), a lymphoma cell line with high expression of CD25, and CD25-CHO cells.

[0201] ​The SU-DHL-1 and CD25-CHO cells in logarithmic growth phase were selected, and the cell density was adjusted to 3 million / mL, and then 100 μL of the cells was added to each well of a 96-well sharp bottom plate (3587), so that the number of cells in each well was 0.3 million, and the mixture was centrifuged at 400 g for 5 min, and the supernatant was discarded. 100 μL of an antibody series diluent (the initial concentration of the antibody was 15 μg / mL, and the dilution was 3 times in gradient, and there were 12 gradients) was added to resuspend the cell precipitate, and the mixture was incubated at 4°C for 1 h. After washing with FACS buffer, the mixture was centrifuged at 400 g for 5 min, 100 μL of a 1:1000 diluent of eBioscience PE (Invitrogen, CAT#12-4998-82) was added to each well, and the mixture was incubated at 4°C for 30 min. After washing with FACS buffer, the mixture was centrifuged at 400 g for 5 min, 150 μL of FACS buffer was added to resuspend the cell precipitate, and the fluorescence intensity (Mean PE-A) was detected by using a flow cytometer.

[0202] The results are shown in FIG. 2, and the EC50 of the antibodies 5A2, 3C4, 2A11 and R5-15 binding to CD25-CHO cells was 3.42 μg / mL, 11.92 μg / mL, 3.35 μg / mL and 1.53 μg / mL, respectively. 50 The results are shown in FIG. 2, and the EC50 of the antibodies 5A2, 3C4, 2A11 and R5-15 binding to CD25-CHO cells was 3.42 μg / mL, 11.92 μg / mL, 3.35 μg / mL and 1.53 μg / mL, respectively.

[0203] The results are shown in FIG. 3, and the EC50 of the antibody 5A2 binding to SU-DHL-1 cells was 0.28 μg / mL. 50 The results are shown in FIG. 3, and the EC50 of the antibody 5A2 binding to SU-DHL-1 cells was 0.28 μg / mL.

[0204] Example 5: Detection of the effect of the antibody on the binding ability of IL-2 to CD25

[0205] 5.1 Detection of whether the antibody blocks the binding of free recombinant human IL-2 protein to CD25 on the surface of CD25-CHO cells by using flow cytometry.

[0206] CD25-CHO cells in logarithmic growth phase were selected, the cell density was adjusted to 3 million / mL, and the cells were added to a 96-well sharp bottom plate (3587) at 100 μL per well, i.e. 300,000 cells per well. Centrifugation was performed at 400 g for 5 min, and the supernatant was discarded. 100 μL of an antibody dilution solution at a concentration of 30 μg / mL was added to each well. The cell pellet was resuspended and incubated at 4°C for 30 min. An appropriate amount of Biotin-IL-2 (Proteintech, CAT#HZ-1015-GMP) was diluted with PBS buffer to prepare a Biotin-IL-2 dilution solution at a concentration of 150 nM. The solution was added to the cells at 100 μL per well, i.e. the final concentration of Biotin-IL-2 in each well was 75 nM, and the mixture was incubated at 4°C for 30 min. FACS buffer was used for washing twice, and 100 μL of a 1:500 dilution of eBioscience streptavidin PE (Invitrogen, CAT#12-4317-87) was added to each well. The mixture was incubated at 4°C for 30 min. FACS buffer was used for washing, and 150 μL of FACS buffer was added to resuspend the cell pellet in each well. The fluorescence intensity (Mean PE-A) was detected by flow cytometry. If the antibody affects the binding of IL-2 to CD25, the fluorescence intensity will decrease.

[0207] The results are shown in FIG. 4. Antibody 5A2 does not block the binding of IL-2 to CHO-CD25 cells and is a non-blocking antibody of IL-2.

[0208] 5.2 Detection of whether the epitopes of antibody 5A2 and recombinant human IL-2 protein binding to CD25 protein are the same by using ForteBio Octet molecular interaction analysis system (Octet Qke)

[0209] A 100 nM Biotin-CD25 protein (Acrobiosystems, CAT#ILA-H82E6) solution was prepared, and the 100 nM Biotin-CD25 protein solution was loaded onto a streptavidin biosensor (Sartorius) for 100 seconds (s). After the baseline step, the sensor was exposed to a 100 nM antibody 5A2 solution for 300 s. After the baseline step, the antibody-captured sensor was exposed to a 400 nM IL-2 solution for 300 s. Whether the CD25 protein that has bound to antibody 5A2 can still bind to IL-2 in the solution was detected.

[0210] A solution of 100 nM Biotin-CD25 protein (Acrobiosystems, CAT# IIA-H82E6) was prepared, and the solution of 100 nM Biotin-CD25 protein was loaded onto a streptavidin biosensor (Sartorius) for 100 seconds (s), after a baseline step, the sensor was exposed to a solution of 100 nM IL-2 for 300 s, after a baseline step, the sensor that captured IL-2 was exposed to a solution of 400 nM 5A2 for 300 s, whether the CD25 protein that had bound IL-2 could still bind 5A2 in solution was detected.

[0211] The results are shown in Figure 5, antibody 5A2 does not block the binding of IL-2 to CD25 protein, suggesting that the epitope of antibody 5A2 binding to CD25 protein is different from the epitope of recombinant human IL-2 protein binding to CD25 protein.

[0212] Example 6: Affinity detection of antibody

[0213] The affinity of antibody 5A2 to human CD25 protein was measured using a biomolecular interaction analysis system (BiaCore):

[0214] Antibody 5A2 was coated onto a Protein A chip at a concentration of 10 pg / mL, and the binding kinetics were measured at 25°C using a biomolecular interaction analysis system, and the equilibrium probe; recombinant human CD25 protein (Acrobiosystems, CAT: IIA-H52H9) was coated onto the chip coated with antibody 5A2 at a concentration of 10-400 nM; only flow through buffer (PBS buffer containing 0.05% Tween-20) was used as a control to subtract background and non-specific binding signals; the kinetic constants (K a Kd is the dissociation rate, K d Kd is the dissociation rate, K D Kd is the dissociation rate, K

[0215] As shown in Table 7, antibody 5A2 has binding to recombinant human CD25 protein.

[0216] Table 7 Affinity constants of antibody 5A2 binding to recombinant human CD25 protein

[0217] Example 7: Detection of simulated killing activity of antibody

[0218] The Jurkat effector cell strain is a modified Jurkat-NFAT-FcγRIIIa 158V stable cell strain. The cell stably expresses FcγRIIIa receptor (expressing V158 high affinity mutant) and firefly luciferase expressed by NFAT response element driven. The biological activity of the antibody in the mechanism of ADCC is quantified by the luciferase produced by the activation of the NFAT pathway, and the luciferase activity in the Jurkat effector cell can be quantified by bioluminescence reading. The construction method of the NFAT-Luc-FcγRIIIa Jurkat stable cell strain is as follows: the PUC57 vector carrying Fc gamma RIIIa-158V cDNA (purchased from Proteintech Group, Inc.) is subjected to EcoRI & NotI double enzyme digestion, and then infected with a lentivirus packaging system to infect Jurkat cells, and a Jurkat-FcγRIIIa 158V stable cell strain is obtained by screening. The luciferase reporter gene plasmid pGL4.30[luc2P / NFAT-RE / Hygro] (Progema, CAT: E848A) is electroporated into the above-mentioned Jurkat-FcγRIIIa 158V cell, and a Jurkat-NFAT-FcγRIIIa 158V stable cell strain is obtained after culture and screening.

[0219] The SU-DHL-1 target cells (ATCC) were collected, and the cell density was adjusted to 1 million / mL. The SU-DHL-1 target cells were added to a white 96-well plate at 50 μL per well, i.e. 50,000 cells per well. The antibody 5A2 and the isotype control antibody IgG1 Isotype were serially diluted with 10% FBS RPMI-1640, and the initial concentration of the antibody was 30 μg / mL, which was diluted by 4 times gradient, and 3 replicate wells were set for each antibody concentration. The gradient-diluted antibody was added to the wells with target cells at 50 μL per well.

[0220] The Jurkat-NFAT-FcγRIIIa 158V effector cells were collected, and the cell density was adjusted to 2 million / mL. The effector cells were added at 50 μL per well, i.e. 100,000 cells per well, and the 96-well plate was placed in a cell incubator after mixing. After 5 hours of culture, the 96-well plate was taken out with Bio-GI chromogenic solution (Promega, CAT#G7940), and was placed at room temperature to restore to room temperature. The chromogenic solution was added to the 96-well plate at 50 μL per well, and the reaction was performed for 5 minutes. The luminescence value was read by a microplate reader.

[0221] As shown in FIG. 6, the antibody 5A2 has a good killing effect on CD25-expressing cells, and the EC50 of the antibody 5A2 is 0.03 μg / mL. 50 is 0.03 μg / mL.

[0222] The heavy chain amino acid sequence of the isotype control antibody IgG1 Isotype is SEQ ID NO: 42, and the light chain amino acid sequence is SEQ ID NO: 43, as shown below:

[0223] The heavy chain amino acid sequence of the isotype control antibody IgG1 Isotype (SEQ ID NO: 42):

[0224] The light chain amino acid sequence of the isotype control antibody IgG1 Isotype (SEQ ID NO: 43):

[0225] Example 8: Detection of cell blocking activity of antibodies

[0226] HEK-Blue TM IL-2 cells were used to detect the signal of intracellular phosphorylation signal transducer and transcription activator 5 (pSTAT5) by flow cytometry to characterize the cell blocking activity of the antibodies. The higher the signal, the lower the blocking rate of the antibodies.

[0227] HEK-Blue TM IL-2 cells (InvivoGen, CAT: hkb-il2) were collected, and the cell density was adjusted to 10 million / mL with RPMI-1640 medium containing 10% FBS, and 100 μL was added to each well of a 96-well U-shaped plate, i.e., 1 million cells per well; the antibodies 5A2, RG6292, Daclizumab, and IgG1 Isotype were diluted with RPMI-1640 medium containing 10% FBS, and the antibody concentration was 15 μg / mL. The control group did not add antibodies, and was labeled as NoAb. The antibody diluent was added to each well of the 96-well U-shaped plate, 100 μL per well, and mixed with the HEK-Blue TMIL-2 cells were mixed and placed in a cell incubator, 30 min later, different concentrations of IL-2 solution (33.3 U / mL, 6.7 U / mL, 1.3 U / mL, 0.3 U / mL, 0.05 U / mL, 0.01 U / mL) were added, 50 μL per well, and placed in a cell incubator. After 10 min, the cells were transferred to a 96-well V-shaped plate and centrifuged at 300 g for 5 min at room temperature, and the supernatant was discarded. 200 μL of Foxp3 fixation / breaking membrane buffer (Invitrogen, CAT#00-5523-00) was added to each well to resuspend the cell pellet, and the mixture was incubated at 4°C for 1 h in the dark; the sample was centrifuged at 500 g for 5 min at room temperature, and the supernatant was discarded. 200 μL of pre-cooled 1X PhosflowTM Perm Buffer III (BD Biosciences, CAT#558050) solution was added to each well, and the mixture was incubated at 4°C for 1 h in the dark, then washed twice with FACS buffer, and 100 μL of 1:100 diluted PE anti-STAT5 Phospho (Biolegend, CAT#936904) diluent was added to each well, and the mixture was incubated at 4°C for 30 min in the dark; the sample was washed twice with FACS buffer, and the fluorescence intensity (Mean PE-A) was detected by flow cytometry.

[0228] As shown in Figure 7, antibody Daclizumab is an IL-2 blocking antibody, and the average fluorescence intensity of pSTAT5 after antibody Daclizumab treatment is significantly reduced, inhibiting IL-2 intracellular signal transduction. Antibody RG6292 is an IL-2 non-blocking antibody, and the average fluorescence intensity of pSTAT5 after antibody RG6292 treatment is equivalent to IgG1 Isotype. The average fluorescence intensity of pSTAT5 after antibody 5A2 treatment is equivalent to antibody IgG1 Isotype, indicating that antibody 5A2 does not block IL-2 binding to HEK-Blue IL-2 cells. TM IL-2 cells do not affect IL-2 intracellular signal transduction, which is an IL-2 non-blocking antibody.

[0229] The heavy chain amino acid sequence of antibody RG6292 is SEQ ID NO: 44, and the light chain amino acid sequence is SEQ ID NO: 45, as shown below:

[0230] The heavy chain amino acid sequence of antibody RG6292 is SEQ ID NO: 44, and the light chain amino acid sequence is SEQ ID NO: 45, as shown below:

[0231] The heavy chain amino acid sequence of antibody RG6292 is SEQ ID NO: 44, and the light chain amino acid sequence is SEQ ID NO: 45, as shown below:

[0232] Example 9: Detection of antibody inhibition of tumor proliferation

[0233] The anti-tumor activity of antibody 5A2 was evaluated in a MC38 colon cancer mouse model of B-hIL2RA humanized mice.

[0234] MC38 colon cancer cells (Shanghai Southern Model Organism Technology Co., Ltd.) were cultured and expanded in vitro, and logarithmic growth phase cells were collected, resuspended in PBS, and adjusted to a cell concentration of 1.0 x 10 7 / mL. The cell suspension was injected into the right subcutaneous tissue of B-hIL2RA humanized mice (Shanghai Southern Model Organism Technology Co., Ltd.) using a 1 mL syringe, with 100 μL injected per animal. When the average tumor volume of the mice was about 129 mm 3 , animals with excessively large, small, or irregular tumor shapes were culled, and the animals were divided into 4 groups using a random block method (Table 8). Drug administration was started according to the grouping scheme, and all animals were administered intraperitoneally, with 2 administrations per week for a total of 6 administrations. Animals with tumor volumes > 2500 mm 3 were euthanized, and animals with tumor volumes < 2500 mm 3 were continued to be administered and observed for survival. Mice were observed twice a week for hair and condition, and body weight and tumor size were recorded. TGI% was calculated.

[0235] Table 8. Grouping and administration scheme

[0236] As shown in FIG. 8 and Table 9, on day 10 after the start of administration, the average tumor volume of mice in control group 1 was 2095.41 ± 211.76 mm 3 . The average tumor volume of mice in group 2 was 1269.23 ± 330.12 mm 3 , and the tumor inhibition rate TGI was 42.0%, which was statistically different from the tumor volume of group 1 (P < 0.05). The average tumor volumes of mice in groups 3 and 4 were 1158.33 ± 230.92 mm 3 and 1084.91 ± 208.08 mm 3 , respectively, and the tumor inhibition rates TGI were 47.64% and 51.39%, respectively, which were significantly statistically different from the tumor volume of group 1 (P < 0.01). Antibody 5A2 inhibited the growth of MC38 colon cancer at dosages of 1, 3, and 5 mg / kg.

[0237] Table 9. Tumor volume inhibition rate TGI of mice TV Note: Data is expressed as Mean ± SEM

[0238] T-test was used to analyze and compare the tumor volumes between group 1 and the treatment groups, and ** indicates p < 0.01 and * indicates p < 0.05.

Claims

1. An antibody or antigen-binding fragment that specifically binds to CD25, said antibody or antigen-binding fragment comprising one or more selected from the following: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3: HCDR1 contains or consists of sequences as shown in SEQ ID NO:1 or 2. HCDR2 contains or consists of sequences as shown in SEQ ID NO:3 or 4. HCDR3 comprises or consists of sequences as shown in any of SEQ ID NO:5-8. LCDR1 contains or consists of sequences as shown in any of SEQ ID NO:9-11 LCDR2 contains or consists of the sequence shown in SEQ ID NO:

12. LCDR3 contains or consists of sequences as shown in any of SEQ ID NO:13-15.

2. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein: (1) HCDR1 comprises or consists of the sequence shown in SEQ ID NO:1, HCDR2 comprises or consists of the sequence shown in SEQ ID NO:3, HCDR3 comprises or consists of the sequence shown in SEQ ID NO:5, LCDR1 comprises or consists of the sequence shown in SEQ ID NO:9, LCDR2 comprises or consists of the sequence shown in SEQ ID NO:12, and LCDR3 comprises or consists of the sequence shown in SEQ ID NO:13; or (2) HCDR1 comprises or consists of the sequence shown in SEQ ID NO:1, HCDR2 comprises or consists of the sequence shown in SEQ ID NO:3, HCDR3 comprises or consists of the sequence shown in SEQ ID NO:6, LCDR1 comprises or consists of the sequence shown in SEQ ID NO:9, LCDR2 comprises or consists of the sequence shown in SEQ ID NO:12, and LCDR3 comprises or consists of the sequence shown in SEQ ID NO:13; or (3) HCDR1 comprises or consists of the sequence shown in SEQ ID NO:2, HCDR2 comprises or consists of the sequence shown in SEQ ID NO:4, HCDR3 comprises or consists of the sequence shown in SEQ ID NO:7, LCDR1 comprises or consists of the sequence shown in SEQ ID NO:10, LCDR2 comprises or consists of the sequence shown in SEQ ID NO:12, and LCDR3 comprises or consists of the sequence shown in SEQ ID NO:14; or (4) HCDR1 contains or consists of the sequence shown in SEQ ID NO:2, HCDR2 contains or consists of the sequence shown in SEQ ID NO:4, HCDR3 contains or consists of the sequence shown in SEQ ID NO:8, LCDR1 contains or consists of the sequence shown in SEQ ID NO:11, LCDR2 contains or consists of the sequence shown in SEQ ID NO:12, and LCDR3 contains or consists of the sequence shown in SEQ ID NO:

15.

3. The antibody or antigen-binding fragment as described in claim 1 or 2, wherein the antibody or antigen-binding fragment is a human antibody, a humanized antibody, or a chimeric antibody.

4. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region, the heavy chain variable region comprising a sequence as shown in any one of SEQ ID NO:16-19, or a sequence having at least 80% identity with a sequence shown in any one of SEQ ID NO:16-19, or a sequence having one or more conserved amino acid substitutions compared to a sequence shown in any one of SEQ ID NO:16-19, or consisting thereof; and / or The antibody or antigen-binding fragment includes a light chain variable region comprising, or consisting of, a sequence as shown in any one of SEQ ID NO:20-22, or a sequence having at least 80% identity with a sequence shown in any one of SEQ ID NO:20-22, or a sequence having one or more conserved amino acid substitutions compared to a sequence shown in any one of SEQ ID NO:20-22.

5. An antibody or antigen-binding fragment that specifically binds to CD25, said antibody or antigen-binding fragment comprising a heavy chain variable region, said heavy chain variable region comprising, or consisting of, a sequence as shown in any one of SEQ ID NO:16-19; and / or The antibody or antigen-binding fragment includes a light chain variable region, which contains or consists of a sequence as shown in any one of SEQ ID NO:20-22.

6. An antibody or antigen-binding fragment that specifically binds to CD25, said antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein: (1) The heavy chain variable region contains or consists of the sequence shown in SEQ ID NO:16, and the light chain variable region contains or consists of the sequence shown in SEQ ID NO:20; or (2) The heavy chain variable region contains or consists of the sequence shown in SEQ ID NO:17, and the light chain variable region contains or consists of the sequence shown in SEQ ID NO:20; or (3) The heavy chain variable region contains or consists of the sequence shown in SEQ ID NO:18, and the light chain variable region contains or consists of the sequence shown in SEQ ID NO:21; or (4) The heavy chain variable region contains or consists of the sequence shown in SEQ ID NO:19, and the light chain variable region contains or consists of the sequence shown in SEQ ID NO:

22.

7. The antibody or antigen-binding fragment according to any one of claims 1-6, wherein the antibody or antigen-binding fragment further comprises a heavy chain constant region, the heavy chain constant region comprising, or having at least 80% identity with, the sequence shown in SEQ ID NO:23, or having, or being composed of, a sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:23; and / or The antibody or antigen-binding fragment further comprises a light chain constant region comprising, or being composed of, a sequence as shown in SEQ ID NO:24, or a sequence having at least 80% identity with the sequence shown in SEQ ID NO:24, or a sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:

24.

8. The antibody or antigen-binding fragment according to any one of claims 1-6, wherein the antibody or antigen-binding fragment further comprises a heavy chain constant region consisting of the sequence shown in SEQ ID NO:23 or thereof, and a light chain constant region consisting of the sequence shown in SEQ ID NO:24 or thereof.

9. The antibody or antigen-binding fragment according to any one of claims 1-6, wherein, The antibody or antigen-binding fragment is selected from the following group: Fab, Fab', F(ab')2, F(ab)2, Fd, Fv, Fab / c, scFv, scFv polymer, disulfide bond stable Fv, (dsFv)2, bispecific dsFv, double-chain antibody, disulfide bond stable double-chain antibody, single-domain antibody, nanobody, domain antibody or divalent domain antibody.

10. The antibody or antigen-binding fragment according to any one of claims 1-6, wherein the antibody or antigen-binding fragment comprises a heavy chain, the heavy chain comprising a sequence as shown in any one of SEQ ID NO:25-28, or a sequence having at least 80% identity with a sequence shown in any one of SEQ ID NO:25-28, or a sequence having one or more conserved amino acid substitutions compared to a sequence shown in any one of SEQ ID NO:25-28, or consisting thereof; and / or The antibody or antigen-binding fragment comprises a light chain, the light chain comprising a sequence as shown in any one of SEQ ID NO:29-31, or a sequence having at least 80% identity with a sequence as shown in any one of SEQ ID NO:29-31, or a sequence having one or more conserved amino acid substitutions as shown in any one of SEQ ID NO:29-31, or being composed of such a sequence.

11. An antibody or antigen-binding fragment that specifically binds to CD25, said antibody or antigen-binding fragment comprising a heavy chain, said heavy chain comprising, or consisting of, a sequence as shown in any one of SEQ ID NO:25-28; and / or The antibody or antigen-binding fragment comprises a light chain, which contains or consists of a sequence as shown in any one of SEQ ID NO:29-31.

12. An antibody or antigen-binding fragment that specifically binds to CD25, said antibody or antigen-binding fragment comprising a heavy chain and a light chain, wherein: (1) The heavy chain comprises or consists of the sequence shown in SEQ ID NO:25, and the light chain comprises or consists of the sequence shown in SEQ ID NO:29; or (2) The heavy chain comprises or consists of the sequence shown in SEQ ID NO:26, and the light chain comprises or consists of the sequence shown in SEQ ID NO:29; or (3) The heavy chain comprises or consists of the sequence shown in SEQ ID NO:27, and the light chain comprises or consists of the sequence shown in SEQ ID NO:30; or (4) The heavy chain contains or consists of the sequence shown in SEQ ID NO:28, and the light chain contains or consists of the sequence shown in SEQ ID NO:

31.

13. Biomaterials, for (1) A nucleic acid molecule encoding an antibody or antigen-binding fragment or a portion thereof as described in any one of claims 1-12; or (2) A vector comprising a nucleic acid molecule or a portion thereof encoding an antibody or antigen-binding fragment as described in any one of claims 1-12; or (3) A host cell containing a nucleic acid molecule or a portion thereof encoding an antibody or antigen-binding fragment as described in any one of claims 1-12.

14. A pharmaceutical composition comprising an antibody or antigen-binding fragment as described in any one of claims 1-12.

15. The pharmaceutical composition of claim 14, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

16. The pharmaceutical composition of claim 14 or 15, wherein the pharmaceutical composition is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection or intralesional injection.

17. A kit comprising an antibody or antigen-binding fragment as described in any one of claims 1-12.

18. The kit of claim 17, wherein the kit further comprises a second antibody that specifically recognizes the anti-CD25 antibody.

19. The kit of claim 18, wherein the second antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, or an enzyme.

20. The kit according to any one of claims 17-19, wherein the kit is used to detect the presence or level of CD25 in a sample.

21. The kit according to any one of claims 17-20, wherein the kit further comprises an antibody or antigen-binding fragment against other antigens.

22. The kit according to any one of claims 17-21, wherein the kit further includes instructions for use.

23. The use of the antibody or antigen-binding fragment according to any one of claims 1-12 or the pharmaceutical composition according to any one of claims 14-16 for treating a disease, or in the preparation of a medicament for treating a disease.

24. A method of treating a disease, comprising administering to a patient in need an effective amount of an antibody or antigen-binding fragment as described in any one of claims 1-12 or a pharmaceutical composition as described in any one of claims 14-16.

25. The use as described in claim 23 or the method as described in claim 24, wherein the disease is a CD25-related disease.

26. The use or method as described in claim 25, wherein the CD25-related disease is an inflammatory disease, an immune-related disease, cancer, or a tumor.

27. The use or method of claim 26, wherein the immune-related disease is selected from transplant rejection, autoimmune diseases, and infectious diseases.

28. The use or method as described in claim 27, wherein the transplant rejection is allogeneic graft rejection or xenograft rejection.

29. The use or method of claim 26, wherein the inflammatory disease or immune-related disease is selected from at least one of rheumatoid arthritis, ankylosing spondylitis, psoriasis, psoriatic arthritis, type 1 diabetes, insulin-dependent type 2 diabetes, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, polymyositis dermatitis, Sjögren's syndrome, arteritis, aplastic anemia, asthma, scleroderma, uveitis, palmoplantar pustulosis, corrosive lichen planus, bullous pemphigus, bullous epidermolysis bullosa, contact dermatitis, and atopic dermatitis.

30. The use or method of claim 26, wherein the cancer or tumor is selected from at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, pancreatic cancer, head and neck cancer, sarcoma, cell tumor, melanoma, myeloma, glioma, leukemia, and lymphoma.

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